ANNUAL MEETING ABSTRACTS 253A tract. KIT is expressed in many human neoplasms, and its activation through gain-of- 1 case showed a direct transition from IDL to a BCA. Focal acinic differentiation was function mutations has been shown to be the underlying genetic event in gastrointestinal common. Most cases had an indistinct myoepithelial layer on H&E, however some stromal tumors (GISTs), germ cell tumors and hematologic malignancies. KIT is also demonstrated a focal clear cell myoepithelial layer. Others had periductal hyalinization. expressed in adenoid cystic carcinomas (ACC). Tyrosine kinase inhibitors were used Immunohistochemically, IDL’s stained diffusely for CK7 (100%) and S100 (71%) by some to treat ACC of the salivary gland (ACCSG) with variable outcomes, but none and focally for lysozyme (100%) and ER (88%). They were negative for PR. Normal of the cases evaluated had c-kit mutations. The aim of our study was to identify c-kit intercalated ducts also showed consistent CK7, lysozyme and focal ER staining but were mutations in primary ACCSG. S100 negative. A thin myoepithelial layer was highlighted with CK14 and calponin in all Design: We identified 14 cases of ACCSG (13 primary, 1 cervical lymph node metastasis) IDL’s (100%) and in normal intercalated ducts. In contrast, BCA’s were larger (average from the pathology files of our institution. KIT protein expression was evaluated by 2.1 cm), did not show significant ER or lysozyme staining and had a more prominent immunohistochemistry (IHC) using formalin-fixed paraffin-embedded tissue. Mutational neoplastic basal/myoepithelial component with these markers. analyses of the c-kit extracellular domain (exon 9), juxtamembrane domain (exon 11) Conclusions: IDL’s have a variety of growth patterns. They share features with normal and the tyrosine kinase domains (exons 13 and 17) were performed by polymerase chain intercalated ducts. They are distinct from BCA’s which have a biphasic neoplastic reaction, T/A cloning, clonal selection and subsequent DNA sequencing. population. However transition to and association with BCA’s is common and appears Results: All 14 cases demonstrated strong KIT expression by IHC. Molecular analysis more common than with EMC. Their frequent occurence in association with salivary was successful in 10/14 cases, and c-kit missense point mutations were detected in tumors lends credence to their role as a precursor lesion. 7/10 cases (70%) including 7 in exon 11, 2 in exon 9, 2 in exon 13 and 2 in exon 17. Eight silent point mutations were detected in 5 cases. Two cases contained missense 1153 Global Epigenetic Alterations in Head and Neck Squamous Cell mutations in multiple exons (exons 11 and 9, exons 11 and 17). Different mutations Carcinoma were found in the primary tumor and the cervical lymph node metastasis of 1 patient. H Zhang, A Stone, C Xie, SA Schichman, C-Y Fan. University of Arkansas for Medical Point mutations in similar domains described in GISTs were detected in our study, Sciences, Little Rock, AR; John L. McClellan Memorial Veterans Hospital, Little including Pro551Leu and Lys558Glu (5’ end of exon 11), Leu576Phe (3’ end of exon Rock, AR. 11), Val643Ala (exon 13) and Asn822Ser (exon 17). Additional novel point mutations Background: Head and neck cancer is the most common epithelial malignancy of the in exons 9, 11, 13 and 17 were also identified. upper aerodigestive tract. Despite multimodality treatment regimens of combined surgery Conclusions: This study is the first to reportc-kit gene mutations in primary ACCSG. and chemoradiation therapies, the overall 5-year survival for head and neck cancer has These potential gain-of-function mutations in exon 11, and less frequently in exons not exceeded 50% for the past 3 decades. Recently, there is growing evidence that in 9, 13 and 17, may play a role in ACCSG. Identification of such activating mutations addition to widespread genetic abnormalities, epigenetic alterations in association with in ACC may be of prognostic value and may also be predictive for response to KIT promoter hypermethylation play significant roles in the development and progression tyrosine inhibitor (imatinib) treatment. of human cancer. In this study, we analyze a global methylation pattern in 4 HNSCC cancer cell lines as compared to normal oral keratinocyte cell line using Illumina DNA 1151 Striated Duct Adenoma: A Report of 3 Cases of a Distinctive methylation microarray. Lesion of Salivary Gland Design: 4 HNSCC cell lines (CRL1623, HEp2, SQ20B and UMSCC1) treated with I Weinreb, B Perez-Ordonez, R Chetty, I Dardick, JL Hunt. University Health Network, or without 5-azacytidine, a DNA demethylating agent and a normal oral keratinocyte Toronto, ON, Canada; Cleveland Clinic Foundation, Cleveland, OH. cell line (HOK16B) were used for extraction of genomic DNA, followed by sodium Background: Benign tumors of salivary gland can be separated into pleomorphic bisulfite modification and global promoter methylation analysis using Illumina adenoma, basal cell adenoma, myoepithelioma and others based on their cell constituents BeadArray (HumanMethylation27) containing 28,544 target CpG sites, representing and architectural patterns. Most of these benign salivary gland tumors contain a a total of 14,956 genes. myoepithelial/basal cell component, with the exception of canalicular adenoma. Normal Results: Among 14,956 genes, there were 1230 genes that showed increased promoter striated ducts in salivary gland show a single ductal layer with only patchy or absent methylation by 2-fold or greater in all 4 HNSCC cell lines CRL1623, HEp2, SQ20B basal/myoepithelial cells. To our knowledge there has been no benign neoplastic ductal and UMSCC1) as compared to the normal keratinocyte line (HOK16B). Following lesion described that is composed primarily of striated ducts with no myoepithelial the treatment of 5-azacytidine, 42 out of these selected 1230 genes showed decreased contribution. methylation in all 4 HNSCC cell lines by at least 10%. Thus, these 42 genes are likely Design: Three cases of striated duct adenomas were collected from the consultation those whose expression is commonly regulated by epigenetic mechanism in HNSCC but files of the authors and reviewed. Each case was stained with various keratins, p63, not in normal oral keratinocytes. Using these 42 genes for cluster analysis, 4 HNSCC actin and S100. cell lines were clustered closely to one another and the promoter methylation pattern for Results: The cases were from parotid (2) and oral cavity (1). They ranged from 0.9-2.4 HNSCC cell lines was distinctively different from that in normal oral keratinocytes. cm. Two patients with clinical data were females of 47 and 57 years of age. All 3 cases Conclusions: Using the powerful Illumina DNA methylation microarray, we have were encapsulated/circumscribed masses. The tumors were made up entirely of back-to- identified a small subset of genes that frequently undergo promoter methylation in back ducts with virtually no intervening stroma. The ducts were of varying size with large HNSCC. This information should be invaluable in helping us to identify novel epigenetic caliber lumina containing eosinophilic secretions. Interspersed ducts formed cysts up to markers for early cancer detection or molecular targets for epigenetic therapy in the 1 mm in diameter. The ductal cells were eosinophilic, had visible striations and bland treatment of HNSCC. basally oriented nuclei. All lesions contained abundant large vessels, some of which were staghorn in shape. One case contained psammoma bodies and focal papillary formations. None of the cases showed the typical epithelial beading pattern with abundant stroma Hematopathology seen in canalicular adenomas. One case had striated duct hyperplasia (SDH) in the background parotid. All 3 cases and SDH were positive for keratins and S100. Normal 1154 Flow Cytometric Panel Including CD10, CD19, CD38, CD58 and salivary elements were S100 negative. No basal/myoepithelial contribution was found CD45 Increases Diagnostic Accuracy for Distinguishing Hematogones with p63 or actin in the tumors or in the background SDH: a pattern identical to normal from Precursor B-Cell Acute Lymphoblastic Leukemia (Pre B-ALL) striated ducts and unlike acini, intercalated and excretory ducts (which all contain basal or myoepithelial cells). CK5/6 in one case demonstrated strong positivity with basal S Alkan, R Veerappan, P Fitting, M Velankar, S Bozkurt. Loyola University Medical accentuation, which was again identical to the background striated ducts. Center, Maywood, IL. Conclusions: Striated duct adenoma is a rare unilayered ductal lesion with virtually no Background: Hematogones are benign precursor B-lineage lymphoid population stroma and no myoepithelial component. These neoplasms recapitulate normal striated that are seen in increased numbers in the bone marrow during of childhood, post bone ducts morphologically and immunohistochemically. Striated duct adenoma is distinct marrow transplant, immune deficiency and autoimmune disorders. They typically show from all other benign salivary gland lesions, including canalicular adenoma. expression of dim CD45, CD10, CD34, CD19, TdT and variable CD20. However, this phenotype is also seen in patients with precursor B-cell acute lymphoblastic leukemia (pre B-ALL) causing a significant problem in distinguishing residual pre B-ALL from 1152 Intercalated Duct Lesions of Salivary Gland: A Re-Appraisal of hematogones in a regenerating marrow. Earlier studies suggested flow cytometric 30 Cases of a Putative Precursor Lesion analysis using the above markers as being useful for differentiating hematogones from I Weinreb, RR Seethala, JL Hunt, R Chetty, I Dardick, B Perez-Ordonez. University pre B-ALL, however, in our experience the use of these limited panel of markers is Health Network, Toronto, ON, Canada; University of Pittsburgh Medical Center, insufficient and may hinder correct interpretation of bone marrow samples. Recent Pittsburgh, PA; Cleveland Clinic Foundation, Cleveland, OH. studies showed that CD58 is commonly expressed in pre B-ALL but not in hematogones Background: Intercalated duct lesions (IDL) are benign ductal proliferations that have and expression of CD38 is different in the two populations. We generated a 5-color been described rarely in association with epithelial-myoepithelial carcinoma (EMC) and antibody panel including the markers mentioned above to assess the feasibility of using are also seen in association with other salivary gland tumors. They have been proposed a novel panel to distinguish pre B-ALL from hematogones. as a putative precursor lesion, but a detailed review of IDL’s has not been performed. Design: Total of 37 cases including 27 cases of pre B-ALL (diagnostic and residual/ Design: 30 cases of IDL were identified and reviewed and immunostaining with CK7, relapsed cases) and 10 cases of hematogones were analyzed by five color flow ER, PR, lysozyme, S100, CK14 and calponin was performed. Due to the morphologic cytometery. Two separate tubes with following markers (Tube1:CD38-PE, CD10-FITC, resemblance to basal cell adenoma (BCA), 8 of these tumors were stained for CD19-ECD, CD20-PC5, CD45-PC7 and tube2: CD58-PE, CD10-FITC, CD19-ECD, comparison. CD34-PC5, CD45-PC7) were used simultaneously with the standard acute leukemia Results: The patients with IDL ranged in age from 19-80 yrs (average 53.2). There was panels. Results were correlated with the bone marrow histologic findings and cytogenetic a 1.8:1 female predominance. The majority were parotid lesions (25/30 - 83%) with the analysis. remaining cases in submandibular gland. Most cases (24/30 - 80%) were small nodular Results: Hematogones typically showed expression of bright CD38, moderate (mod) lesions ranging from 1-8 mm (average 3.2 mm). The remainder were diffuse/multifocal CD19, mod CD10, variable CD20 and no CD58; in contrast pre B-ALL showed weak lesions. 16 IDL’s (53%) were seen in conjunction with another salivary gland tumour, the to mod CD38, mod to strong CD19, mod to strong CD10 and mod CD58. Without the most common being BCA/BC adenocarcinoma (7 cases). The IDL’s showed a spectrum combination of both CD38 and CD58 together, some cases would have been difficult from irregular ductal proliferations to round encapsulated “adenomas” with hybrid forms. to interpret. 254A ANNUAL MEETING ABSTRACTS

Conclusions: Combination of CD38 and CD58 increases the diagnostic accuracy in 1157 Gene Expression Profile of Nodal Marginal Zone Lymphoma. differentiation of hematogones from pre B-ALL. Based on this analysis, we recommend The Search for Diagnostic Markers diagnostic laboratories to include these markers in their routine evaluation of bone JA Arribas, Y Campos-Martin, M Sanchez-Beato, G Roncador, P Algara, G Kanellis, S marrow samples that are submitted for acute leukemia evaluation especially post Montes-Moreno, MA Piris, M Mollejo. Hospital Virgen de la Salud, SESCAM, Toledo, treatment samples in order to prevent misdiagnosis. Spain; CNIO, Madrid, Spain. Background: Nodal marginal zone lymphoma (NMZL) is a small B-cell neoplasm 1155 Plasma Cell Myeloma with MYC Rearrangement Involving whose molecular pathogenesis is still essentially unknown. The differential diagnosis Immunoglobulin Genes with other small B cell lymphomas, as Follicular Lymphoma (FL) or other MZL M Almiski, A Maleki, R Garcia, W Chen. UT Southwestern Medical Center at Dallas, arising in MALT or spleen is hampered by the lack of consistent diagnostic markers. Dallas, TX. The purpose of this study is to improve the knowledge of the molecular mechanism Background: Rearrangement (R) of the MYC gene has been suggested as a secondary of this lymphoid neoplasia, selecting a set of possible NMZL markers to be explored oncogenetic event in the pathogenesis of plasma cell myeloma (PCM). While this in paraffin sections. aberration was reported in 15% of PCM, a comprehensive clinicopathologic study of Design: Frozen tissue blocks from 17 NMZL and 8 reactive lymph nodes were analyzed. cases with MYC R involving the immunoglobulin (Ig) partner genes is largely absent. For comparison purposes, lymph nodes infiltrated by Follicular Lymphoma (16 cases), We describe the morphologic, immunophenotypic and cytogenetic features of this Splenic marginal zone lymphoma (4 cases) and MALT Marginal zone Lymphoma (5 subtype of PCM. cases) were included. Cases were diagnosed according to WHO classification criteria. Design: An institutional database was searched for cases of PCM with abnormal ADN was amplified for studying the IgVH somatic mutation. High-quality total RNA karyotypes that included MYC R involving Ig genes, which yielded 18 bone marrow was purified and amplified for microarray hybridization on Human Whole Oligo cases. Immunophenotype (IP) was analyzed by flow cytometry (FC). These results were Microarray (4x44k, Agilent Technologies). Statistical analyses were performed by correlated with available morphologic and clinical data for 4 cases. In addition, IP of using GEPAS, Babelomics and GSEA. 17 cases without MYC R was analyzed for comparison. Results: The NMZL signature contains genes related to BCR signaling, memory B-cell Results: Patients were 6 males and 12 females, median age 58 years (range 37-90). signature, apoptosis, inflammation, Rho GTPases, NFkB and TNF pathway, such as: Neoplastic cells expressed CD19 in 1 case (5.6%), CD20 in 2 (11%), CD38 in all, CD45 CXXC5, ATP13A1, LYN, KCNN4, BTG2, CD82, CD81, SMARCB1, PTPN1, BBC3, in 5 (28%), CD56 in 11 (61%); dim surface and intracellular Ig restriction in 10 (55%) REL A, SYK, UBC, TGFB1, COL9A3, GRB2, NFKBIB, TACI, PIK4CB, BLK, RAC2. and all cases, respectively. This IP was not different from the cases without MYC R that Of the interest, the NMZL signature include genes associated to marginal zone, like expressed CD19, CD20, CD38, CD45, CD56, surface and intracellular Ig restriction in TACI, RAC2, COL9A3. Additionally, comparision of the gene expression profiles of 5.9%, 5.9%, 100%, 35%, 65%, 53% and 100% of cases, respectively. Most common NMZL and FL subtypes revealed a set of genes differentially expresed in both groups, MYC R was t(8;22) [12 cases, 67%] followed by t(8;14) [5] and t(2;8) [1]. The MYC R such as TACI or MNDA for NMZL and BCL6 and CD10 (MME) for FL. occurred in the context of a complex karyotype in all cases, with an average of 8.8±1.0 Conclusions: Our study identified new candidate diagnostic molecules for NMZL, which additional cytogenetic aberrations including recurrent abnormalities such as t(11;14)(3 could provide useful diagnostic and prognostic markers for improving the diagnosis cases) and -13(5). Most cases (15/18) had single but complex clone, and only 3 cases had accuracy in these tumors. 2 or 3 related clones that all had MYC R. Clinical and morphologic data were available in 4 cases. All had a de novo diagnosis. Neoplastic cells had intermediate-grade cytology. 1158 JunB Oncogenic Activity in Anaplastic Large Cell Lymphoma Patients presented at various clinical stages (IA in 1, IB in 1, IIIA in 2 cases), and beta-2 V Atsaves, L Lekakis, M Feretzaki, V Leventaki, C Liakou, E Drakos, FX Claret, microglobuin level was from normal (1 case) to higher than 3.5 mg/L in 2. All patients D Jones, LJ Medeiros, E Patsouris, GZ Rassidakis. The University of Texas M.D. received treatment; 2 are alive and 2 have died of disease. Anderson Cancer Center, Houston, TX; National and Kapodistrian University of Conclusions: The overall features of PCM with MYC R are similar to those without Athens, Athens, Greece. MYC R with respect to morphology, immunophenotype, and clinical presentation. Background: JunB is a member of the jun family of AP-1 transcription factors These data combined with a predominance of single complex clone do not appear to involved in cell proliferation and survival. Recent evidence suggests that JunB is support the view of MYC R as a secondary oncogenetic event. However, a large cohort overexpressed in CD30+ lymphomas and positively regulates CD30 gene expression of patients is needed to determine the place of MYC aberrations in PCM pathogenesis. at the transcriptional level in Hodgkin lymphoma and anaplastic large cell lymphoma Interestingly, most MYC rearranged cases had t(8;22), in contrast to common t(8;14) (ALCL) cells.We hypothesized that JunB operates as an oncogene in ALCL and may in high grade B-cell lymphomas. contribute to ALCL oncogenesis, at least in part, through activation of CD30 signalling leading to uncontrolled cell cycle progression. 1156 T-Regulatory Cells in Lymph Nodes Correlate with HIV Status, Design: To investigate the underlying mechanism of JunB overexpression in ALCL, Gender, Viral Load and Morphologic Features of Castleman’s Disease we used real-time quantitative PCR to assess JunB gene copy number in primary J Arce, M Levin, Q Xie, J Albanese, H Ratech. Montefiore Med Ctr/Albert Einstein Coll tissue samples obtained prior to treatment from 6 patients with ALCL. Transfection Med, Bronx, NY; Creighton Univ Med Ctr, Omaha, NE. experiments were performed in human embryonic kidney (HEK) cell line 293T Background: Lymph nodes from many HIV infected individuals show Castleman’s and in 2 ALCL cell lines, Karpas 299 and SU-DHL1. Protein expression, promoter disease (CD)-like changes. These changes could be caused by either immune activity and DNA binding were assessed by Western blot analysis, luciferase assay dysregulation or viral infection. The association of human herpes virus-8 (HHV8) with and EMSA method, respectively. BrdU incorporation and cell cycle were analyzed multicentric CD supports the viral hypothesis. On the other hand, there are only a few by flow cytometry. studies that have correlated immune dysregulation with lymph node changes. Since Results: We show significant JunB gene amplification in the majority of ALCL tumor FoxP3+ CD4+ T-regulatory cells (T-regs) can impair HIV-specific responses and limit samples. Forced expression of JunB gene in HEK 293T resulted in CD30 upregulation. immune activation, we evaluated T-regs in a large number of reactive lymph nodes Inversely, silencing of JunB using siRNA in ALCL cell lines resulted in decreased CD30 from both HIV+ and HIV- individuals. expression and CD30 promoter activity. Silencing of JunB also resulted in decreased Design: Reactive lymph nodes from 48 HIV+ (20 males; 28 females) and 106 HIV- (40 AP-1 binding activity in ALCL cells and significant decrease of the S-phase fraction of males; 66 females) individuals were studied for T-regs and T-helper cells by FoxP3 cell cycle. These changes were associated with increased levels of the CDK inhibitors and CD4 antibody staining on tissue microarrays and by counting positive cells in the p21 and p14 with simultaneous decrease of the unphosphorylated fraction of Rb and paracortical T-zone that crossed the lines of a 1 mm2 10x10 grid. Lymph nodes were expression levels of cyclins A, D2 and D3. In addition, silencing of CD30 using siRNA scored for 6 hyaline vascular and 6 plasma cell morphologic features of CD. in ALCL cells led to decreased AP-1 binding activity assessed by EMSA, which was Results: The mean FoxP3+ count per grid, which is proportional to the total number of associated with cell cycle arrest linked to decreased levels of p21 and p14 similar to FoxP3+ cells in the lymph node, was 3 times greater in HIV+ males compared to HIV+ those seen after JunB silencing. females (23.5 vs 7.8; p=0.0006). Also, HIV+ males compared to HIV- males had almost Conclusions: Our data suggest that JunB functions as an oncogene in ALCL. JunB twice as many FoxP3+ cells (23.5 vs 13.1; p=0.03). On the other hand, HIV+ females gene amplification might represent the initial genetic event that triggers uncontrolled compared to HIV- females had fewer FoxP3+ cells (7.8 vs 13.6; p=0.03). HIV+ males cell cycle progression, at least in part, through activation of CD30 signaling and its compared to HIV+ females had higher viral load (239000 vs 73000; p=0.02), lower downstream effectors in ALCL. peripheral blood CD4 count (262 vs 466; p=0.01), but higher lymph node CD4 count (73.3 vs 28.8; p=0.02), and shorter interval from HIV diagnosis to lymph node biopsy 1159 Immunoglobulin VH Gene Analysis of Splenic Marginal Zone (28 mos vs 93 mos; p=0.04). More morphologic features of the plasma cell variant of Lymphoma Histologic Variants CD were seen in lymph nodes from HIV+ compared to HIV- individuals (p=0.007). DW Bahler, P Szankasi, SD Dufresne, RE Felgar, SH Swerdlow. University of Utah, High FoxP3 counts were associated with plasma cell features in lymph nodes from both Salt Lake City, UT; University of Pittsburgh, Pittsburgh, PA. HIV+ and HIV- individuals. In 8 lymph nodes, HHV8 was associated with interfollicular Background: Splenic marginal zone lymphomas (SMZL) are typically CD5-, CD10- plasma cells and increased vascularity (p<0.01). B-cell neoplasms that classically display a biphasic growth pattern in the white pulp Conclusions: High T-regs in lymph nodes are associated with high viral load and with marginal zone-like cells merging with a more central zone of smaller lymphocytes. low peripheral blood CD4 counts. Furthermore, elevated T-regs are associated with However, many cases lack a biphasic pattern but show nodules composed mostly of the morphologic features seen in CD plasma cell variant. We hypothesize that gender marginal zone-like cells. Other less easily classified cases have mostly just small differences in lymph node T-regs might be explained by worse HAART compliance lymphocytes. To evaluate whether there may be molecular correlates to these histological and more high risk behavior in males than females. differences related to direct antigen stimulation, lymphoma immunoglobulin heavy chain variable (VH) genes were analyzed. Design: 23 splenic small B-cell lymphoma cases were identified that had available frozen tissue and were not HCL, CLL, FL, or MCL. Diagnoses were established by morphologic analysis and immunohistochemical staining. Rearranged lymphoma VH genes were amplified from the isolated DNA and the resultant PCR products could be directly sequenced in 19/23 cases. ANNUAL MEETING ABSTRACTS 255A

Results: The 19 successfully sequenced cases consisted of 5 having a biphasic pattern, of engraftment syndrome and GVHD to MDS is intriguing. It is conceivable that 7 having monophasic marginal zone-like nodules, and 7 unclassifiable cases. A single perturbation to the host immunity caused by prior chemotherapy and/or conditioning functional VH gene without stop codons was identified in all 19 sequenced cases. VH1 regimens in the elderly may play a role in the development of MDS after autologous family gene segments were used in 4 cases, VH3 segments in 5, and VH4 family gene transplant. segments in 10. All 4 VH1 cases used the same V1-2 segment, and 5/10 VH4 cases used the V4-34 gene segment. Although cases using V4-34 were distributed among 1162 Correlation of Flow Cytometry (FC) and Fluorescence In- all 3 morphologic types, most (3/4) cases using V1-2 showed a biphasic pattern. Situ Hybridization (FISH) in Precursor T-Cell Lymphoblastic Leukemia The lymphoma VH genes were mutated from the most closely related germline gene (T-ALL) segments (98% or less homology) in 13 cases, with 4 being heavily mutated (mean DS Bosler, CA Hanson, RA Knudson, RP Ketterling, JD Hoyer. Mayo Clinic, Rochester, germline homology 83.0% ± 5.9%), and unmutated (greater than 98% homology) in 6 MN. cases. The presence or absence of somatic mutations did not appear to correlate with Background: T-ALL is phenotypically diverse. CD7 and c-CD3 are uniformly the use of specific VH gene segments or histologic types. expressed, while expression of other T-cell markers varies, and myeloid or B-cell Conclusions: Splenic marginal zone lymphomas appear to preferentially express VH4 antigen expression can present diagnostic challenges. Integrating genetic findings family genes and specific VH gene segments, V1-2 (4/4 VH1 cases) and V4-34 (5/10 provides additional information and may help clarify lineage in these cases. The aim VH4 cases). These findings are consistent with direct antigen stimulation playing an of our study was to correlate FC and FISH findings in T-ALL. important role in lymphoma growth and recognition of similar antigens. In addition, Design: We identified 64 T-ALL cases with diagnostic FC and concurrent archived cell our study suggests the high use of V1-2 previously reported in SMZL may be mostly pellets for FISH. Histograms were reviewed when available. FISH performed included: restricted to those cases showing a classic biphasic growth pattern. SIL/TAL1 (1p32), TCRAD (14q11.2), p16/D9Z1 (9p21), BCR/ABL, t(9;22)(q34;q11.2) dual fusion (D-FISH), MLL (11q23), HOX11L2/BCL11B, t(5;14)(q35;q32) DFISH, 1160 Plasmablastic Lymphomas (PBL) Are Genetically Characterized TCRB (7q34), and AF10/PICALM, t(10;11)(p13;q13) DFISH. by Frequent MYC Translocations Results: c-CD3, TdT, CD5 and CD7 were almost always uniformly expressed, while O Balague, S Valera, A Martinez, L Colomo, J Delabie, A Macolscy, E Campo. Hospital CD2 (77%), s-CD3 (42%), CD10 (48%), CD34 (32%), HLA-DR (18%), and CD56 Clínic, University of Barcelona, Barcelona, Spain; Rikshospitalet-Radiumhospitalet (23%) were each expressed in some cases. Dual CD4/CD8 positivity (54%) and CD4/ Medical Center, Oslo, Norway; Semmelweis University, Budapest, Hungary. CD8 negativity (20%) were also prevalent. CD13 was expressed in 27%, CD33 in 15%, Background: PBL is an aggressive lymphoma characterized by a diffuse proliferation of CD15 in 13% and CD117 in 21%. At least 2 myeloid markers were coexpressed in 18%. large cells with predominant immunoblastic morphology and a plasma cell phenotype. Of cases expressing a myeloid antigen, 70% were s-CD3 negative. The clinical and pathological features of this lymphoma have been relatively well % FISH Pos % CD2 Pos % s-CD3 Pos % CD4/8 Dual Pos characterized but its genetic alterations are not well known. The aim of this study was All Cases 72 77 42 54 to identify recurrent cytogenetic aberrations that may be involved in the pathogenesis TCRAD 25 94 40 80 of PBL. TCRB 6 100 75 100 p16 41 92 50 63 Design: We investigated 31 PBL, 4 primary effusion lymphomas (PEL) and 5 SIL/TAL 13 100 63 40 plasmablastic plasmacytomas (PBL-PC). Genetic alterations involving BCL2, BCL6, TCRAD,TCRB,p16,SIL/TAL Neg - 64 32 46 MALT1, PAX5, MYC and IGH genes were assessed in paraffin sections by interphase TCRAD, TCRB, p16 and SIL/TAL positivity was more likely to be associated with CD2 and fluorescence in situ hybridization (FISH) using split-apart probes. In cases carrying a s-CD3 expression compared to other cases. CD4/CD8 expression was slightly more common in MYC translocation FISH for MYC/IGH translocation was additionally investigated with a TCR rearranged cases. dual color-dual fusion probe. MYC expression was examined by immunohistochemistry CD10 was expressed more often in TCRAD (73%) and ABL (80%) cases. While in 12 PBL. TCRAD and TCRB often coexisted with p16 loss (75% and 100%), MLL (8% of Results: Structural alterations of MYC were detected in 11 of 28 PBL (39%), 9 of cases) was always a sole abnormality, and was associated with CD34 (60%). FISH and them corresponded to split signals (32%) and 2 were additional copies of the locus. cytogenetics (CG) were concordant in 65% of cases, with FISH positive/CG negative The translocation t(8;14) was confirmed in 5 of the 9 cases with a 8q24 rearrangement (30%) > CG positive/FISH negative (5%). No differences in age or WBC count were using the MYC/IGH dual color-dual fusion probe. Two of the four cases in which observed between various FISH findings. the fusion probe failed were investigated with the IGH split-apart probe that showed Conclusions: CD5 and CD7 are the most consistently expressed T-cell markers in also a rearrangement of the this locus suggesting the presence of a t(8;14). BCL2, T-ALL, while s-CD3 is expressed in <50%. TCR rearranged T-ALL correlates with BCL6, MALT1 and PAX5 were examined in 26, 25, 23, and 17 cases respectively. CD2, s-CD3 and dual CD4/CD8 expression. FISH abnormalities are present in 72% No rearrangements were observed but extra copies of these genes were observed in of cases, 39% of which have normal karyotypes, making FISH a useful adjunct to CG. 3 (12 %), 3 (16%), 4 (21%) and 4 (21%) of the cases respectively. Two cases showed In cases with myeloid and T-cell marker expression, FISH abnormalities may provide gains of BCL2 and MALT1. Two PEL showed a gain of MYC and PAX5, respectively, potentially useful complimentary information. whereas only a gain of MYC was detected in one of the PBL-PC. No rearrangements in any of these loci were detected in the PEL or PBL-PC cases. Nuclear MYC protein 1163 Molecular Prognostic Factors in Splenic Marginal Zone expression was detected in 4 PBL with gene translocations but was negative in 8 cases Lymphoma (SMZL): Correlation of Clinical Behavior with Immunoglobulin without genetic alterations. Heavy Chain (IgH) Somatic Mutation Status, Array Comparative Genomic Conclusions: PBL are genetically characterized by frequent structural alterations of Hybridization (aCGH) and Gene Expression Profiling (GEP) the MYC locus that in most cases correspond to the t(8;14) translocation. The lack of DS Bosler, ED Remstein, E Braggio, WJ Chng, G Huang, SP Menon, RF McClure, translocations and the low number of gains in the BCL6, MALT1, BCL2 and PAX5 loci R Fonseca, DX Sun, MJ Maurer, A Dogan. Mayo Clinic, Rochester, MN; National distinguishes genetically PBL from other types of diffuse large B-cell lymphomas. University Hospital, Singapore, Singapore. Background: Unmutated IgH status has been correlated with adverse outcome in SMZL. 1161 Possible Role of Engraftment Syndrome in Myelodysplastic The aim of this study was to examine IgH variable (V) region usage, somatic mutation, Syndrome after Autologous Transplants GEP and aCGH in SMZL to analyze the relationships with clinical outcome. MW Beaty, M Pettenati, DH Hurd, Y Keung. Wake Forest University School of Medicine, Design: We studied 35 splenectomy confirmed SMZL cases. IgHV analysis was Winston-Salem, NC. performed from RNA by reverse transcription, PCR and sequencing. Mutation status Background: Autologous graft versus host disease (GVHD) and engraftment syndrome was scored for productive rearranged sequences (cutoff=98% homology) using the (ES) probably result from host immune dysfunction during the recovery from high Immunogenetics (IMGT/V-QUEST) database. The Human Agilent 244A platform was dose chemotherapy and radiation. Since impaired immunity has been associated with used for aCGH analysis. GEP was performed using Affymetrix Human Genome U133 myelodysplastic syndrome, we explore the risk factors of post-transplant myelodysplastic plus 2.0. Progression free survival (PFS) and overall survival (OS) were determined syndrome (MDS), specifically, in relation to the GVHD and ES. from review of medical records. Design: Review of clinical features and diagnostic material in consecutive patients Results: IgH status: Of 31 cases with productive sequences, IgHV1-2*04 was utilized with Hodgkin (HL) and non-Hodgkin lymphoma (NHL) undergoing autologous in 11, IgHV3-23*01 in 3, and 3-7*01, 1-69*01 and 4-34*01 each twice. Mean sequence transplantation in our institution 1991 to 2006. homology was 96% (89.1-100%). There were 10 unmutated and 21 mutated. There Results: 452 lymphoma patients underwent autologous transplants in this period; was no association between IgHV usage and mutation status. GEP: Del7q correlated median age of 50 years (range 16-76). 85 patients with HL and 367 NHL, of which, with hexokinase 2 expression and negatively correlated with UBE1L expression in 291 are B-cell, 47 T-cell and 29 unknown. Total of 277 received TBI-based and 175 unsupervised clustering analysis. aCGH: 91% of cases showed genetic imbalance, with chemotherapy-alone conditioning regimens; 98 patients received transplantation of a median total imbalance of 54.0 (0-782.4) Mb and a median of 4 (0-55) gains/losses. the bone marrow, 343 peripheral blood stem cells and 11 both. 32 patients (7%) died Nine of 35 had del7q. Clinical data/follow-up: Median age=67 years (42-85), 11 males of regimen-related toxicity within 100 days of transplant. Eleven patients developed : 24 females. Median follow-up for living patients was 56 months (11-152). 83% were severe engraftment syndrome (high fever, skin rash ± pulmonary infiltrate requiring alive at last follow-up, and 28% had progressed or recurred. Increased total genetic systemic steroid); 27 patients had skin and 2 patients had gastrointestinal biopsies imbalance was associated with shorter PFS (per unit HR=1.006, 95% CI=1.002-1.01, consistent with GVHD. Twenty-four patients (5.3%) developed MDS with median time p=.004) and OS (per unit HR=1.005, 95% CI=1.001-1.009, p=.02). Mutated IgHV of onset of 4.2 years (range 8 months-7.5 years). Additional 5 patients developed clonal status was associated with decreased total imbalance (p=.03), though association with karyotypic abnormalities in the bone marrow without clinical MDS. The incidences of PFS and OS was not significant (p=.35 and p=.24). There were no differences in PFS MDS are similar in HL and NHL. Significant risk factors of developing MDS include or OS by IgHV region or del7q status. older age, advanced stage, onset of ES or GVHD, and longer intervals between the Conclusions: Frequency of IgHV1-2*04 (35%) and del7q (26%) are similar to previous initial diagnoses to transplant. studies. As increased total genetic imbalance showed the only significant association Conclusions: Overall incidence of MDS is 5.3%. The actuarial risk at 8 years is up to with shorter PFS and OS, quantification of genetic imbalances by aCGH may be a better 15% and may be higher in selected patients such as older age, and prolonged interval marker of adverse outcome than IgHV region usage, mutation status, or del7q. from initial diagnosis to transplant (a surrogate for prior chemotherapy). The association 256A ANNUAL MEETING ABSTRACTS

1164 Mycosis Fungoides/Sezary Syndrome Staging: Potential Pitfalls Conclusions: CD34 expression by megakaryocytes may be predictive of the in Analyzing Bone Marrow and Lymph Nodes Using Flow Cytometric and presence of underlying MDS and is usually associated with complex (non-favorable) Molecular Diagnostic Methods cytogentics. None of the cases of potential ITP or MPD demonstrated significant CD34 KT Bradley, SS Parker, DL Jaye. Emory University Hospital, Atlanta, GA. immunoreactivity by megakaryocytes. The prognostic or other clinical significance of Background: Staging of Mycosis Fungoides/Sezary Syndrome (MF/SS) often includes CD34-expressing megakaryotes in MDS is currently unknown. lymph node (LN) biopsy and for some patients bone marrow (BM) evaluation, and is crucial for determining prognosis and treatment. Histologic recognition of atypical 1166 Analysis of Karyotype Complexity and Chromosome Aneuploidy lymphocytes is the gold standard for involvement by MF/SS. Flow cytometry (FC) in Burkitt Lymphoma/Leukemia and PCR for T-cell receptor gene rearrangements (TCR) aid in the diagnosis of blood KE Capocelli, G Kotnis, S Pearson, K Swisshelm, B Greffe, M Wang, N Dirks, X involvement, but whether these tests provide accurate staging data for BM and LNs is Liang. University of Colorado Health Sciences Center, Aurora, CO; The Children’s unclear, particularly in patients with circulating neoplastic cells. Recently, the ISCL/ Hospital, Aurora, CO. EORTC proposed new subgroups for LN staging based on the results of TCR, and Background: Burkitt lymphoma/leukemia (BL) is an aggressive B-cell neoplasm recommended tracking TCR data for BM specimens (Olsen et al, Blood 2007). In characterized by medium-sized malignant lymphoid cells, high degree of apoptosis response, we sought to determine the incidence of discordant test results between and high mitotic index. Atypical BL is a morphologic variant of BL with greater morphology and FC/TCR in BM and LN specimens from MF/SS patients. pleomorphism in nuclear size and shape, and more prominent and fewer nucleoli Design: We identified patients treated at our institution from 2000-2006 who had MF/ when compared with classical BL. Although all types of BL show deregulation of the SS and blood involvement. Data were reviewed for those who underwent a LN or BM c-myc gene, other cytogenetic features and their associations with different morphology biopsy for morphologic evaluation and FC and/or TCR. FC and TCR results were subtypes have not been well investigated. We examined the karyotype complexity and compared among blood, BM, LN, and skin biopsy specimens to confirm similarities in chromosome aneuploidy to investigate if there is any association with morphologic phenotype and base pair sizes of clonal peaks. subtypes in BLs. Results: 24 specimens (10 LN, 14 BM) from 12 patients were identified. For each Design: 37 cases of BL (7 cases of atypical BL, 30 cases of classical BL) with c-myc gene patient, the phenotype and clonal peak sizes were the same among specimen types. 10 rearrangement and complete cytogenetic karyotype were selected from the pathology of 14 BM (71%) and 1 of 10 LN (10%) specimens showed discordance between the archives of The Children’s Hospital, Denver and University of Colorado from 1/1999 morphologic diagnosis and the FC or TCR results. In all discordant cases, morphology to 8/2008. The difference of frequency in karyotype complexity (≥ 4 chromosomal was negative while FC and/or TCR was positive (Figure). aberrations) and chromosome aneuploidy (chromosome # > or < 46) between atypical BL group and classical BL group was analyzed by Fisher’s Exact test. Results: Atypical BLs Classical BLs P values Complex karyotype 4 cases 5 cases 0.045 Non-complex karyotype 3 cases 25 cases

Aneuploidy 5 cases 6 cases 0.016 Diploidy 2 cases 24 cases Conclusions: Complex karyotypes and aneuploidy of chromosome are associated with atypical BL. We speculate that complex genetic aberration and aneuploidy of chromosome may play a role in causing pleomorphic morphology of tumor cells in atypical BL. The biologic significance of these findings is under investigation.

1167 Prognostic Impact of Immunohistochemical Profile and Microvessel Density (MVD) in Diffuse Large B Cell Lymphoma (DLBCL) Treated with R-CHOP T Cardesa, G Gutierrez-Garcia, F Climent, L Arenillas, S Serrano, V Romagosa, E Gonzalez-Barca, E Campo, A Lopez-Guillermo, Ll Colomo. Hospital Clínic, Barcelona, Spain; Hospital de Bellvitge, Barcelona, Spain; Hospital del Mar, Barcelona, Grup Estudi Limfomes Catalunya i Balears (GELCAB), Spain. Background: The adittion of rituximab (R) to the classic schemes of treatment of Conclusions: Our data raise the possibility that FC and TCR are detecting blood-derived DLBCL has significantly improved the course of the disease. As new treatments may clonal cells contaminating BM and, to a lesser extent, LN samples rather than identifying change the known prognostic parameters it is necessary to validate them and to describe true parenchymal disease. Thus, for MF/SS patients with blood disease, these findings potential new ones. will likely limit the ability to interpret FC- and TCR-based staging data for LNs and Design: We have investigated the prognostic impact of germinal center (GC) and non-GC viscera in the absence of a test to resolve this issue. Given the rarity of MF/SS, multi- subgroups of DLBCL defined by the Hans classifier and the microvessel density (MVD) institutional studies investigating the prognosis of patients with blood disease and FC/ in a series of 53 de novo DLBCL treated with R-CHOP. Immunohistochemical staining TCR-positive, morphology-negative tissues are needed. for CD10, BCL6, MUM1, BCL2 and CD31 was performed on tissue microarrays. Microvessel areas, defined as CD31+ lumen areas or CD31+cell clusters, were marked and quantified using an Olympus Cell B Basic Imaging Software. For each case, MVD 1165 CD34 Expression by Megakaryocytes in Myelodysplastic was assessed as the quotient of the total microvessel areas, divided by the total area of Syndromes the core analyzed. A MVD above percentile 25 (p25) was considered high and a MVD SL Butler, P Boonsakan, DD Grier, Y Li, RC Braylan, SZ Al-Quran. University of Florida, below the p25 was denoted low. Gainesville, FL; Wake Forest University, Winston-Salem, NC. Results: CD10, BCL6, MUM1, BCL2 expression and the immunohistochemical Background: CD34 is a glycosylated surface protein expressed on immature subgroups (GC and non-GC DLBCL) did not have an impact on the overall survival hematopoietic precursor cells, endothelial cells and dendritic cells. CD34 expression (OS) of the patients. However, comparing the current series with our previously cohort is observed in early megakaryocytic precursors; however, megakaryocytes generally of patients that had not received Rituximab (Blood 2003;101:78-84), R-CHOP had a lose this surface CD34 as they mature. Immunohistochemical staining for CD34 significant effect on the OS of the patients with non-GCB DLBCL (p=0.009) whereas is used to assess the blast count in myelodysplastic syndromes (MDS) and acute no differences were observed in patients with GC-DLBCL. Interstingly, a low MVD leukemias. In our experience and, as observed by other investigators, CD34 expression was a predictive factor for better survival in patients with DLBCL treated with R-CHOP by megakaryocytes is increased in association with MDS. The aim of our study is (mean OS 5.6 vs 4 years, p=0.027). Moreover, MVD also correlated with the clinical to evaluate the CD34 expression of megakaryocytes in MDS and its relationship to significant parameters such as the IPI score (p=0.032) and LDH (p=0.001). cytogenetic abnormalities. Conclusions: These results suggest that treatment with R-CHOP may modify the Design: We reviewed 47 bone marrow (BM) cases, diagnosed with MDS between prognostic significance of some biological markers. Moreover, patients with GCB 2005 and 2008, from the Hematopathology files at two separate institutions. Each case DLBCL defined by the currently used immunohistochemical markers seem to have a had CD34 (Qbend/10 clone) immunohistochemistry performed on the core biopsy and scarce benefit from the use of R-CHOP, but this observation should be confirmed in cytogenetic analysis. Staining in 10% or more of the megakaryocytes was considered further prospective studies. In addition, the role of angiogenesis in DLBCL treated with significant. The percentage of immunoreactive megakaryocytes and percentage of R-CHOP may be of significant interest and may have a prognostic impact. blasts was assessed for each case. The megakaryocyte morphology was recorded, along with the cytogenetic abnormalities. Twenty cases each of normal marrow/idiopathic thrombocytopenic purpura (ITP) and chronic myelproliferative disorders (MPD), with 1168 IgM Plasma Cell Myeloma: Clinicopathologic Characterization CD34 immunohistochemistry, were examined as controls. of a Rare Entity Results: Megakaryocytes expressed CD34 in 21/47 cases (44.6%) of MDS. Expression N Cetin, I Bansal, W Bellamy, M Santra, B Barlogie, RB Lorsbach. University of of CD34 was seen in morphologically “dysplastic” or immature megakaryocytes. In 6 Arkansas for Medical Sciences, Little Rock, AR; Myeloma Institute of Research and cases, more than 90% of megakaryocytes expressed CD34 in each BM specimen, while Technology, Little Rock, AR. only 7 cases demonstrated immunoreactivity in less than 50% of the megakaryocytes. Background: Plasma cell myelomas expressing immunoglobulin (Ig) M are rare, The percentage of blasts in these cases ranged from 1-25%. CD34+ megakaryocytes were comprising less than 1% of all myeloma. Given their rarity, relatively little is known seen in a higher percentage (71.4%) of cases with cytogenetic abnormalities (p=0.04, regarding the clinical and pathologic features of IgM myeloma. In this study, we provide Fisher exact test). The majority of the cytogenetic abnormalities were complex, including a detailed profile of 18 cases of IgM myeloma, the largest reported series to date. monsomy 7 and trisomy 8. None of the normal or ITP bone marrows expressed more Design: We retrospectively evaluated the clinical and laboratory data from 18 patients than 10% CD34+ megakaryocytes. with IgM myeloma between 1998 and 2007. A diagnosis of IgM myeloma was made ANNUAL MEETING ABSTRACTS 257A on the basis of IgM paraproteinemia, infiltration of bone marrow by >10% plasma vs 33%; p<0.001) while BCL6 (44% vs 53%) and MUM1 (48% vs 59%) were seen in cells and lack of a morphologically or immunophenotypically detectable B-lymphoid less AR cases. The number of BCL2 positive AR-DLBCLs was lower (64; 58%) than IC component. Immunohistochemistry (IHC) for CD138, CD56, CD20 and BCL-1(cyclin cases (125; 85%; p<0.001). The largest difference was in the GCs: only 47% of AR-GCs D1) was performed on the paraffin embedded sections of the bone marrow biopsies were BCL2 positive compared to 76% of IC-GCs (p<0.005). BLIMP1 was positive in (BMB). The morphologic and cytogenetic findings were also reviewed. <20% of AR and IC-DLBCLs; however 48% AR compared to 21% IC non-GCs were Results: The median age at diagnosis was 58 years (range 36-70 years), 67% of patients positive (p<0.05). There was no difference in p53 expression between AR and IC cases were male and 78% were Caucasian. The serum IgM protein level at diagnosis was or histogenic types. 45 (39%) AR-DLBCLs and only 3 (2%) IC-DLBCLs were EBV 352-8996 (median 4040) mg/dL in 16/18 cases. Ten and 8 patients had IgM kappa positive (p<0.001). More non-GC AR cases were EBV positive (69%) than GC (27%) and IgM lambda paraproteins, respectively. Radiologic studies identified lytic bone or null (40%) cases (p<0.001). Only 6% of the EBV negative ARs expressed BLIMP1 lesions in 11/16 patients. Random BMB were available in all patients. In each case an compared to 37% of the EBV positive cases (p<0.001). interstitial or diffuse infiltrate of plasma cells comprising 10 to >80% was present in Conclusions: AR-DLBCLs are more likely of GC origin, EBV positive and BCL2 15/18 patients. The remaining 3 patients had either osseous or extramedullary disease negative than IC-DLBCLs. Correlation of EBV with non-GC cases and BLIMP1 (EMD) at diagnosis and no significant plasma cell infiltrate in the random BMB. Three expression suggest that EBV may play a role in the development of these cases although patients developed significant EMD during subsequent stages of their disease. IHC the high frequency of the GC phenotype in AR-DLBCL is surprising. These data provide revealed that the myeloma cells in all 18 cases were positive for CD138 and negative new insights into differences between AR- and IC-DLBCLs. for CD56. CD20 was variably positive in 2/16 cases. BCL-1 was expressed in 7/17 cases. Cytogenetics was performed in 17 cases, 5 contained the t(11;14) all of which 1171 Genetic Instability and RAS Mutations Characterize Leukemic were positive for BCL-1 by IHC. Five had complex karyotypes lacking the t(11;14), Progression in t(9;11)-Bearing Acute Myeloid Leukemia and 7 were normal diploid. P Chandra, R Luthra, G Garcia-Manero, D Jones. UT MD Anderson Cancer Center, Conclusions: IgM myeloma is a rare entity that is characterized by negativity for CD56 Houston, TX. and CD20 in most instances. Most cases are negative for the t(11;14), although this Background: Acute myeloid leukemias (AML) harboring translocation of the mixed- genetic lesion is somewhat overrepresented in IgM myeloma. There is good correlation lineage-leukemia (MLL) gene at chromosome 11q23 have a poor prognosis. Genetic between BCL-1 detectable by IHC and the presence of the t(11;14) in these cases. factors that may synergize with MLL to produce this aggressive phenotype are poorly While IgM lacks distinctive clinical features, a significant minority of patients with studied. To identify interacting factors in leukemic progression, we compared the flow IgM myeloma develop EMD. cytometric, cytogenetic and molecular features at diagnosis and relapse in 61 patients with t(9;11)(p21-22;q23) AML. 1169 Hematologic Manifestations of Copper Deficiency: One Year Design: Patients included 22 men and 39 women, with a median age of 50 years (range Experience of a Single Institution 1-81), including 32 de novo and 29 therapy related (t-AML) t(9;11) cases, with 0.2 N Cetin, K Oza, Z Singh, J Bornhorst, L Chen, R Lorsbach. University of Arkansas for to 87.5 months followup (median 12.2). Flow cytometric (FC) phenotype, G-banded Medical Sciences, Little Rock, AR; UT Medical School, Houston, TX. karyotype and MLL-FISH analysis, were compared with codon 12, 13, and 61 KRAS Background: Copper deficiency can cause pancytopenia and dyserythropoesis in and NRAS mutation status performed by PCR-based pyrosequencing, and codon 835/836 the bone marrow (BM) mimicking myelodysplastic syndrome (MDS). After copper FLT3-ITD mutation status mostly determined by PCR-based methods. supplementation, hematological parameters rapidly improve. However, copper levels Results: FAB subtypes at diagnosis were M5 (41%), M4 (26%), M2 (13%), M1 (15%), are not routinely checked in patients with cytopenias. The literature on the hematological and M0 (5%). Most patients displayed some degree of monocytic differentiation by manifestations of copper deficiency and its awareness amongst clinicians is limited. The cytochemistry or FC phenotype. RAS mutations were found at diagnosis in 32% (14/44), purpose of this study was to investigate the prevalence, laboratory and hematological with FLT3 alterations in 9% (4/43) and were mutually exclusive; RAS mutations were manifestations of copper deficiency. more frequent in t(9;11)-AML as compared to t(6;11)-bearing AML from our database Design: The laboratory database at UAMS was searched for patients with a serum (1/17, p = 0.04). Fifty-one patients (84%) had relapsed/refractory disease, with 45 copper determination in the year 2005. Patients with low copper levels (<80mcg/dL; deaths due to disease (median survival 11.8 months), 7 alive with disease, and 9 in normal range 85-155mcg/dL) were selected for further analysis. The hematologic complete remission. t-AML patients (p = 0.023) and those with cytochemical evidence data and BM aspirates were reviewed and correlated with clinical characteristics and of monocytic differentiation did worse (p = 0.05), by Kaplan-Meier analysis. Only 6 laboratory parameters. cases (10%) had t(9;11) as the sole cytogenetic aberration throughout disease. High Results: A total of 185 tests for serum copper were performed. Forty patients (22%) with rates of aneuploidy (48% overall, +8 in 18%), and/or structural chromosomal changes hypocupremia were identified; the primary diseases were: cirrhosis 13, malignancies (43%) were common at diagnosis or upon relapse. Shifts in FC phenotype were also 8, gastric surgery 7 neurological diseases 4, nutritional deficiencies 2, diabetes 2, noted in 7/19 cases. Changes in HLA-DR, CD34, CD52, and CD64 correlated with autoimmune 2, cystic fibrosis 1, and familial polyposis 1. Laboratory studies revealed increasing aneuploidy and disease progression. decreased white blood cell (WBC) count, hematocrit, and platelet counts in 19, 26, and 27 Conclusions: AML with t(9;11) (p21-22;q23) often shows complex cytogenetic patients respectively. The mean corpuscular volume was decreased in 4/40 and increased changes including shifting patterns of aneuploidy and genomic progression upon in 7/40 patients. Serum ceruloplasmin level was low in 3/17. High serum zinc level was relapse, factors which may contribute to its extremely poor outcome. Frequency of observed in 4/17 patients. BM evaluation performed in 6 patients typically documented RAS mutation is similar to other leukemias with monocytic differentiation. Therefore, dyserythropoesis with erythroid vacuolation, ringed sideroblasts, and presence of iron strategies targeting RAS pathway activation may be helpful in treatment of this within plasma cells. In 2 patients the trilineage dyspoesis was significantly pronounced aggressive AML subtype. so as to mimic primary MDS. Hemophagocytosis and marrow hypoplasia were noted in one case each. 1172 Evaluation of 6q Deletion in Waldenstrom’s Macroglobulinemia, a Conclusions: Our findings demonstrate that copper deficiency frequently manifests Multicenter Collaborative Study as clinically significant peripheral blood cytopenias and is often associated with H Chang, C Qi, Y Trieu, A Jiang, W Xu, D Reece, C Chen, KH Young, A Chesney, P dyserythropoiesis. This study highlights the importance of evaluating serum copper level Jani, C Wang, R Padmore. University Health Network, Toronto, Canada; University of to exclude hypocupremia as the cause in cytopenic patients to avoid misinterpretation Wisconsin, Wisconsin; Sunnybrook Health Sciences Center, Toronto, Canada; Thunder of the peripheral blood and BM findings as indicative of primary MDS. A high index of Bay Health Sciences Center, Thunder Bay, Canada; Mount Sinai Hospital, Toronto, suspicion for copper deficiency is particularly warranted in patients with liver disease, Canada; Ottawa General Hospital, Ottawa, Canada. in those who have undergone gastrectomy, or in the presence of typical BM findings Background: Waldenstrom macroglobulinemia (WM) is a rare clinical syndrome as described above. characterized by a clonal lymphoplasmacytic bone marrow infiltrate and a serum IgM paraprotein. The deletion of the long arm of chromosome 6 (del (6q)) is the most common 1170 Comparison of AIDS-Related Diffuse Large B-Cell Lymphomas cytogenetic abnormality in WM, but its prognostic significance is unclear. (AR-DLBCLs) and Immunocompetent (IC) DLBCLs Suggest Biologic Design: A total of 89 cases diagnosed with WM from the participating institutions were Differences entered into the study. Lymphoplasmacytic cells from bone marrow aspirates of the A Chadburn, S Silver, L Pasqualucci, R Dalla-Favera, K Merati, M McGrath, L Ayers, patients were assessed for 6q deletions by interphase fluorescence in situ hybridization JM Orenstein, DM Knowles, E Cesarman. Pathology & Laboratory Medicine, Weill (FISH) with 6q21 and 6q25 probes. Clinical and laboratory data were reviewed. Cornell Medical College; George Washington University, University of California- Results: Overall, interphase FISH detected hemizygous 6q deletions in 37 (41.6%) of San Francisco, Ohio State University; Pathology & Cancer Genetics, Herbert Irving the 89 WM cases. The 6q21 locus was deleted in 31 (34.8%) of 89 cases, and 6q25 in 25 Comprehensive Cancer Center, Columbia University; Acupath Laboratories, Plainview, (33.8%) of 74 cases. Both 6q21 and 6q25 were deleted in 19 (51%) of 37 deleted cases. NY. Patients with 6q deletions had higher C-reactive protein levels than non-deleted patients Background: DLBCL accounts for 30-40% of non-Hodgkin lymphoma (NHL) in (p= 0.02). There was no correlation between del (6q) and other biological factors such as western countries and about 40% of AIDS-related NHL. Although previous studies have age, gender, hemoglobin, platelet count, viscosity, beta-2 microglobulin, albumin, IgM compared AR-DLBCLs with those in IC patients, no large study comparing histogenic level and degree of bone marrow lymphoplasmacytic infiltration. The median follow-up origin, Epstein-Barr virus (EBV) positivity and prognostic marker expression in AR- was 57.5 months with median overall survival of 163 months and a 10-year survival rate DLBCLs with IC-DLBCLs has been done. of 63%. Twenty-eight (93%) of 30 patients with 6q deletion received treatment, while Design: Tissue microarrays of 151 IC-DLBCLs and 115 AR-DLBCLs were studied 32 (80%) of the 40 patients without the deletion were treated (p=0.17). There was no for expression of CD10, BCL6, MUM1, BLIMP1, BCL2, p53 and EBER (EBV) using significant difference in time to the initial treatment between deleted and non-deleted standard protocols. Cases were classified as germinal center (GC), non-GC and not groups (median 5.6 months vs. 2.6 months, p=0.46), or overall survivals in patients GC/non-GC (null) based on the schema of Hans, et al. SPSS software was used for with and without del (6q) (163 months vs. not reached, p= 0.83). statistical analysis. Conclusions: Our data indicat that 6q deletions are frequently detected in WM but do Results: 71/115 (62%) AR-DLBCLs were of GC, 29 (25%) of non-GC and 15 (13%) not appear to influence the clinical outcome. The 6q deletion extends beyond 6q21 to of null origin; the IC-DLBCLs were evenly divided into GC and non-GC cases (each involve at least the 6q25 locus. Further studies with clinical trials and longer follow-up 65; 43%; p<0.01). A larger proportion of AR than IC-DLBCL was CD10 positive (57% are indicated to confirm its prognostic relevance, identify the minimal deleted region 258A ANNUAL MEETING ABSTRACTS on 6q, and search for candidate tumor suppressor gene(s) potentially involved in the chromatin, abundant cytoplasm, cytoplasmic projections, and numerous TRAP-positive pathogenesis of WM. cytoplasmic granules. The BM were extensively involved (range: 60-95%) and showed a typical interstitial infiltrative pattern. Monotypic B-cells in all cases expressed CD5 1173 Expression of Survival Signals of Endoplasmic Reticulum Stress (dim to bright), CD20, CD22/CD11c, and CD103. IHC stains for TRAP and DBA- in Hodgkin Lymphoma 44 were all positive. Four patients, whose neoplastic B-cells coexpressed dim CD5, received 2-chlorodeoxyadenosine (2-CDA) treatment. All these 4 patients achieved KC Chang, WC Chen, YT Jin, PCH Chen, D Jones. National Cheng Kung University, excellent initial responses, 3 of which were confirmed by follow-up BM biopsies. Tainan, Taiwan; Veterans General Hospital-Taipei, Taipei, Taiwan; University of Texas Nevertheless, all of these 3 patients eventually had recurrent disease. In 1 case, neoplastic M.D. Anderson Cancer Center, Houston. cells coexpressed bright CD5 and cyclin D1, and were also positive for CCND1/IGH Background: Hodgkin lymphoma (HL), originated from germinal center B cells, is translocation. This patient was initially treated as mantle cell lymphoma without associated with Epstein-Barr virus (EBV). EBV-encoded latent membrane protein 1 response. Because its pathologic features and relatively indolent course were most (LMP1) rescues and reprograms germinal center B cells towards a Reed-Sternberg cell- consistent with HCL, this patient recently received 2-CDA treatment. like phenotype. In addition, LMP1 induces endoplasmic reticulum (ER) stress-related Conclusions: Rare CD5+HCLs do exist and have the common clinicopathologic features unfolded protein responses in EBV-infected B cells. of HCL. They respond to initial 2-CDA treatment but may have a higher recurrence Design: To test the expression of ER stress signals (PERK, GRP78, XBP1, CHOP) in HL rate. It remains to be seen if the patient with the CCND1/IGH translocation will have and the association with EBV, 156 cases of HL were analyzed by immunohistochemistry a response to 2-CDA treatment as did the other CD5+HCLs. and EBV in situ hybridization. Results: ER stress survival signals (GRP78 and XBP1 in 49% and 34% of cases, respectively) were expressed in all histologic subtypes of HL and in both EBV-positive 1176 Complete Absence of KSHV/HHV8 in Post-Transplant and –negative cases at a similar level. In contrast, HL rarely expressed ER death signal Lymphoproliferative Disorders: An Immunohistochemical and Molecular (CHOP, 9%). There was agreement of EBV in situ hybridization with LMP1 stain (p Study of 52 Cases < 0.0001) and GRP78 with XBP1 expression (p = 0.006). In vitro LMP1 transfection W Chen, Q Huang, CW Zuppan, EH Rowsell, JD Cao, LM Weiss, J Wang. City of Hope increased expression of GRP78 and XBP1 in HL cell line. Expression of ER signals National Medical Center, Duarte, CA; Loma Linda University Medical Center, Loma did not bear prognostic significance. Linda, CA; Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Conclusions: It implies that near half cases of HL adapted and survived ER stress, which Hangzhou, Zhejiang, China. may be associated with overexpression of LMP1 and may be a decision-making step Background: Kaposi sarcoma-associated herpesvirus (KSHV), or human herpesvirus accounting for the relatively rare occurrence of HL in more prevalent EBV infection. 8 (HHV8), is lymphotropic and has been associated with primary effusion lymphoma, multicentric Castleman disease (MCD), and MCD-associated plasmablastic lymphoma. 1174 A Flow Cytometric Protocol for Differential Diagnoses of Acute KSHV/HHV8-associated lymphomas occur mostly in HIV-positive and other Monocytic Leukemia, Chronic Myelomonocytic Leukemia and Reactive immunosuppressed patients, suggesting an association of immunosuppression, KSHV/ Monocytosis HHV8 infection and lymphomagenesis. Post-transplant lymphoproliferative disorders (PTLDs) are a heterogeneous group of lymphoproliferative disorders that occur C Chen, RM Braziel, J Huang, G Fan. University of Maryland, Baltimore, MD; Oregon exclusively in iatrogenically immunosuppressed recipients following solid organ or bone Health & Science University, Portland, OR. marrow transplantation, and represent a spectrum of diseases ranging from early EBV Background: Leukemias with a prominent monocytic component include acute driven polyclonal B-cell proliferation to full-fledged lymphoma. The clear relationship myelomonocytic leukemia, acute monocytic leukemia (AMoL), and chronic between KSHV/HHV8 infection and pathogenesis of PTLDs remains largely unknown. myelomonocytic leukemia (CMML). Accurate diagnosis of these diseases is crucial In the present study, we investigate the role of KSHV/HHV8 in various forms of PTLD for correct clinical management. Although flow cytometry (FC) is a highly sensitive in 43 organ transplant patients. and efficient method for evaluation of leukemias, its utility in diagnosing AMoL and Design: 52 formalin-fixed, paraffin-embedded PTLD tissue specimens from 43 patients CMML has to date been limited. The aim of this study is to identify a practical protocol were analyzed for EBV status by EBER in situ hybridization and for expression of that can distinguish immature from mature monocytes, and distinguish neoplastic from KSHV/HHV8 LNA-1 protein by immunohistochemistry, as well as for KSHV/HHV8 non-neoplastic monocytes. genome by PCR analysis on most cases. Design: Using 4-color FC, we characterized monocytic cells from 75 leukemia samples Results: The 43 patients ranged from 1 to 64 years old (median age 9 years). Most (43 AMoL and 35 CMML), 37 non-neoplastic bone marrow or blood samples (15 from PTLDs developed after heart (79%), kidney (11.5%), and liver (9.5%) transplantation. normal individuals and 22 from various reactive conditions). Monocytic differentiation Diagnosis and classification were based on the current WHO criteria. The PTLD and phenotypic features were determined with a panel of 11 antibodies, including CD13, subtypes included 12 early changes (1 plasmacytic hyperplasia and 11 infectious- CD14, CD15, CD34, CD36, CD45, CD56, CD64, CD91, CD117, and CD163. monoinucleosis-like), 10 polymorphic, 23 monomorphic (5 Burkitt, 14 diffuse large Results: We compared expression patterns of various markers in AMoL, CMML and B cell lymphoma, 1 plasmacytoma, 1 multiple myeloma, and 2 T-cell), 1 Hodgkin non-neoplastic monocytes, and found that (1) CD36 and CD64 were expressed by all lymphoma (HL), 5 HL-like lesions, and one unclassified. 92% of tested cases (46/50) monocytic cells and 50% of maturing granulocytes; the expression levels were higher in were EBER+. None of the tested specimens (0/51) showed expression of KSHV/HHV8 most but not all AMoL cells as compared to granulocytes; (2) absence or partial absence LNA-1 protein. Furthermore, all specimens tested (46/46) were negative for KSHV/ of CD14 or CD91 was characteristic of immature monocytes, and was found in 91% of HHV8 genome by PCR analysis. AMoL but only in 14% of CMML; (3) diminished CD163 was seen in 78% of AMoL Conclusions: Despite the strong association of KSHV/HHV8 with lymphoma in and 50% of CMML; (4) expression of CD56 was detected in 70% of AMoL and 33% patients with HIV infection and other forms of immune dysregulation, our results of CMML; (5) under-expression of CD13 or CD15 was identified in 83% and 43% of indicate that KSHV/HHV8 is not associated with lymphoproliferative disorders in the AMoL, and 15% and 45% of CMML, respectively; (6) an increase in CD34+ or CD117+ post-transplantation setting. cells (more than 7%) was seen in 38% of AMoL and 30% CMML. Although no single marker can reliably distinguish neoplastic from reactive monocytes, a combination of 11 markers showed that 98% of AMoL and 85% of CMML samples had at least two 1177 High Frequency of Clonal Immunoglobulin Receptor Gene abnormal FC features whereas only 3% of non-neoplastic monocytes did. Rearrangements in Sporadic Histiocytic/Dendritic Sarcomas Conclusions: Our studies show that a relatively simple FC protocol with 11 antibodies W Chen, SK Lau, D Fong, J Wang, E Wang, DA Arber, LM Weiss, Q Huang. City of can distinguish neoplastic monocytes in AMoL and CMML from non-neoplastic Hope National Medical Center, Duarte; Stanford University, Stanford; Loma Linda monocytes in normal or reactive conditions with high sensitivity and specificity. In University, Loma Linda; Duke University, Durham. addition, loss of CD14 and/or CD91 is reliably identified in CD45dim immature Background: True histiocytic/dendritic cell (H/DC) sarcomas are extremely rare but monocytes, indicating that CD14 and CD91 are valuable markers in differentiation of aggressive hematopoietic neoplasms. The diagnosis of H/DC sarcomas is based on AMoL and CMML. morphology and the presence of immunophenotypic features of D/DC differentiation. The current WHO definition of H/DC sarcoma requires the absence of clonal B/T cell 1175 CD5-Positive Hairy Cell Leukemia: A Rare but Distinct Variant receptor gene rearrangements. Nonetheless, rare case of H/DC sarcoma with detectable IGH gene rearrangements have been described. Recently, Feldman et al provided D Chen, WG Morice, DS Viswanatha, CA Hanson. Mayo Clinic, Rochester, MN. compelling evidence that patients with follicular lymphoma and concurrent H/DC Background: CD5-positive hairy cell leukemia (CD5+HCL) is a rare B-cell sarcoma share identical genotypic features, suggesting the trans- or de-differentiation lymphoproliferative disorder. Its existence has been controversial due to overlapping of two otherwise morphologically and immunophenotypically distinctive neoplasms. phenotypic features with other mature B-cell neoplasms. Its clinicopathologic features Here, we investigate the molecular characteristics of 23 patients with sporadic H/DC have not been reported. In this retrospective study, we confirmed the existence of this sarcomas. rare HCL variant, and characterized its clinicopathologic features. Design: PCR on genomic DNA from formalin-fixed, paraffin-embedded tumor tissues Design: We searched our bone marrow (BM) database for HCL cases that had CD5 with primer sets designed to detect IGH and IGK gene rearrangements was performed. coexpression by neoplastic B-cells. Out of a total of 157 HCL cases, we identified Q-PCR for major and minor break points of IGH/BCL2 fusion was also measured. All 5 CD5+HCL patients. Clinical data from each case were reviewed. Blood and BM positive cases were verified by direct DNA sequencing. Furthermore, a t(14;18) positive specimens were examined including Tartrate-resistant acid phosphatase (TRAP) histocytic sarcoma case was confirmed by targeted FISH analysis. cytochemical studies, flow cytometric studies using a panel of B and T cell antibodies, Results: Twenty-five specimens from 23 patients with a confirmed diagnosis of H/DC and immunohistochemical (IHC) stains on BM biopsy sections using antibodies against sarcoma (14 H and 9 DC) were included. None had a history of or concurrent diagnosis CD20, TRAP, DBA-44, and cyclin D1. FISH analyses for CCND1/IGH translation were of lymphoma. All cases were negative for CD20. Nine of 23 cases showed clonal IGH performed using BM aspirate samples. (with or without IGK) gene rearrangements, while 2 cases demonstrated only clonal Results: All 5 patients (median age: 59, range: 30-77; M:F = 4:1) had anemia, IGK gene rearrangements. All 11 (48%) cases with gene rearrangements were further monocytopenia, circulating neoplastic lymphocytes, splenomegaly, and absent sequenced to determine immunoglobulin gene VDJ or VJ assemblies and segment lymphadenopathy at presentation. All of the patients are alive (median follow-up in usage. Among those, one histiocytic sarcoma case showed IGH/BCL2 by PCR, which months: 32, range: 6-116). The neoplastic lymphocytes in all cases had reticulated was further confirmed by FISH analysis. Notably, all IGH and/or IGK positive H/DC ANNUAL MEETING ABSTRACTS 259A sarcomas were negative for B-cell associated transcription factors PAX-5 and Bob-1, 1180 Clonal X Chromosome Inactivation Suggests That Splenic Cord while 4/7 histiocytic sarcoma cases were positive for OCT-2. Capillary Hemangioma and Myoid Angioendothelioma Are True Neoplasms Conclusions: This study provides evidence that clonal IG receptor gene rearrangements and Not Subtypes of Splenic Hamartoma may be detected at a high frequency in sporadic H/DC sarcomas, challenging the current A Chiu, M Czader, L Cheng, M Wang, DM Knowles, H Al-Ahmadie, A Orazi. Weill definition of H/DC sarcoma by the WHO. The finding of one “de novo” histiocytic Cornell Medical College, New York, NY; Indiana University, Indianapolis, IN; The sarcoma with a IGH/BCL2 fusion in neoplastic histiocytes is intriguing and requires University of Chicago, Chicago, IL. further investigation. Background: Splenic hamartoma (SH) is a rare tumor-like proliferation composed of structurally disorganized red pulp elements. It has been hypothesized that other red 1178 Distinct Cytogenetic Profiles of Diffuse Large B-Cell Lymphoma pulp lesions, such as cord capillary hemangioma (CCH) and myoid angioendothelioma in Chinese and Western Population (MA), may fall within the spectrum of SH, simply representing morphological variants. Y Chen, BJ Dave, X Zhu, WC Chan, R Irons, K Fu. University of Nebraska Medical We utilized immunohistology and histochemistry to compare the vascular and stromal Center, Omaha, NE; Shanghai Cancer Hospital, Shanghai, China; Fudan University, composition of classical SH (C-SH) with cases of CCH and MA. In addition, we Shanghai, China. ascertained their clonal vs. polyclonal nature. Background: Diffuse large B-cell lymphoma (DLBCL) derived from germinal center Design: Paraffin sections from 11 cases (6, CCH; 3, MA; 2, C-SH) were assessed B cells (GCB) have a better prognosis. Studies from Western countries have shown by reticulin and trichrome stains and immunohistology for stromal (Collagen that cytogenetically GCB-DLBCL frequently exhibits the t(14;18) and gain of 12q, IV,SMA,NGFR), vascular (CD34,CD31,CD8,D2-40), histiocytic (CD163), and whereas non-GCB-DLBCL commonly shows gain of 3q, 18q and loss of 6q. However, proliferation (MIB-1) markers, and EBV-RNA (EBER). Clonality by HUMARA assay GCB-DLBCL and the t(14;18) are significantly less frequent in DLBCL from Chinese was performed in 7 female cases by laser-assisted microdissection (PixCell II System, patients. We studied cytogenetic profiles in Chinese patients with DLBCL to determine Arcturus Engineering, Mountain View, CA), sampling areas of hamartoma and normal possible distinct alterations and whether there are any differences between the Chinese spleen. Samples with/without HhaI restriction endonuclease digestion were analyzed by and western cases. PCR on polyacrylamide gels by autoradiography. The samples were informative if the Design: Conventional cytogenetic analysis was performed on 134 Chinese patients control samples displayed two alleles without HhaI digestion. Nonrandom inactivation with de novo DLBCL. Immunohistochemical analysis of CD10, BCL6, and MUM1 of the X chromosomes was defined as a complete or near complete absence of one or expression was used to categorize the GCB or non-GCB DLBCL (Hans, et al, 2004, the other allele after HhaI digestion, indicating predominance of one X-linked human Blood 103:275-282). Interphase fluorescence in situ hybridization (FISH) was used to androgen receptor allele. determine the incidence of the t(14;18). Results: All cases showed increased reticulin and collagen content. The 2 C-SH cases Results: Thirty-two of the 134 (24%) cases showed near-triploid or near-tetraploid showed a CD8(+) vasculature, while CCH and MA cases contained many CD34(+) with high chromosome number (CN) ranging from 58 to 101. The remaining 102 CD31(+) vessels but little CD8(+) vasculature. All cases were D2-40(-), EBV(-), and (76%) cases were near-diploid. Only 4 of 134 (3%) cases had the t(14;18). The most had <10% MIB-1. CCH and MA cases lacked perisinusoidal expression of collagen frequent genetic imbalances included whole chromosome gains of 18 (17%), 3 (14%), IV or NGFR. Increased CD163(+) histiocytes were found in 2 cases (1 MA; 1 CCH). 5 (14%), X (11%), 7 (10%), and partial gains of 1q (16%), 7p (10%), 7q (10%). Whole Nonrandom X-chromosome inactivation was informative in all 7 cases. Three cases chromosome losses of Y (21%), 15 (11%) and partial losses of 6q (20%), 17p (15%), showed a non-random X-chromosome inactivation pattern, indicating clonality. All and 1p (13%) were also frequently noted. We divided the 134 cases into 3 groups: GCB- clonal cases had non-classical SH morphology. DLBCL with near-diploidy (n=24), non-GCB DLBCL with near-diploidy (n=78) and Conclusions: In spite of considerable morphologic heterogeneity and overlapping DLBCL with high CN (n=32). Gain of 11q was more commonly seen in near-diploid features, C-SH and non-classical SH lesions termed CCH and MA are different in GCB DLBCL. Gain of 7p and loss of 17p were more frequently seen in near diploid terms of vascular/stromal composition. Clonality analysis supports a true neoplastic non-GCB DLBCL. Multiple ploidy-related gains and losses were noted in high CN- origin for the two latter diseases. DLBCL, including frequent gains of 5 (34%), 20 (27%), 6p (21%), and losses of 17p (40%), 1p (35%), and 6q (35%). 1181 Most Primary Intestinal T and NK/T-Cell Lymphoma in Taiwan Are Conclusions: Our studies revealed distinct cytogenetic profiles of DLBCL in Chinese Unspecified Peripheral T-Cell Lymphomas Showing Cytotoxic Phenotype, compared to the reported Western patients, specifically gain of 11q in GCB-DLBCL and NK Lineage and Perforation Predict Poor Survival and loss of 17p in non-GCB DLBCL cases. A very low frequency of the t(14;18) is S-S Chuang, S-T Chang, W-T Huang, P-P Hsieh, M-H Tsou, Y-C Jung. Chi-Mei Medical observed in GCB-DLBCL among Chinese patients. Center, Tainan, Taiwan; Chang Gung Memorial Hospital-Kaohsiung, Kaohsiung, Taiwan; Veterans General Hospital-Kaohsiung, Kaohsiung, Taiwan; Koo Foundation Sun 1179 Differential Expression of L-Plastin in Primary Mediastinal Large Yat-Sen Cancer Center, Taipei, Taiwan; Sin-Lau Christian Hospital, Tainan, Taiwan. B-Cell Lymphoma (PMBCL) and Classical Hodgkin Lymphoma (cHL) Background: Most primary intestinal T and NK-cell lymphomas (ITNKL) in the West MD Chiselite, V Basrur, D Fermin, K Conlon, J Teruya-Feldstein, KSJ Elenitoba- are enteropathy-associated T-cell lymphomas (EATL), a rare complication of celiac Johnson, MS Lim. University of Michigan, Ann Arbor, MI; Memorial Sloan-Kettering disease (CD) which is common in the West but is extremely rare in the East including Cancer Center, New York, NY. Taiwan. Type A EATL is definitely CD-associated, with typical HLA types and serology, Background: PMBCL is a rare subtype of extranodal diffuse large B-cell lymphoma and large tumor cells that are usually CD56-negative. The rare type B variant is probably (DLBCL) that is difficult to distinguish from other types of DLBCL and cHL. Recent unrelated to CD, and the small tumor cells usually express CD56. studies reveal similar gene expression profiles in PMBCL and cHL suggesting a common Design: We retrospectively collected 22 cases of ITNKL, the largest series from Taiwan, cell of origin. We hypothesized that quantitative mass spectrometry-based proteomic for a clinicopathological and molecular study. strategies would be useful in identifying potential protein biomarkers that may help in Results: There were 14 males and 8 females with a median age of 56. Seventeen (77%) the distinction of these lymphomas. presented with perforation. Seventeen (77%) patients were at stage I, and 5 (23%) at Design: We performed proteomic analysis of three cell lines: PMBCL (Karpas 1106P), stage II. Eighteen (82%) tumors were of T cell lineage and 4 (18%), NK cell. The tumor cHL (L428) and DLBCL (SUDHL-9) using a differential isotopic strategy, iTRAQ cells in 18/22 (82%) cases were small to medium-sized including 15/18 T and 3/4 NK (isobaric tag for relative and absolute quantitation) and tandem mass spectrometry. After cell tumors. The T-cell tumors included 16 unspecified peripheral T-cell lymphomas normalization, quantitative data were subjected to false discovery rate (FDR) calculation (uPTL) without enteropathy and 2 with features of enteropathy mimicking Western type to identify differentially expressed proteins through Mixture Modeling. Significantly B EATL (Chuang SS et al. Histopathology 2008; 53: 234). These 18 tumors expressed differentially expressed proteins were scored at a false discovery rate (FDR) cut-off CD3 (100%), T-cell intracellular antigen-1 (89%), CD7 and CD56 (83%), CD8 and of ≤0.13. L-plastin was selected and validated by immunoblotting (clone LPL4A.1, granzyme B (67%), CD2 (56%), and CD5 (6%), but not CD4. All tumors expressed at NeoMarkers, Fremont, CA) on PMBCL and cHL cell lines and immunohistochemistry least one cytotoxic marker. Seven (39%) tumors were positive for EBV-encoded mRNA of tissue microarrays (TMAs) of PMBCL (n=37), cHL (n=94) and NLPHL (n=16). (EBER). All but two tumors with poor DNA quality were clonal for T-cell receptor Results: Several proteins that have been previously reported to be differentially (TCR) gene rearrangement. The 4 NK cell lymphomas expressed at least one cytotoxic expressed between cHL and PMBCL were identified including Fascin, Galectin-1, marker. All 4 tumors were positive for EBER and were polyclonal for TCR. Three of Galectin-3, STAT1 and SWAP70. We selected one protein, L-plastin for validation. these expressed CD56. The 1-yr and 2-yr survival rates of these 22 patients were at Western blotting showed high L-plastin expression in the PMBCL cell lines K1106P 38% and 21%, respectively. NK cell lineage (log rank test; P = 0.012) and perforation and MedB1, relative to cHL cell lines confirming the mass spectrometry data. The (P = 0.026) were associated with poor survival. prevalence of L-plastin expression in TMAs was as follows: Conclusions: ITNKL in Taiwan is predominately uPTL, with a high frequency of perforation at presentation, lack of association with CD, small to medium tumor cells L-plastin (-) weak(+) mod/strong(+) PMBCL 1/37 4/37 32/37 expressing cytotoxic marker, CD56 and EBER, and poor prognosis. Furthermore, NK cHL 32/94 37/94 25/94 cell lineage and perforation predict poor prognosis. NLPHL 1/16 3/16 12/16 Conclusions: We identified L-plastin, a 65 kDa leukocyte-specific actin bundling protein 1182 FISH Panel Testing as a Screening Tool for MDS in Elderly Patients to be differentially expressed in PMBCL and cHL. The strong expression of L-plastin in with Anemia 32/37 (86%) of tissue samples of PMBCL and 25/94 (27%) cHL suggest that it may be KT Codispoti, L DePalma. The George Washington University Medical Center, useful in the distinction of these two neoplasms (P<0.0001). This study demonstrates the Washington, DC. utility of large-scale mass spectrometry-based proteomics for the discovery of proteins Background: A substantial proportion of the American elderly population is anemic and that may serve as potential diagnostic marker panels for the distinction of PMBCL and the presence of anemia has been associated with adverse outcomes, including increased cHL, as well as candidate therapeutic targets. mortality. The most common causes of anemia in the elderly are nutritional deficiency and chronic disease/inflammation. However, over 1/3 of cases are unexplained. A significant proportion of these unexplained anemias could represent early or undiagnosed myelodysplastic disorders (MDS). We assessed the utility of using a FISH panel for common chromosomal abnormalities seen in MDS as a screening test in elderly patients 260A ANNUAL MEETING ABSTRACTS with unexplained anemia who underwent bone marrow biopsy. Table 1: Clinical, PB and BM findings Design: We included 102 elderly patients (defined as ≥ 65 years old) who presented # of cases (%) with anemia (<13g Hb/dL for men and <12g Hb/dL for women) in the outpatient setting. Clinical features Patients did not have other clinical or laboratory findings which would explain their Cases 137 Patients 83 cytopenia(s) and as a result had a bone marrow biopsy performed. Pathology reports, Male 54 (65) conventional cytogenetic analysis (CC), and FISH panel including DNA probes specific Female 29 (35) for chromosome regions 5p15.2, 5q31, 7cen, 7q31, 8cen, and 20q12 were reviewed. Splenomegaly 48 (35) Results: 22 (21.5%) of the 102 patients had MDS. A combination of CC and FISH Thrombocytosis 42 (31) identified characteristic chromosomal abnormalities in 18 (82%) of the patients with BM morphology Hypercellular for age 28 (20) MDS. The remaining 4 (18%) patients were diagnosed with MDS based solely on Megakaryocyte hyperplasia 47 (34) WHO morphologic criteria. Two (11%) of the patients had incongruent CC and FISH Megakaryocyte clustering No clusters 30 (22) results. Except in one case, FISH did not reveal abnormalities not already detected by 1-4 clusters 75 (55) cytogenetics. Of the 80 patients who did not have morphologic or chromosomal findings 5+ clusters 32 (23) of MDS, 61 (76.3%) had anemia of chronic disease, 10 (12.5%) had iron deficiency, Megakaryocyte cytology Large 28 (20) Small 18 (13) and 9 (11.3%) had an unknown cause for their anemia. Atypical 12 (9) Conclusions: A FISH panel for common chromosomal abnormalities in MDS is not Lymphoid aggregate 49 (36) useful as a screening diagnostic test in the elderly population with unexplained anemia. PC rimming 42/49 (86) FISH results confirmed the morphologic findings and conventional chromosome PC clonality results in the majority of the patients with MDS. Most cases of anemia in patients Monotypic λ 59 (43) Monotypic κ 3 (2) without positive CC or FISH could be explained as a result of chronic disease or iron Polytypic 72 (53) deficiency. A minority of patients in our study did not have a definitive cause for their Too few PC (by FC) 3 (2) anemia identified after evaluation of their bone marrow and neither CC nor FISH added Conclusions: The BM histopathology of POEMS is complex with features which additional diagnostic information. may be associated with other disorders. For example, many of the features may evoke a suspicion of a MPN. Likewise the PC clonality of POEMS may be difficult to detect 1183 FOXP2 Deletion May Represent a Candidate Biomarker for due to the presence of a polytypic PC background. The PC-rimmed LAs are distinctive. Myelodysplastic Syndromes POEMS should be considered when these unusual features are encountered in the setting CV Curry, I Nandi, WT Huang, FA Monzon, L Novoa-Takara, CC Chang. The Methodist of monoclonal gammopathy and/or unexplained neuropathy. Hospital, Houston, TX; Rice University, Houston, TX; Chang Gung University College of Medicine, Kaohsiung, Taiwan; Weill Medical College of Cornell University, New 1185 Acute Promyelocytic Leukemia at Time of Relapse: Clinicopathologic York, NY; Medical College of Wisconsin, Milwaukee, WI. Findings and Results of Ancillary Testing Background: The pathogenesis of Myelodysplastic Syndromes (MDS) is poorly ND Dimov, C Bueso-Ramos, LJ Medeiros. MD Anderson Cancer Center, Houston, understood and no reliable biomarkers are available. Our previous study using high TX. resolution 250K single-nucleotide polymorphism (SNP) array in 12 flow cytometry Background: Although the therapy for acute promyelocytic leukemia (APL) is effective, (FCM) sorted MDS bone marrow samples identified 2 cases with deletion of theFOXP2 up to 40% of patients relapse. The biological characteristics of APL at time of relapse gene located at 7q31.1 (cytogenetically cryptic deletion of 7q31.1 in only erythroid are not well described. In this study we evaluated the morphologic, immunophenotypic, fraction and whole chromosome 7 deletion in both erythroid and blastic fractions, molecular and cytogenetic characteristics of APL at time of relapse and compared these respectively). Deletion of FOXP2 was confirmed by real-time quantitative PCR findings with initial diagnostic specimens before therapy. (RQ-PCR). Since 7q31.1 is frequently deleted in MDSs, cryptic deletion of FOXP2, Design: We retrospectively reviewed clinicopathologic data and the results of ancillary particularly in the erythroid fraction, may represent a potential biomarker for MDS testing in all APL patients treated in our institution between 2001 and 2008. patients without cytogenetic abnormalities. Results: Clinical and/or hematologic relapse occurred in 12 of 112 (10.7%) patients. Design: Twenty-six cryopreserved bone marrow samples consist of 8 controls and 18 Time to relapse ranged from 6 to 49 months (mean 19 months). Two patients relapsed MDS cases (8 low-grade, 10 high-grade), including 8 cases from the previous SNP twice and 1 patient had three relapses. The sites of relapse were bone marrow (BM) study. The samples were fractionated into erythroid (CD71 bright/CD34-) and lymphoid (n=8), BM with extramedullary sites (n=3), and central nervous system (CNS) (n=1). fractions (CD45 bright/low side-scatter) using multicolor FCM. FOXP2 deletion status Two patients died from intracranial bleeding and CNS relapse. While in second was analyzed by performing RQ-PCR from erythroid and lymphoid fractions. In each remission 2 patients died from meningitis and sepsis, respectively. BM blasts varied case, FOXP2 to β-actin ratio of erythroid fraction was normalized to the same ratio of from 71 to 96% (mean 83.3%) at initial diagnosis and from 0 to 82% (mean 39.2%) lymphoid fractions. The FOXP2 gene deletion was correlated with cytogenetics and at relapse. Peripheral blood blasts ranged from 1.44 to 126.1 x 109/L (mean 47.29) at SNP analysis results. initial diagnosis and from 0 to 46.78 x 109/L (mean 5.87) at relapse. Slides of the initial Results: None of the control cases showed FOXP2 deletion. Four out of 18 (22%) MDS BM specimens were available in 8 patients; blast cell morphology was similar in 7 and samples demonstrated FOXP2 deletion; three were low-grade (2 refractory anemia, 1 changed from microgranular to typical in 1 patient. Flow cytometry immunophenotyping 5q- syndrome) and one was high-grade (therapy-related). All three low-grade MDSs was performed on both the initial and relapse specimens in 6 patients: at relapse 2 cases showed normal karyotype for chromosome 7 (2 cases with normal karyotype, 1 with expressed CD33 and HLA-DR, 1 case expressed CD34, 1 case showed increased CD117 5q-). The high-grade MDS had complex cytogenetic abnormalities including monosomy intensity, and 1- decreased CD13 intensity. Cytogenetic studies, performed on both the 7. The RQ-PCR results were consistent with SNP array data in all cases studied by initial and relapse specimens in 9 patients, showed 5 identical karyotypes, additional SNP array previously. aberrations at relapse in 3 cases (including +8, t(9;11) and complex), and diploidy in 1 Conclusions: Our studies suggested that deletion of the FOXP2 gene, a member of the case. All 11 patients tested by PCR at time of relapse were positive. FOX transcriptional factor family, may be an early event in MDS and may represent Conclusions: Changes in blast cell morphology (from microgranular to typical), a candidate biomarker. Further studies are needed to confirm our observation and to immunophenotype (gain or loss of antigens), and cytogenetics (diploid or complex characterize the function of FOXP2 in hematopoiesis for its role in the pathogenesis karyotypes) can occur at time of APL relapse. Although conventional cytogenetic studies of MDS. may not be adequately sensitive to detect all APL relapses, they are helpful for detecting additional aberrations that occur in a subset of patients. These findings underline the 1184 Bone Marrow (BM) Histopathology of POEMS Syndrome: Analysis importance of a multidisciplinary approach incorporating morphological and ancillary of 134 Cases Reveals Features Which May Obscure the Diagnosis and studies for evaluating APL patients in follow up BM specimens. Mimic a Myeloproliferative Neoplasm (MPN) LD Dao, WG Morice, PJ Kurtin, A Dispenzieri, JD Hoyer. Mayo Clinic, Rochester, 1186 The Density of Regulatory FOXP3+ T-Cells Is Associated with MN. Caspase-3 Activation in Hodgkin/Reed-Sternberg Cells of Classical Background: POEMS is a rare syndrome comprised of polyneuropathy, organomegaly, Hodgkin Lymphoma endocrinopathy, monoclonal plasma cell (PC) dyscrasia, skin changes, sclerotic bone E Drakos, GZ Rassidakis, EJ Schlette, TP Vassilakopoulos, C Kittas, AH Sarris, LJ lesions, Castleman disease (CD), edema, papilledema and thrombocytosis. The BM Medeiros. The University of Texas M.D. Anderson Cancer Center, Houston, TX; histopathology of POEMS remains poorly characterized. National and Kapodistrian University of Athens, Athens, Greece; Hygeia Hospital, Design: Peripheral blood (PB) smears, BM aspirates and biopsies from 137 clinically Athens, Greece. documented POEMS cases were reviewed. PC clonality was analyzed by 2 or 6 color Background: FOXP3+ regulatory T-cells (T-regs) are critical for suppressing destructive flow cytometry (FC) (100 and 37 cases respectively), BM immunohistochemistry (IHC) overactivation of the immune system. Thus, T-regs might provide immunologic privilege (66 cases) and in situ hybridization (3 cases). JAK2 mutational analysis was performed to neoplastic cells facilitating their survival. By contrast, recent reports suggest that high on 43 BM aspirates or PBs. numbers of FOXP3+ T-regs, which are prominent among the reactive population in Results: Table 1 summarizes the clinical, PB and BM findings. Megakaryocyte classical Hodgkin lymphoma (cHL) are associated with favorable prognosis. However, hyperplasia and clusters were frequent, however none of the POEMS cases tested the relationship between FOXP3+ T-regs and intrinsic biologic parameters of neoplastic had detectable JAK2 mutation. Erythroid and granulocytic dysplasia was not present. Reed-Sternberg/Hodgkin (HRS) cells remains unknown. Lymphoid aggregates (LA) were also common and in most cases were surrounded at Design: Expression of FOXP3, granzyme B and activated caspase-3 (aC3) were assessed the periphery by PCs. One case had diagnostic features of CD. Monotypic PCs were by immunohistochemistry using specific monoclonal antibodies in 85 cHL tumors detected in 62 cases (45%) by either IHC and/or FC; the vast majority of monotypic PCs of previously untreated patients with available clinical data. FOXP3+ T-regulatory expressed λ light chain. The monotypic PCs were typically present in a background of T-cells, granzyme B+ cytotoxic T-cells and aC3+ HRS cells were counted manually polytypic PCs, and in many cases only polytypic PCs were identified. and FOXP3+ T-regs /HRS (FOXP3/HRS) and Granzyme B+ T-cells/HRS (GB/HRS) ratios were calculated. ANNUAL MEETING ABSTRACTS 261A

Results: FOXP3+ T-regs, or cytotoxic Granzyme B+ T-cells were present in all cHL Design: After a small pilot study, 47 biopsies of non-transformed MF (36 first diagnostic tumors. The FOXP3/HRS ratio in the study group ranged from 0,5 to 10,1 (mean 3.7, bx, 11 first bx at UPMC) were stained for Ki67 and CD30. Up to 1000 dermal (D) and median 3.4, SD 2.1). The GB/HRS ratio ranged from 0,1 to 8 (mean 1, median 0.6, SD epidermal (Ep) lymphocytes were separately counted and % Ki67+ and CD30+ cells 1.4). The percentage of aC3+ HRS cells reflecting the rate of spontaneous apoptosis calculated. Results were correlated with stage at dx (44 patients), therapy types and ranged from 0% to 4,3% (mean 1.3, median 0.8, SD 1.2). FOXP3/HRS or GB/HRS survival from onset of rash, definitive dx, and bx date. ratios were not correlated with clinical parameters including, gender, age, or stage of Results: All cases had at least rare D-CD30+ cells. Higher %’s of D-CD30+ and D-Ki67+ disease, or the presence of Epstein-Barr virus in HRS cells. Importantly, FOXP3/HRS lymphoid cells were associated with a significantly higher stage at dx. CD30 positivity ratio and percentage of aC3+ HRS cells were positively correlated (Spearman R=+0.58, did not correlate with the proportion of Ki67+ cells. There were no correlations with p<0.001). However, FOXP3/HRS and GB/HRS ratios were not associated with failure- patients’ therapies. free or overall survival in this cHL study group. Ki67 & CD30 vs stage at Dx Conclusions: High numbers of FOXP3+ T-regs seems to influence negatively the Ki67+, mean±SD,% CD30+, mean±SD,% survival of HRS cells in cHL. The underlying mechanisms and potential therapeutic Epidermis Dermis Epidermis Dermis implications of this association merit further investigation. Overall % 15.5±12.0 14.8±10.3 21.5±21.9 7.5±10.2 Stage at Dx ns p<0.05 ns p=0.040 Low 13.7±11.6 12.8±10.2 20.6±22.5 7.1±11.9 1187 Splenic Small B-Cell Lymphomas (SSBCLl) of Marginal Zone (MZ) High 20.3±13.4 18.9±10.0 24.6±21.0 8.8±6.8 and Unclassifiable Types: A Cytogenetic (CG) and Molecular Study Survivals from time of dx, time of bx, and onset of rash were all significantly worse if SD Dufresne, RE Felgar, DW Bahler, JR Cook, U Surti, RL Sargent, SM Gollin, SH Ep or D-Ki67 or D-CD30 was greater than the median (14%, 13%, 4.7%). D-Ki67 as a Swerdlow. U of Pittsburgh Sch. of Med., Pittsburgh, PA; U of Utah Med. Center, Salt continuous variable was an independent prognostic indicator (p<.001), as were D-Ki67 Lake City, UT; U of Pittsburgh Grad. Sch. of Pub. Health, Pittsburgh, PA; Cleveland (p=.004) and D-CD30 (p=.027) but not stage analyzed as dichotomous variables. Clinic Foundation, Cleveland, OH. Background: Splenic MZ lymphomas (SMZL) classically have a biphasic (BP) pattern, but others have a monophasic (MP) MZ-like proliferation. The 2008 WHO also recognizes unclassifiable (UC) SSBCL. Del(7q) is the most characteristic CG marker for SMZL but is only found in a minority of cases. IGH@ mutational status also varies in SMZL and in limited studies is of prognostic value. Design: FISH for del(7q) was performed on tissue microarrays with 43 SMZL and other UC SSBCL not of CLL, MCL, FL, or HCL type. Positive cases had ≥1 core with ≥20% positive cells. IGH@ mutation status was assessed in 17 cases. Findings were correlated with morphologic type, survival, and phenotype. Results: The patients (med. age 66 yr, 18M:25F) had a 5 yr survival of 82%. The SSBCL included 15 SMZL-BP, 12 SMZL-MP and 16 heterogeneous UC. 16% of cases had del(7q) (2 BP, 2MP, 3UC). IGH@ was mutated in 7/17 cases (2 BP, 3 MP, 2UC). Cases with del(7q) showed some phenotypic heterogeneity but were all IGD+, CD23- and MUM1-. There was a trend for IGH@ mutated cases to be MUM1+ (p=0.13) and to have a better prognosis. 1/1 tested case with del(7q) was IGH@ unmutated. Cytogenetic & Genotypic Correlations del(7q) (%) No del(7q) (%) Mutated (%) Unmutated (%) Pathologic Group BP 13 87 40 60 MP 17 83 43 57 UC 19 81 40 60 Phenotype IgD+ 100 71 86 80 MUM1+ 0 36 86 40 CD43+ 17 15 14 13 CD5+ 14 9 0 11 CD23+ 0 11 14 20 Alive 100 86 100 70

Conclusions: CD30+ cells are present in non-transformed MF often with <10% in the dermis and usually greater proportions in the epidermis. D-CD30% greater than the median and, to a greater extent, D-Ki67% are independent adverse prognostic indicators.

Conclusions: Although splenic small B-cell lymphomas with del(7q) appear to be 1189 Activation of mTORC1 Signaling Pathway in AIDS-Related among the more phenotypically homogeneous and typical cases, they are not associated Lymphomas (ARL) with a specific morphologic appearance. The results also suggest that some SSBCL- M El-Salem, PN Raghunath, M Marzec, M Kasprzycka, X Liu, MA Wasik. University UC probably represent SMZL. IGH@ mutation status may have some phenotypic of Pennsylvania, Philadelphia, PA. associations and is probably a prognostic indicator. Additional CG studies may help Background: ARL typically represent the proliferation of enlarged, transformed B further clarify the spectrum of SMZL and elucidate the heterogeneous UC cases. lymphocytes from the category of diffuse large cell lymphoma (DLBCL), Burkitt lymphoma (BL), and Hodgkin lymphoma (HL). The pathogenesis of ARL is poorly 1188 Ki67 and CD30 Predict Survival in Non-Transformed Mycosis understood and treatment results unsatisfactory. mTOR serine/threonine kinase is Fungoides involved in key cellular functions including protein synthesis and proliferation. mTOR JT Edinger, BZ Clark, BE Pucevich, LJ Geskin, SH Swerdlow. U. of Pittsburgh Sch. associates with several proteins including raptor to form the mTORC1 complex which of Med., Pittsburgh, PA. activates p70S6 kinase 1 (p70S6K1) and inhibits 4E binding protein 1 (4E-BP1). In turn, Background: Mycosis fungoides (MF) has a usually indolent but sometimes aggressive p70S6K1 phosphorylates an S6 protein (S6rp). The highly potent and specific inhibitors course. CD30 is usually assumed to be associated with transformation and its extent from the rapamycin family can functionally inactivate mTORC1. and significance in non-transformed MF is uncertain. The prognostic impact of Ki67 Design: By immunohistochemistry with antibodies against the phospho-serines in S6rp staining is also uncertain. and 4EBP1 proteins, we examined tissues from 29 ARL cases and 8 HIV-associated lymphadenopathy (HAL) cases. In addition, we evaluated by Western blotting 6 cell 262A ANNUAL MEETING ABSTRACTS lines developed from DLBCL and BL as well as by infection with Epstein-Barr virus turn-around time and can detect non-neoplastic clonal rearrangement. We assess the (EBV) for activation of the mTORC1 pathway before and after exposure to inhibitors clinical usefulness of TCR-Vβ flow cytometry analysis as a replacement technique for of mTORC1 and Syk kinase. CTCL-blood staging. Results: mTORC1 pathway activation was identified in all ARL cases regardless of Design: We studied the blood samples of 88 CTCL patients and 18 controls using a panel their histologic classification. TORC1 was also activated in the hyperplastic but not in of TCR-Vβ antibodies covering 70% of the TCR-Vβ segments. To improve sensitivity, the involuted follicles of the HIV-associated lymphadenopathy, supporting the notion TCR-Vβ was analyzed on abnormal T-cells with aberrant CD3, CD4 or CD26 expression. that mTORC1 activation is a common feature of transformed lymphocytes irrespective TCR-γ and TCR-β chain PCR testing were conducted in 40 cases in parallel. Circulating of their reactive or malignant phenotype. Finally, we found that inhibition of not only CTCL was determined by morphology, immunophenotype and clonality. mTORC1 but also Syk kinase resulted in at least partial suppression of the mTORC1 activation in all types of the analyzed cell lines. Conclusions: These findings indicate that ARL universally display constitutive activation of the mTORC1 signaling pathway as demonstrated in situ in ARL tissues using antibodies specific for serine phosphorylated mTOR target proteins. Furthermore, ARL and possibly other lymphomas derived from transformed lymphocytes represent a potential therapeutic target for mTORC1 inhibitors as well as inhibitors of its upstream activators including Syk kinase.

Results: Of the 88 samples, Sezary cells identified by manual differential were of 0-5% in 54(61%); 5-19% in 17(19%) and >20% in 17(19%) patients. Immunophenotypic aberrancies were detected in 23/54, 14/17 and 14/17 of these patients. Abnormal TCR- Vβ was detected in 32/54, 17/17 and 17/17 of the CTCL cases but not in the controls, demonstrating a 92% concordance with blood involvement, similar to TCR gene PCR but with superior specificity (100% vs 75%). 44 cases had detectable tumor-associated Vβ expression permitting the use for follow-up studies. Table 1. Correlation of Abnormal Cells Detected by Morphology, Immunophenotyping, Vbeta, and TCR Gene Rearrangement Activation of mTORC1 signaling pathway in ARL, DLBCL type. %Sezary cells by morphology Control(n=18) <5% (n=54) 5-19% (n=17) >20% (n=17) SS by CD4 CD3dim CD7- CD26- • POS 0 23 14 14 1190 New Flow Cytometry Markers for Distinguishing Acute Myeloid • NEG 13 13 0 0 • Intermediate 5 6 3 3 Leukemia, Acute Myelomonocytic Leukemia and Acute Monoblastic/ Tumor cells by V beta Monocytic Leukemia • POS 0 32 17 17 G Fan, J Huang, R Braziel, J Li, C Chen. Oregon Health and Science University, • NEG 18 22 0 0 TCR gene rearrangement Portland, OR; Maryland University School of Medicine, Baltimore, MD. • POS 2 14 3 7 Background: Acute myeloid leukemia with monocytic differentiation represents • NEG 9 10 1 3 pathologic entities of great clinical importance. Identification of monocytic components • ND 6 30 13 7 is traditionally based on morphology and cytochemical findings. However, morphologic Conclusions: TCR-Vβ chain analysis in combination with immunophenotyping evaluation is subjective. Cytochemical stain with nonspecific esterase (NSE) has a improves the detection sensitivity and allows a definite identification of tumor cell- relatively high specificity, but 10-20% of acute monocytic leukemia (AMoL) cases are defining blood involvement. The Vβ flow cytometry analysis is rapid, quantitative and negative for NSE. Flow cytometry using CD14, CD36 and CD64 as monocytic markers suitable for monitoring tumor load during therapy. had been shown to be useful. However, significant portion of AMoL show absence of CD14, low CD36 and low CD64 expression, which pattern is indistinguishable 1192 Assessing Clonality and Disease Burden of T-Cell Large Granular from granulocytic precursors. The aim of this study is to identify markers that allow Lymphocyte Leukemia (T-LGL) by TCR-Vβ Flow Cytometric Analysis differentiation between graunulocytic and monocytic precusors. B Feng, JL Jorgensen, LJ Medeiros, SA Wang. UT MD Anderson Cancer Center, Design: Eight markers (CD49a, CD54, CD86, CD91, CD115, CD116, CD163 and Houston, TX. CD172) as well as well-known monocytic/myeloid markers (CD14, CD36, and CD64) Background: T-large granular lymphocyte leukemia (T-LGLL) is characterized by were chosen for 4-color flow cytometry study. We analyzed all markers on monocytic a monoclonal proliferation of LGLs of T-cell lineage. Because increased T-LGL are and granulocytic precusors/blasts from 15 normal bone marrow, 43 AMoL, 7 acute commonly seen in reactive conditions as well as in monoclonal T-LGL proliferations of myelomonocytic leukemia (AMML), and 24 non-monocytic acute myeloid leukemia uncertain significance, the diagnosis of T-LGLL requires assessment of both clonality (NM-AML) samples. and tumor burden. In this study we assessed the utility of flow cytometry analysis Results: The studies show that all tested markers were expressed on normal monocytes. (FCM) of T-cell receptor b chain gene variable region (TCR-Vβ) repertoires in the CD14, CD91 and CD163 had the highest specificity since they are not expressed by any diagnosis of T-LGLL. of the NM-AML nor by normal maturing granulocytes; but they are highly expressed Design: Peripheral blood samples of 20 T-LGLL patients and 18 controls were analyzed in AMoL cases (CD14 in 70%, CD163 in 80%, and CD91 in 93%). In addition, they by 4-color FCM assessing CD2, CD3, CD4, CD5, CD8, CD7, CD16, CD26, CD56, often show abnormal expression pattern (dim or partial positivity) in AMoL. CD36 and CD57, CD158a, 158b, and 158e. Vb expression was studied by using 24 antibodies CD64 had the highest sensitivity as they were expressed by all AMoL and AMML cases. reactive to 70% of the TCR-Vβ repertoire on the cell population of interest. TCRγ However, their specificity is low since CD36 was detected in 36% of NM-AML and and TCRβ gene rearrangement were assessed by PCR in 20/20 T-LGLL and 12/18 CD64 in 45% of NM-AML samples. Furthermore, CD64 shows same low expression controls. in monocytic precursors as in granulocytes for about 20% of AMML/AMoL cases. Results: All 20 cases of T-LGLL demonstrated morphologic evidence of bone CD49a, CD54, CD86, CD115, CD116 and CD172 showed no significant different marrow involvement. By FCM immunophenotyping, 19/20 T-LGLL cases were staining pattern between NM-AML and AMoL. CD3+CD8+CD57+ and 1 case was CD3+CD4+CD57+.15/20 (75%) T-LGLL cases Conclusions: Our studies have identified CD91 is a sensitive (100% sensitivity) and demonstrated at least an aberrant immunophenotype, with altered CD5 expression specific (93% specificity) monocytic marker. Its expression appears to be earlier than most frequent. Abnormal TCR-Vβ expression was detected in 17/20(85%) T-LGLL CD14 and represents the earliest monocytic lineage specific marker. CD91 should prove cases, showing 95% concordance with TCR gene rearrangement results. Strong to be a useful marker for diagnosing leukemias with monocytic differentiation. correlations between absolute counts of the expanded Vβ clone and cells with a T-LGLL immunophenotype cells was observed (r=0.8). Although an abnormal CD4:CD8 ratio 1191 The Utility of Flow Cytometry TCR-Vβ Chain Analysis in was sensitive for detecting T-LGLL, its correlation with the disease burden was poor. Combination with Immunophenotyping for Assessing Blood Involvement by Cutaneous T-cell Lymphoma (CTCL) B Feng, JL Jorgensen, DM Jones, LJ Medeiros, SAA Wang. UT MD Anderson Cancer Center, Houston, TX. Background: Blood involvement by tumor cells has adverse prognostic significance in patients with CTCL. Since the tumor-cells frequently show nonspecific morphology, clonality testing is required for confirmation. Commonly used TCR gene PCR has long ANNUAL MEETING ABSTRACTS 263A

Table 1. Summary of TCR-Vβ Flow Cytometric and Hematologic 1195 Characterization of TCRδ Expression in Paraffin Embedded Tissue Parameters from 20 T-LGLL and 18 Control Cases Sections: A New Diagnostic Tool for γδT-Cell Lymphomas T-LGLL CONTROL A Garcia, N Villamor, P Gaulard, E Jaffe, E Campo, A Martinez. Hospital Clinic, PARAMETERS median(range) median(range) WBC X10(9)/L 6.20(1.4-30.3) 4.60(1.40-34-50) Barcelona, Spain; Hospital Henri Mondor, Creteil, France; National Cancer Institute, %Lymphs 69.00(26-98) 42.6(14-90) Bethesda. ABS Lymphs X10(9)/L 3.53(0.81-23.94) 2.08(0.67-23.12) Background: Human γδ T lymphocytes constitute a small proportion (1-5%) of the % of Vβ+ Lymphs 74.00(28-91) NA peripheral blood (PB) lymphocytes, but represent up to 50% of T-cells in the skin and ABS Vβ+ X10(9)/L 2.77(0.228-17.65) NA in the mucosa of the gastrointestinal (GI) tract. Malignant neoplasms derived from CD4/CD8 0.14(0.028-0.713) 1.22(0.31-12.97) % of Bone marrow involvement 30.00(8.00-62.00) NA these cells are uncommon. The precursor T-cell lymphoblastic leukemia/lymphoma and hepatoesplenic T-cell lymphoma are the more frequent subtypes. γδ T-cell lymphomas Conclusions: FCM analysis of the TCR-Vβ repertoire is a fast, reliable and quantitative (γδ TCL) involving mucosal surfaces and skin also have been recognized. The use of method for assessing T-LGLL clonality and tumor burden. Its clinical utility is not limited chain specific monoclonal antibodies (MAbs) against TCRδ in pathology is hampered to the initial diagnosis but also the disease monitoring during therapy. by their restricted application to frozen tissue. The purpose of this study was to validate the utility of MAb for human TCRδ constant region on paraffin-embedded samples. 1193 Significant CD5 Expression on Normal Stage III Hematogones and Design: We performed immunohistochemical (IHC) staining for TCRδ expression Mature B-Lymphocytes in Bone Marrow in normal human tissues and a series of 33 T-cell lymphomas including 27 tumors FS Fuda, NJ Karandikar, W Chen. UT Southwestern Medical Center, Dallas, TX. suspicious of γδ T-cell differentiation. Five cases diagnosed of peripheral T-cell Background: Hematogones (HG) are normal maturing B-lineage precursors that reside lymphoma unspecified (PTCL) and 1 extranodal NK/T cell lymphoma were used as in bone marrow (BM) and exhibit a typical consistent, reproducible, complex spectrum negative control. A 10% neutral buffered formalin was used in 26 cases and 7 were of sequential antigen expression. This defines HG into three stages of maturation. We fixed in Bouin’s solution (BS). We used a 5A6.39 MAb against δ-chain. β-chain was had observed CD5 expression on a subset of HG and mature B cells, particularly in cases also analyzed in all cases. A cell pellet of negative sorted PB δ T-cells was used as a with HG hyperplasia. The expression pattern of CD5 on B-cell subsets at different stages control. of maturation has not yet been systematically evaluated and is the focus of this study. Results: A normal population of intraepithelial lymphocytes with expression of δ-chain Design: Twenty-one BM specimens with HG hyperplasia (>3.5% of total events) were was observed in the GI tract and the splenic red pulp. An optimal staining was obtained prospectively studied by 4-color flow cytometry for≥ 25 lymphoid and myeloid antigens. in 20 formalin fixed samples and the IHC failed in 13 cases including all samples fixed Cluster analysis was performed using BD Paint-a-Gate software. HG were divided into in BS. A δ-chain expression was observed in 12 out of 20 cases (60%) priory diagnosed 3 stages of maturation: Stage 1 HG (CD10+bright/CD45+dim/CD20-/CD22+/CD34+), of γδ TCL. In two of them we corroborated the IHC results using flow cytometry. Two Stage 2 (CD10+/CD45+moderately/CD20+variably/CD22+/CD34-) and Stage 3 (CD10+/ cases priory diagnosed of γδ TCL were negative with a good internal control and also CD45+moderately bright/CD20+bright/CD22+bright). The proportion of cells with CD5 expression lack β-chain expression. In addition, 5 PTCL and 1 extranodal NK/T cell lymphoma, was determined for total B-lineage cells, mature B cells and each stage of HG. nasal type were studied for δ-chain expression. All of them were negative for-δ chain Results: Patients ranged in age from 9 months to 63 years; 15 were male and 6 were and 4 showed β-chain expression. female. Submitted clinical histories included 17 hematolymphoid neoplasia and Conclusions: The detection of γδ T cells in paraffin sections is possible and allow the 4 cytopenias. Flow cytometric findings were unremarkable in 14 of 21 cases. The recognition of tumors that although clinically, phenotypically and morphologically remaining specimens showed residual previously diagnosed neoplasia in 6 cases and mimics a γδ TCL lacks a γδ T-cell differentiation. Nevertheless, the standardization of acute myeloid leukemia in 1 case. Of total events, the percentage (%) of mature B cells the IHC for δ-chain in the clinical settings is challenged by the preanalytical variability ranged form 0 to 6.1 (mean +/- SD: 1.6 +/- 1.9) and HG from 3.7 to 17.0 (7.4 +/- 3.8). and requires a good fixation in 10% neutral formalin. Within the HG population, the % of stage 1 HG ranged from 1.2 to 70.0 (14.2 +/- 15.8), stage 2 from 22.0 to 87.0 (66.0 +/- 17.0), and stage 3 from 0 to 45.0 (19.6 +/- 12.0). The 1196 Morphologic Features in Small Lymphocytic Lymphoma % of CD5 expression by entire B-lineage cells ranged from 6.7 to 66 (25.4 +/- 16.0), by Are Predominantly Not Predictive of Underlying Prognostic FISH or mature B cells from 16.0 to 83.0 (53.6 +/- 20.2), and by stage 3 HG from 31.0 to 91.0 Immunophenotypic Markers (69.7 +/- 14.4). Virtually all CD5(+) B cells expressed polytypic surface light chain. C Garcia, K Hunt, H Kang, J Gale, MA Vasef, K Reichard. U. of Pittsburgh, Pittsburgh, Interestingly, expression of CD5 was not observed on stage 1 and stage 2 HG. PA; U. of New Mexico, Albuquerque, NM; Tricore Reference Lab, Albuquerque, Conclusions: CD5 is expressed on normal subsets of B cells in BM at different stages NM. of maturation, specifically on stage 3 HG and mature B cells. Awareness of this normal Background: Common recurring genetic abnormalities with prognostic relevance are pattern of expression would have clinical implications in the differential diagnosis of detected by FISH in ∼80% of SLL/CLL cases. The presence of ZAP-70 and an unmutated HG and CD5(+) neoplastic processes. Furthermore, exploration on the mechanism of immunoglobulin heavy chain gene variable region (IgVH) each also independently this highly regulated CD5 expression may shed light on the pathogenesis of CD5(+) predicts an overall worse survival. Given the heterogeneity in outcome, we evaluated B-cell lymphoma. SLL lymph nodes for morphologic clues that may predict a more or less favorable genetic or immunophenotypic profile. Thus, the pathologist could devise a directed 1194 Nuclear Staining of SOX11 Is a Reliable Immunohistochemical approach to performing these ancillary studies. Marker for Mantle Cell Lymphoma Design: We identified 41 cases of conventional SLL diagnosed by a hematopathologist. J Gao, L Peterson, Y Chen. Northwestern University Feinberg School of Medicine, Cases with “atypical” morphology or those with a component of transformation were Chicago, IL. excluded. H&E sections were evaluated for three morphologic features: expanded Background: SOX genes encode a family of transcriptional factors involved in proliferation centers (EPC) comprising >35% of surface area, >10 large cells per 40X regulating neural crest development. Overexpression of SOX11 has been reported in hpf outside of proliferation centers (LC) and marked nuclear contour irregularities malignant glioma. SOX11 shares sequence homology with SOX4, a transcription factor (NCI) in tumor small and large cells (KR, CG). ZAP-70 IHC, FISH for del13q14, +12, crucial for B cell development. A recent study reported specific nuclear expression of del11q22 (ATM), del17p (p53) and IgVH mutational status (MS) were performed on SOX11 in mantle cell lymphoma (MCL). The findings indicate that SOX11 may serve paraffin sections and interpreted in a blinded fashion (KR, CG, KH, JG). Statistical as a marker for diagnosis of MCL. So far, the data reported is very limited. analysis was performed using R and Stata. Design: In the current study, we evaluated 43 cases with a confirmed diagnosis of Results: FISH, ZAP-70 and IgVH MS were determined in 100%, 100% and 65% of MCL and compared SOX11 expression in these cases with cyclin D1 expression and cases, respectively. 27/41 cases (66%) had FISH abnormalities with the following FISH analysis for t(11;14). We also tested SOX11 expression in non-MCL lymphomas frequencies: del 13q14 (63%) (sole abnormality in 55%), del 11q (30%), +12 (26%) including 50 cases of CLL/SLL, 22 follicular lymphomas (FL) and 20 diffuse large B cell and del 17p (4%). There was no association between FISH markers and EPC or LC. lymphomas (DLBCL). Expression of SOX11 was examined by immunohistochemical There was however an association between NCI in small cells and the presence of del stain on formalin fixed, paraffin embedded whole tissue sections. 11q (p=0.0055), while NCI in large cells predicts against finding del 13q (p=0.0013). No Results: The diagnosis of MCL was confirmed either by flow cytometric significant association was found between morphology and ZAP-70+. ZAP-70+ however immunophenotyping and positive cyclin D1 staining, and/or FISH for t(11;14). Of the 43 did correlate with a less favorable FISH abnormality (p=0.04), and lacks the favorable cases, 39 (90.7%) showed positive nuclear staining of SOX11 in a diffuse homogeneous del 13q14 (p=0.006). 13/14 ZAP-70+ cases were IgVH unmutated (p=0.001). pattern, including 5 cases of CD5-negative MCL and 2 cases of blastoid variant. The Conclusions: Nuclear contour irregularities in SLL may signal the presence of an positive nuclear staining was seen in >50% cells in 34 cases (87.1%), 20-30% cells underlying adverse FISH abnormality (del 11q). However, given that SLL morphology in 4 cases, and 10% in 1 case. Of the four SOX11 negative MCL cases, two were does not substantially predict FISH, ZAP-70 or IgVH mutational status, performance of also negative for cyclin D1; but FISH showed positive t(11;14). The SOX11 staining these specialized tests should be directed by clinical parameters as needed. was in agreement with cyclin D1 staining in 95% of the cases. We also tested SOX11 expression in non-MCL lymphomas. None of the CLL/SLL (50 cases) or FL (22 cases) 1197 CD52 Expression in T Cell Lymphomas showed positive homogeneous nuclear stainging, but demonstrated perinuclear granular Z Ghorab, JW Wong, R Buckstein, M Cheung, S Nofech-Mozes. Sunnybrook Health staining most prominent in prolymphocytes and centroblasts. The staining pattern was Sciences Centre, Toronto, Canada. different from that in MCL. Of the 20 DLBCL, 19 were negative for nuclear staining Background: Alemtuzumab (CAMPATH-1H) is a humanized monoclonal antibody but showed perinuclear granular staining. One DLBCL demonstrated both cytoplasmic that targets CD52, a cell surface glycoprotein, and is currently used in the treatment of and nuclear staining. several hematologic malignancies. The drug has an antitumor effect mediated by human Conclusions: Our study demonstrated that positive nuclear staining of SOX11 is an complement and via antibody dependent cellular cytotoxicity, but is also associated immunohistochemical marker for MCL. It may be useful as a complement to cyclin with significant toxicity. Given the poor response to conventional chemotherapy in T D1 for the diagnosis of MCL. cell lymphomas, there has been increasing interest in evaluating the addition of novel therapies such as CAMPATH for these conditions. However, limited data on the level of expression of the CD52 target antigen are available in T cell neoplasms. 264A ANNUAL MEETING ABSTRACTS

Design: We evaluated CD52 expression using immunohistochemistry (Rat anti-human Results: 33 of 81 cases (40.7%) had demonstrable hematologic malignancy. These clone YTH34.5, Serotec, Oxford, UK1:400) on formalin fixed paraffin embedded included 15 cases of T-cell lymphoma, 14 cases of B-cell lymphoma, 3 cases of tissue from 52 consecutive cases accessioned between March 2005 and July 2008 in precursor B-ALL, 1 Hodgkin lymphoma and 1 acute myeloid leukemia. The positive the department of Anatomic Pathology at Sunnybrook Health Sciences Centre. Tumors predictive value for a diagnosis of T-cell lymphoma was 18.5%. Of 34 cases with were classified by a hematopathologist according to the WHO classification. The CD52 follow-up specimens but without a neoplastic diagnosis when TCR PCR testing was stain was considered positive when the neoplastic cells exhibited strong cytoplasmic performed, 9 developed subsequent hematologic malignancy including 4 cases of or membranous staining. Staining in less than 30% of the cells was reported as focal mycosis fungoides, 2 cases of T-cell lymphoma, 2 cases of B-cell lymphoma and one positive. Nuclear staining was considered nonspecific. plasma cell neoplasm. Results: CD52 was expressed in the cytoplasm/membrane of 24 (46.2%) cases. Conclusions: TCR gamma gene rearrangement studies using PCR with visible The staining was focal in 7 out of 24 (29.1%). CD52 was positive in: 11/23 (47.8%) homoduplex bands <10% positive controls are not infrequent. The low PPV for peripheral T cell lymphoma- unspecified; 1/6 (16%) anaplastic large cell lymphoma; hematologic malignancy, and specifically T-cell malignancy, supports caution in the 3/5 angioimmunoblastic T-cell lymphoma; 2/5 (40%) mycosis fungoides involving interpretation of such findings. At least some of these cases may represent oligoclonal lymph nodes; 1/4 (25%) precursor T lymphoblastic lymphoma; 3/3 (100%) adult T proliferations. cell leukemia/lymphoma; 0/2 (0%) NK/T-cell lymphoma; 1/2 (50%) enteropathy-type T- cell lymphoma; and 2/2 (100%) subcutaneous panniculitis-like T-cell lymphoma. 1200 Amyloidogenic Nature of Circulating Transthyretin Variants, The expression of CD52 was not associated with disease site (12/28 nodal vs. 12/24 V122I and V30A, and the Effect of the NSAID, Diflunisal extranodal: p = 0.78). MJ Greene, K Laporte, E Klimtchuk, D Seldin, LH Connors. Boston University School Conclusions: CD52 expression is variable among T cell neoplasms. Almost half of all of Medicine (BUSM), Boston, MA; BUSM, Boston. T-cell non-Hodgkin’s lymphomas are CD52 positive and PTCL unspecified are CD52 Background: The systemic amyloidoses are diseases caused by protein misfolding, positive. The role of CD52 expression as a predictor of response to CAMPATH targeted aggregation, and deposition in tissues throughout the body. One familial type of amyloid therapy in T cell lymphoma should be further evaluated. disease is associated with variant forms of transthyretin (TTR), a plasma protein which normally circulates as a homotetramer. More than 80 pathologic TTR point mutations 1198 Oncogenic Kinase NPM/ALK Induces Immunosuppressive have been identified, each giving rise to an amyloidogenic variant with destabilized Protein CD274 (PD-LI, B7-H1) through STAT3 Expression quaternary and tertiary structure. A Goradia, M Marzec, Q Zhang, P Raghunath, X Liu, M Paessler, H Wang, M Wysocka, Design: The aim of this study was to investigate the tetrameric stabilities and amyloid M Wasik. Hospital of the University of Pennsylvania, Philadelphia, PA. fibril forming propensities of TTR-V122I and TTR-V30A, two amyloidogenic variants; Background: The mechanisms of malignant cell transformation caused by the V122I/T119M, a double mutant containing a putative protective residue; and wild oncogenic, chimeric NPM/ALK tyrosine kinase remain partially understood with most type (wt) TTR. In addition, we tested the ability of diflunisal, a non-steroidal anti- studies focusing on the impact of NPM/ALK on cell survival and proliferation. Here inflammatory drug (NSAID) currently being tested in a clinical trial at our institution, to we show that the NPM/ALK+ T-cell lymphoma (ALK+ TCL) cells strongly express inhibit TTR tetramer dissociation. Using recombinantly-generated proteins, we analyzed the immunosuppressive cell-surface protein CD274 (PD-L1, B7-H1) as determined on TTR tetramer dissociation (acid or urea denaturation) by SDS-PAGE and amyloid fibril the mRNA and protein level. formation (acid-mediated) by Congo red (CR) binding spectroscopy. Design: A multifaceted approach was employed using the ALK+TCL cell lines and Results: In the acid denaturation studies, our results showed that V122I and V30A tissues. NPM/ALK-dependent gene expression was determined using CEP-14083 had reduced tetramer stabilities compared to wt TTR across a decreasing pH gradient. ALK inhibitor and U133 Plus 2.0 array chips (Affymetrix) microarray analysis and Incubation of V122I and V30A with 50x molar excess diflunisal prior to acid addition confirmatory RT-PCR (on the RNA level) and flow cytometry and immunohistochemistry increased tetramer stability of each protein. In the amyloid fibril formation assays, V122I (on the protein level). The association of the NPM/ALK-activated STAT3 transcription and V30A each demonstrated enhanced fibril formation over 5 days incubation relative factor with the PD-L1 gene promoter was determined using electromobility shift assay to the wt protein as evidenced by red shifts (Amax 512nm-V122I; Amax 502nm-V30A; (EMSA) and chromatin immunoprecipitation (ChIP). Amax 491-wt) in the absorbance spectrum of CR (Amax 485nm). The formation of Results: The CD274 expression is strictly dependent on the expression and enzymatic amyloid fibrils was verified by polarized light microscopic examination of the CR-bound activity of NPM/ALK as demonstrated by inhibition of the NPM/ALK function in samples which exhibited the characteristic green birefringence. ALK+ TCL cells by the small molecule ALK inhibitor CEP-14083 and by documenting Conclusions: Our data shows that the TTR variants, V122I and V30A, have a greater CD274 expression in IL-3-starved BaF3 cells transfected with the wild-type NPM/ propensity than wt TTR to form amyloid fibrils and that the addition of diflunisal ALK but not the kinase-inactive NPM/ALK K210R mutant or empty vector alone. reduces tetramer dissociation of these variants, thereby inhibiting fibril formation. It is Immunohistochemical analysis with anti-CD274 antibodies demonstrated selective interesting that these variants with conservative amino acid substitutions are destabilized expression of PD-L1 in ALK+ TCL tissues. and cause amyloid formation in cardiac tissue, with V122I being almost exclusive to the African American population.

1201 Neither Cell of Origin Algorithms or LMO2 Expression Predict Survival in DLBCL Treated with Autologous Hematopoietic Stem Cell (HSC) Transplantation K Gu, K Fu, WC Chan, TC Greiner, P Aoun, LM Smith, Z Liu, PN Meyer, WW Choi, G Bociek, JM Vose, DD Weisenburger. University of Nebraska Medical Center, Omaha, NPM/ALK acts through activation of its key signal transmitter, transcription factor NE. STAT3. STAT3 binds to the CD274 gene promoter in vitro and in vivo as shown in the Background: Diffuse large B-cell lymphoma (DLBCL) includes at least two EMSA and ChIP assays and is required for PD-L1 gene expression as demonstrated by prognostically-important subtypes, the germinal center B-cell-like (GCB) type siRNA-mediated STAT3 depletion. and the non-GCB type, which can be identified by the immunostaining algorithm Conclusions: These findings identify a novel mechanism of NPM/ALK-induced of Hans et al. (Blood 103:275, 2004). A new immunostaining algorithm using five malignant cell transformation, based on the induction of CD274 expression and represent antibodies was recently developed to improve the accuracy of this classification (Mod the first documented direct link between an oncoprotein and an immunosuppressive Pathol 21:250A, #1144, 2008). LMO2 expression is also reported to be a predictor cell-surface protein. of survival in DLBCL. The aim of this study is to evaluate the use of these three immunohistochemical approaches in predicting survival in DLBCL following autologous 1199 T-Cell Receptor Gamma Chain Gene Rearrangement by HSC transplantation. Homoduplex Analysis, Clinical Significance of Low Intensity Clonal Design: We selected 54 DLBCL patients who received CHOP chemotherapy, including Bands 46 patients who also received Rituximab, as the first line treatment. The patients JF Gradowski, MA Melan, SH Swerdlow, JA Kant. University of Pittsburgh Medical subsequently relapsed or had primary refractory chemosensitive disease and were Center, Pittsburgh, PA. treated with high-dose therapy and autologous HSC transplantation. Tissue microarrays Background: Demonstration of T-cell clonality is a powerful tool used to support the were prepared and immunostained with monoclonal antibodies against GCET1, CD10, often complicated diagnosis of T-lymphoid neoplasms. PCR approaches to clonality BCL6, MUM1, FOXP1 and LMO2. The positive cutoffs for the new algorithm were assessment most frequently target the T-cell receptor (TCR) gamma chain gene. ≥30% staining for CD10 and BCL6, and ≥80% for the other stains. A positive cutoff of Homoduplex analysis of PCR products is a sensitive approach to detect clonal fragments ≥30% was used for LMO2. The Kaplan-Meier method was used to estimate the overall which avoids interpretative difficulties encountered when evaluating PCR products by survival (OS) and event-free survival (EFS) distributions. size. We chose a 10% positive control as an initial cut-off to report results as positive. Results: Using the Hans algorithm, we classified 56% of the cases as GCB type and Samples with less intense homoduplex bands were reported as indeterminate; here we 44% as non-GCB type. Using the new algorithm, we classified 52% of the cases as assess the clinical significance of those samples. GCB type, 48% as non-GCB type. LMO2 was positive in 70% of the cases. However, Design: Cases from a 15 month period were studied. Amplification was performed no statistically significant differences were found in the five-year OS and EFS of the with TCR gamma chain V1-8, V9, V10 and V11 forward and Jg1 /Jg2 reverse primers. transplantation with any of these approaches. Table1: Estimated five-year OS and EFS Products were analyzed on 20% PAGE gels following formamide treatment. 118 in GCB and non-GCB DLBCL classified by cell of origin algorithms, and by LMO2 Samples with a visable homoduplex product <10% positive controls (Jurkat/ HSB-2 expression alone. T-cell leukemia lines diluted in tonsil DNA) and no positive bands >10% with any Hans Algorithm New Algorithm LMO2 other primers were identified. 47 displayed a distinct band <10% of the positive control GCB, non-GCB P= GCB, non-GCB P= Positive, Negative P= % of cases 56%, 44% 52%, 48% 70%, 30% while 71 displayed low intensity bands. All available hematopathology diagnoses and OS (%) 44%, 36% 0.95 48%, 31% 0.71 42%, 36% 0.34 reports from subsequent biopsies were reviewed. 81 cases had a concurrent pathologic EFS (%) 33%, 37% 0.50 36%, 33% 0.70 39%, 26% 0.16 diagnosis with the majority of remaining cases peripheral blood specimens that were not further analyzed. ANNUAL MEETING ABSTRACTS 265A

Conclusions: Neither of the cell of origin algorithms or LMO2 expression predict Results: 58 patients with lymphoid neoplams associated with serum IgA paraprotein the survival outcome in relapsed or refractory DLBCL treated with autologous HSC were identified. Patient age ranged from 21 to 80 years; there were 33 men and 25 women transplantation. (Table 1). 50 patients had B-cell and 8 patients had T-cell lymphomas. The most common B-cell neoplasms were chronic lymphocytic leukemia/small lymphocytic lymphoma 1202 The MicroRNA Expression Signature of Lymphoplasmacytic (CLL/SLL)(n=12;21%) followed by lymphoplasmacytic lymphoma/Waldenstroms Lymphoma Is Distinct from Chronic Lymphocytic Leukemia macroglobulinemia (LPL/WM)(n=9; 16%). In the T-cell subgroup, mycosis fungoides (n=6; 10%) was most common. Serum IgA paraprotein levels ranged from 0.1 to 3.4 I Gurevich, M Johnson, C Eiken, S Vadhan-Raj, M Fernandez, M Nguyen, H Kantarjian, g/dL, with the highest levels in patients with LPL/WM (median 1.1 g/dL; range, 0.1 to LJ Medeiros, CE Bueso-Ramos. The University of Texas MD Anderson Cancer Center, 3.3). A subset of patients with LPL/WM presented with symptoms of hyperviscosity. Houston, TX. All cases of LPL/WM histologically resembled cases of LPL/WM associated with Background: MicroRNAs are a newly discovered class of short (19-25 nt), naturally IgM paraprotein. occurring, single-stranded RNA molecules that regulate the expression of target genes Conclusions: A wide range of lymphoid neoplasms can be associated with an IgA post-transcriptionally, mostly by repressing translation or inducing mRNA degradation. paraprotein. Low-grade B-cell lymphomas represent approximately two thirds of cases. MicroRNAs can function as both oncogenes or tumor suppressor genes, or as both at LPL/WM cases associated with an IgA paraprotein resemble IgM paraprotein-associated the same time, playing an important role in the development of solid and hematopoetic cases of LPL/WM. The association of IgA paraprotein with mycosis fungoides is malignancies. To date, the microRNA expression signature of lymphoplasmacytic unexplained, but is probably not simply a chance occurrence. lymphoma (LPL) has not been assessed. The goal of this study was to identify the microRNA expression signature of LPL and compare this signature to that of chronic Table 1. Lymphoid Neoplasms Associated with IgA Paraprotein Histologic type No. IgA Paraprotein (median, Range g/dl) lymphocytic leukemia (CLL). Unique patterns of altered microRNA expression may CLL/SLL 12 0.2(0.1-1.2) allow a better understanding of the molecular pathogenesis of LPL, and also may be LPL/WM 9 1.1(0.1-3.3) useful in differential diagnoses, assessment of prognosis, and prediction of therapeutic MALT 8 0.2(0.1-3.4) response. Follicular lymphoma 7 0.2(0.1-0.6) Design: 10 lymphoma samples, 5 LPL and 5 CLL, were the study group. All patients Diffuse large B-cell 6 0.4(0.1-1.2) Mycosis fungoides 6 0.25(0.1-0.6) were adults, had diffuse bone marrow involvement, and were treated at M. D. Anderson Mantle cell lymphoma 2 0.1(0.1) Cancer Center. The patients with LPL all had a serum IgM paraprotein and also fit the Classical Hodgkin lymphoma 2 1.4(0.1-2.8) criteria for Waldesntrom macroglobulinemia. Flow cytometric immunophenotypic Splenic marginal zone lymphoma 2 0.35(0.2-0.5) analysis of both the LPL and CLL cases showed a mature B-cell immunophenotype; the Pre-B acute lymphoblastic leukemia 1 0.1 CLL cases were CD5+ and CD23+. The recoverAll TM Total Nucleic Acid Isolation Kit Peripheral T-cell lymphoma unspecified 1 0.3 Angioimmunoblastic T-cell lymphoma 1 0.1 (Ambion, Inc) was used for microRNA isolation from formalin-fixed, paraffin-embedded EBV+PTLD 1 0.4 bone marrow biopsy specimens. New high performance microfluidic custom microarray platforms were used to generate microRNA signatures. MicroRNA quality was assessed by TaqMan reverse transcriptase real time PCR for ubiquitous microRNAs. 1205 Histiocytic Differentiation and Loss of B-Cell Identity in Follicular Results: MiR-155 and miR-423-5p were significantly down-regulated in LPL compared Lymphoma with CLL (p-value< 0.01), and miR-638 was strongly up-regulated in LPL compared E Haralambieva, T Nedeva, H Horn, E Hartmann, EM Maggio, HK Mueller-Hermelink, with CLL with an 8-fold statistically significant difference (p-value<0.01). The miR-155 A Zettl. University of Wuerzburg, Wuerzburg, Germany. and miR-638 results were confirmed by RT- PCR. Background: Hematopoetic lineage differentiation is considered a stepwise process of Conclusions: Our results indicate that specific microRNA signatures exist for LPL and irreversible lineage commitments. In disagreement with this, however consistent with CLL that can be helpful in discriminating LPL from CLL in difficult cases. In particular, in vitro reprogramming experiments, Feldman et al. reported recently on follicular miR-155, miR-423-5p, and miR-638 are differentially expressed in LPL and CLL. lymphoma (FL) patients developing genetically related tumors with histiocytic differentiation. 1203 Clinicopathologic Analysis of 18 Cases with Refractory Anemia Design: Similarly, we identified in our pathology files four patients with meta- or with Ringed Sideroblasts and Thrombocytosis synchronous histiocytic/dendritic cell sarcoma (HS) and FL. One patient had medical I Gurevich, R Luthra, LJ Medeiros, P Lin. The University of Texas MD Anderson records of FL and only HS sample was available for further studies.Six of the Cancer Center, Houston, TX. coupled samples were submitted to comparative genomic hybridization (CGH) and Background: The provisional MDS/MPD-U entity refractory anemia with ringed sequence analysis of immunoglobulin heavy chain (IgH) rearrangement. Extensive sideroblasts(RS) and thrombocytosis (RARS-T) in the WHO classification is reported immunohistochemistry, fluorescence in situ hybridization (FISH) for detection of to be associated with a platelet count of > 600x109/L, frequent JAK2 V617F mutation translocation breakpoints in BCL2 gene and IgH clonality PCR and GenScan assays and megakaryocytic hyperplasia morphologically resembling essential thrombocytosis. were performed in all samples. Ten randomly selected samples of histiocytic lesions Recent studies show that RARS with thrombocytosis or RARS with JAK2 V617F are not were used as a control group. necessarily RARS-T, suggesting that clinicopathological correlation is required for the Results: All HS lacked CD20 and CD79a and expressed variably CD68, CD163 and diagnosis of this entity. The aim of this study is to evaluate the morphological features CD123.Only one tumor showed weak PAX5 expression in occasional cells, however of cases presenting with RARS and thrombocytosis to better characterize RARS-T. B-cell transcriptional factor Oct2 was positive in all HS. In keeping with the data of Design: Cases of RARS (>15% RS) and thrombocytosis (>500x109/L) were identified Feldman et al., all HS harboured a chromosomal breakpoint into BCL2 gene as well from our clinical database. CBC and the bone marrow aspirate and biopsy were reviewed. as clonally rearranged IgH genes. In contrast, no clonal IgH rearrangement and BCL2 JAK2 and MPL mutations were analyzed by pyrosequencing in cases with available chromosomal breakpoints were identified in control group. CGH analysis indicated DNA. Cases with inv(3) or 5q- were excluded. that at least in two out of three patients HS and FL shared common chromosomal Results: 18 patients were identified, 10 male and 8 female, age range from 40 to 83 imbalances. (median 63). 13 patients had a platelet count >600x109/L. Only 2 patients presented N sex/age tumor CGH with a mildly elevated WBC count (12.7-12.9x109/L), all other patients had a normal gains losses 1 m/36 FL 2p22-pter, 2cen-q14 13q14-q22 WBC count. The JAK2 mutation was identified in 10 of 15 cases analyzed with the HS 1q41-qter, 2cen-p14, 2cen-q22, 8p22-pter 2q24-q33, 9p21-pter, 13q14-q22 mutant alleles ranging from 18-55%. MPL was germline in all 9 cases tested. 8 of 2 f/55 FL 2q36-qter, 6p, 11p, 11cen-q13 6cen-q21 10 patients in the JAK2 mutated group had a platelet count >600x109 /L, and 2 had HS 6p, 6q25-qter 2q24-q33, 6cen-q21, 13q14-q21 platelet counts of 550 and 580x109/L, respectively. There was a morphologic spectrum 3 m/50 FL 2p23-pter, 6p, 12q24-qter 6q24-qter of atypical megakaryocytes among different cases ranging from large forms, as in HS - - 4 f/67 FL NA NA essential thrombocytosis, to pleomorphic forms as in chronic myelofibrosis, as well HS NA NA as non-specific forms associated with variable degree of fibrosis. 3 cases that had a FL - follicular lymphoma, HS - histiocytic sarcoma, NA - not applicable 9 platelet count ranging from 529-568x10 /L and were negative for JAK2 mutation; Sequence analysis of IgH gene confirmed in the coupled samples of 2 patients sequence these cases were not considered to be diagnostic of RARS-T despite ringed sideroblasts homology with the same VH family usage and similar mutation rate. and thrombocytosis. Conclusions: On genetic level all four HS are consistent with mature B cell neoplasia, Conclusions: RARS-T with JAK2 mutation is more likely to display morphologic demostrating however loss of B cell phenotype and morphological, immunophenotypic 9 features of a myeloproliferative disease with a platelet count > 600x10 /L. However, and functional (phagocytosis in one case) features of histiocytic differentiation. The 9 occasional cases may present with a platelet count <600x10 /L. There is a spectrum of patients had progressive disease with significantly longer overal survival than common megakaryocytic morphology. MPL mutation is infrequent. HS patients.

1204 Lymphoid Neoplasms Associated with IgA Paraprotein 1206 The Specificity of Immunophenotypic Alterations in Blasts in Non- SA Gustafson, BC Handy, P Lin, LJ Medeiros. MD Anderson Cancer Center, Houston, Acute Myeloid Disorders TX. AM Harrington, H Olteanu, SH Kroft. Medical College of Wisconsin, Milwaukee, Background: Lymphoid neoplasms can be associated with immunoglobulin (Ig) WI. paraprotein. Most studies in the literature have focused on IgM-associated neoplasms. We Background: Existing data regarding flow cytometric (FC) abnormalities in non-acute reviewed our experience with lymphoid neoplasms associated with IgA paraprotein. myeloid disorders is confounded by variable gating strategies and control groups Design: Serum protein electrophoresis and immunofixation results were reviewed generally limited to normal bone marrows (BMs). CD45/side scatter gating, the most over a 25 year period. The study group included all patients with lymphoid neoplasms common approach to blast isolation, is imprecise, with blasts typically representing associated with IgA paraprotein, both treated and untreated. Plasma cell neoplasms were a minority of gated events. Using an approach designed to delineate all relevant BM excluded. Clinical and pathologic findings were reviewed and lymphoid neoplams were populations, we sought to precisely isolate and immunophenotypically characterize classified using WHO criteria. blasts in MDS, MPD, and CMML. We compared the findings to both normal BMs 266A ANNUAL MEETING ABSTRACTS and non-neoplastic cytopenias and cytoses, in order to determine the specificity of the Results: The diagnoses by classification system are shown in Table 1. immunophenotypic (IP) changes. Classification of 59 cases Design: Diagnostic BMs of 16 MDSs, 12 MPDs, and 7 CMMLs were compared to Diagnosis WHO 2001 WHO 2008 20 non-neoplastic cytopenias/cytoses (CCs) and 10 (-) lymphoma staging BMs, using RA 0 0 cluster analysis of 4-color FC tubes designed to delineate all relevant populations RCUD NA 2 RARS 10 8 using the following antigens: CD7, CD10, CD11b, CD13, CD14, CD15, CD16, CD20, RCMD 21 41 CD22, CD33, CD34, CD36, CD38, CD45, CD56, CD64, CD117, and HLA-DR. Blasts RCMD-RS 6 NA were recognized as distinct clusters, after exclusion of all other populations. Antigen MDS with isolated del(5q) 2 2 alterations were defined as at least a half-log shift. MDS,U 19 2 Results: Blasts in 10/20 CCs (50%) showed IP differences (1-2/case) vs controls, Not MDS 1 4 including over- and underexpression of CD13 and CD117, overexpression of HLA-DR, RA=refractory anemia; RC=refractory cytopenia; UD=unilineage dysplasia; RS=ring sideroblasts; and variability in CD33 and CD38. IP alterations were identified in 12/16 (75%) MDSs MD=multilineage dysplasia; U=unclassifiable; NA=not applicable By 2008 criteria, 4 cases were not MDS, including 2 cases of RARS with marked , 9/12 (75%) MPDs, and 7/7 (100%) CMMLs vs controls, and 8/16 (50%) MDSs, 5/12 thrombocytosis, 1 case of myelodysplastic/myeloproliferative neoplasm, unclassifiable, (41.7%) MPDs, and 3/7 (42.9%) CMMLs compared to CCs. There were 1-4 changes/ and 1 case that had insufficient dysplasia. There was no significant correlation between case in MDSs, 1-3/case in CMMLs, and 1-2/case in MPDs. IP changes exclusive to the presence of dysplasia and cytopenia in the erythroid, granulocytic, or megakaryocytic neoplastic blasts included expression of CD7 (3/16 MDSs, 4/12 MPDs, 1/7 CMMLs), lineages. The presence of any ring sideroblasts correlated with anemia (p=0.003), lack CD15 (1/16 MDSs, 1/7 CMMLs), CD16 (1/16 MDSs), CD36 (1/16 MDSs), CD56 (1/16 of thrombocytopenia (p=0.01), and lack of granulocytic dysplasia (p=0.01), and was MDSs, 1/7 CMMLs), and CD64 (1/16 MDSs, 1/7 CMMLs); variable HLA-DR (3/16 borderline correlated with lack of neutropenia (p=0.05). Granulocytic dysplasia was MDSs, 1/12 MPDs, 2/7 CMMLs); underexpression of CD33 (3/16 MDSs), CD38 (1/16 borderline correlated with poor prognosis IPSS karyotype score (p=0.10). MDSs), and CD45 (3/16 MDSs, 1/12 MPDs, 1/7 CMMLs); and partial loss of CD117 Conclusions: Lineage-specific dysplasia in MDS does not correlate with cytopenia (1/12 MPDs). In all cases, the entire blast population was CD34(+). in any lineage, although granulocytic dysplasia is borderline associated with a poor Conclusions: Several blast IP alterations were seen in both non-acute myeloid prognosis karyotype. The allowance of RCMD with a single cytopenia and non-erythroid malignancies and non-neoplastic cytopenias and cytoses and are therefore not neoplasia- RCUD in the 2008 Classification has markedly reduced the number of cases that would specific. Approximately 40-50% of MDSs, MPDs, and CMMLs exhibited aberrancies previously be classified as MDS,U. not seen in reactive BMs, including expression of CD7 and CD56, variability of HLA- DR, and underexpression of CD33 and CD45. Of note, the entire blast population was CD34 (+) in all neoplastic cases. 1209 Interphase FISH on TEL/AML1 Positive ALL Relapses – Possible Clinical Relevance of TEL and AML1 Copy Number Changes and Extra TEL/AML1 Copies 1207 Distinct Expression Patterns of CD123 and CD34 on Hematogones and Blasts in Precursor B-Cell Acute Lymphoblastic Leukemia T Heiden, A Peter, T Taube, G Korner, K Seeger. Otto-Heubner-Center for Pediatrics, Charité Campus Virchow-Klinikum, Berlin, Germany. NM Hassanein, PJ Buckley, AS Lagoo. Duke University Medical Center, Durham, Background: TEL/AML1 fusion resulting from the translocation t(12;21)(p13;q22) NC. constitutes the most frequent genetic rearrangement in initial childhood B-cell precursor Background: Bone marrow derived normal B-cell precursors (hematogones) (BCP) ALL (19-27%), and has been associated with good prognosis. Three secondary and blasts in precursor B-cell acute lymphoblastic leukemia (B-ALL) share many copy number aberrations in TEL/AML1 positive ALL have been suspected to negatively immunophenotypic properties. Distinguishing between these two populations following influence outcome: deletion of the second TEL allele, gain of the second AML1 allele treatment or during early relapse of precursor B-ALL can be challenging. and duplication of the derivative chromosome 21. Design: Using four color flow cytometry, we examined the expression patterns of two Design: In this study, samples from 38 children with relapsed TEL/AML1 RT-PCR hematopoietic stem cell antigens, CD34 and CD123 (interleukin-3 receptor alpha) on positive and negative BCP-ALL were analyzed retrospectively for these mutations by hematogones in 75 regenerating bone marrow samples without a diagnosis of precursor interphase fluorescence in situ hybridization (iFISH). B-ALL and in 40 cases of precursor B-ALL. The hematogones were identified by their Results: Among 11 TEL/AML1 negative cases, the frequencies of loss and gain of characteristic low side scatter, and CD45, CD19 and CD10 expression. CD34 and untranslocated TEL (T) as well as loss and gain of untranslocated AML1 (A) were 18%, HLA-DR expression on these cells was also analyzed. 27%, 9% and 45% respectively. Among 27 TEL-AML1 positive relapse cases, those Results: CD123 was expressed only on the more mature (moderate CD45, CD34-, rates were 52%, 22%, 0% and 27% and gain of the derivative chromosome 21 (TA) DR+) fraction of hematogones and the less mature hematogone fraction (dimmer CD45, was found at a frequency of 26%. These results were compared with published data. In CD34+, DR+) did not express CD123 on most cells. In contrast to this discordant children with TEL/AML1 positive relapse, additional TEL loss (a), AML1 and fusion expression of CD34 and CD123 on hematogones, leukemic blasts in most cases of gene gain (b), TEL loss and AML1 gain (c) and the occurrence of a subpopulation with precursor B-ALL showed concordant expression of these antigens. Thus in 31 of 40 the genotype 1T/3A/1TA (d) appear to be related to higher PBCs at relapse diagnosis cases (77.5%) the blasts expressed both CD34 and CD123 and in 5 of 40 cases (12.5%) (in a and d) or a tendency towards the occurrence of a subsequent relapse (b and c) neither antigen was expressed. Only in 4 additional cases in which the blasts did not (p-values <0.05). Those results were compared with reported data as well. express CD34, a variable proportion of blasts (7 to 96%) expressed CD123. Conclusions: Hematogones show a discordant expression patterns of CD34 and CD123 while blasts in most cases of precursor B-ALL show concordant expression patterns of these antigens. Among the CD19+ B-cells in the bone marrow, CD34+/CD123+ (“double positive”) or CD34-/CD123- (“double negative”) patterns can be used to identify leukemic blasts and distinguish them from the hematogones with their CD34+/ CD123- or CD34-/CD123+ discordant expression patterns.

1208 Application of 2008 WHO Classification Criteria for Myelodysplastic Syndromes (MDS) without Excess Blasts: Observations from Cancer and Leukemia Group B Study 10105 RP Hasserjian, P Gupta, K Donohue, K Owzar, RA Larson, F Racke, CD Bloomfield. MGH, Boston, MA; University of Minnesota, Minneapolis, MN; Duke University, Durham, NC; University of Chicago, Chicago, IL; Ohio State University, Columbus, OH. Background: The new 2008 WHO Classification of MDS emphasizes the number of lineages affected by dysplasia, but its impact on the classification of MDS entities has not yet been assessed, nor has the correlation between dysplasia and cytopenia in each lineage been well-characterized. Design: A single observer reviewed bone marrow biopsies, aspirates, and peripheral smears from 59 cases diagnosed as MDS without excess blasts (<5%) and scored percentage of dysplastic forms in each lineage and ring sideroblasts. In combination with peripheral counts and cytogenetics results, cases were classified according to both the 2001 and updated 2008 WHO criteria. We also assessed the relationship of dysplasia and ring sideroblasts to cytopenias and International Prognostic Scoring System (IPSS) score. Conclusions: Our results suggest a poor prognostic significance of loss of the second TEL allele, gain of the second AML1 allele and duplication of the derivative chromosome 21in children with relapsed TEL/AML1 positive BCP ALL in particular when those aberrations occur in a combined manner. We would consider it wortwhile to pursue such studies in larger patient populations. ANNUAL MEETING ABSTRACTS 267A

1210 B-Cell Neoplasms with Aberrant T-Cell Antigen Expression Design: Formalin-fixed, paraffin embedded sections from 54 DLBCLs, were Show Plasmacytic Differentiation immunostained by automated methods (Ventana Medical Systems, Inc, Tucson, AZ) JL Herrick, KL Grogg, WR Macon, A Dogan, AL Feldman. Mayo Clinic College of with polyclonal antibodies to Notch1, Notch2, and Notch4 (Santa Cruz Biotechnology, Medicine, Rochester, MN. Inc, Santa Cruz, CA). Cytoplasmic and nuclear immunoreactivity of each protein was Background: Phenotyping for B- and T-cell lineage-associated antigens is an integral semiquantitatively assessed in all cases. Scoring was based on staining intensity (weak, part of lymphoma diagnosis and classification. While CD20-positive T-cell lymphomas moderate, intense) and percentage of positive cells (focal <= 10%, regional 11-50%, are well-documented, B-cell neoplasms (BCN) expressing CD3 or other T-cell antigens diffuse >50%). Results were correlated with clinicopathologic variables. (TCAs) (excluding CD5 and CD43) have been reported only rarely. Such cases can Results: Immunoreactivity was predominantly cytoplasmic for the Notch1, Notch2 represent a diagnostic pitfall, since unifying pathologic features of such cases have and Notch4 proteins, while nuclear immunoreactivity was also observed. Notch not been identified. We compiled a series of BCNs with aberrant TCA expression and overexpression was noted as follows: 78% Notch1, 65% Notch2, and 44% Notch4. assessed morphologic and immunophenotypic features to provide a clearer understanding Notch1 overexpression correlated with nodal involvement [95% nodal vs 67% of these rare neoplasms. extranodal, p=0.01] and lengthened survival [100% alive vs 63% expired, p=0.04]. Design: BCNs evaluated at the Mayo Clinic were reviewed for aberrant TCA expression Notch2 overexpression correlated with nodal involvement [89% nodal vs 50% on initial immunohistochemical panels. All identified cases were evaluated for CD2, extranodal, p=0.007] and HIV negative status [72% HIV negative vs 40% HIV positive, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD30, CD56, CD79a, PAX-5, TIA-1, p=0.05]. Notch4 overexpression correlated with nodal involvement [86% nodal vs 22% BetaF1, and MUM1/IRF4 by immunohistochemistry. EBV encoded RNA was assessed extranodal, p<0.0001] and lengthened survival [83% alive vs 20% expired, p=0.003]. On using in situ hybridization. PCR studies for T-cell receptor and immunoglobulin gene multivariate analysis, HIV positive status and lack of cytoplasmic Notch1 overexpression rearrangements were performed in 7 cases. were independent predictors of shortened survival. Results: 14 BCNs expressed TCAs including 10 diffuse large B-cell lymphomas Conclusions: Our data demonstrates that expression of Notch proteins in DLBCLs (DLBCL) (3 nodal, 7 extranodal), 3 plasmacytomas (all extranodal), and one nodal correlates with favorable prognostic variables including nodal disease, lengthened follicular lymphoma, grade 3A (FL). 9/14 cases showed some degree of plasmacytic survival and negative HIV status. The findings suggest that altered Notch expression differentiation (6/11 lymphomas and 3 plasmacytomas). 8/14 (57%) were positive for may play a role in the pathogenesis of DLBCLs and its detection may aid in predicting CD20. 12/14 (86%) showed CD3 expression, 4/13 (31%) expressed CD4 and 3/13 clinical outcomes. (23)% expressed CD8. Most cases expressed only one TCA, predominantly CD3. One case expressed two TCAs. Three cases expressed multiple TCAs (2 DLBCL and 1 FL). 1213 Detection of Monoclonal B-Cell Lymphocytoses by Flow Cytometry All cases were positive for MUM1/IRF4. 2 cases were EBV positive (DLBCLs). All in Patients with T-Cell Large Granular Lymphocyte Leukemia tested cases showed clonal immunoglobulin gene rearrangements with no clonal T-cell MT Howard, J Maciejewski, ED Hsi. Cleveland Clinic, Cleveland. receptor gene rearrangement detected. Background: T cell large granular lymphocyte leukemia (T-LGL) has been associated Conclusions: Most BCNs with aberrant TCA expression show plasmacytic with monoclonal gammopathy of undetermined significance (MGUS) and low grade differentiation and express the transcription factor MUM1/IRF4. Since lymphomas B cell lymphoma. Routine flow cytometry (FC) may identify patients with a low grade with plasmacytic differentiation often lack CD20, T-antigen expression may falsely lead B-cell lymphoma or with a monoclonal B-cell lymphocytosis (MBL). We retrospectively to an impression of T-cell lymphoma. To avoid this pitfall, lineage should be assessed reviewed our experience with T-LGL patients who underwent FC during their disease with a panel of B- and T-cell antigens and molecular studies performed in ambiguous course to determine the incidence of detecting an abnormal B-cell population. cases. Aberrant antigen expression might result from disrupted B-cell transcriptional Design: 45 patients with T-LGL were studied based on available 4-color FC data. FC programming, a hypothesis that merits further study. For example, B-cell programming data files from tubes with antibodies to CD5/CD19/CD23/CD45 and CD19/CD45/κ/λ is disrupted in classical Hodgkin lymphomas and primary effusion lymphomas both of were reviewed retrospectively. The tube containing surface immunoglobulin (sIg) was which express MUM1/IRF4 and may aberrantly express TCAs. acquired with a “live-gate” on CD19+ events, collecting 20,000 events or 0.2 ml of sample. Specimens were considered positive for a B-cell dyscrasia if one of the following 1211 Expression of CD304 (Neuropilin-1) in Acute Leukemia and criteria were met: 1) B-cells with a κ:λ > 10:1 or κ:λ < 0.2; 2) > 80% of B-cells were Hematopoietic Cells sIg negative; 3) B-cells with a specific disease phenotype were found as a subset of a LC Ho, HJ Meyerson. University Hospitals Case Medical Center, Cleveland, OH. sIg polytypic B-cell population, though not in sufficient quantity to cause an abnormal Background: CD304 (neuropilin-1 or BDCA-4) is a transmembrane C-type lectin that overall κ:λ ratio. In addition, the population of abnormal B-cells must have been a plays an important role in angiogenesis and neuronal guidance. CD304 and another cluster of greater than 100 events. C-type lectin, CD303 (BDCA-2), are useful in the immunophenotypic identification Results: 45 patients were included, with a mean age of 62 years. The mean number of of plasmacytoid dendritic cells (PDCs); however, the expression of CD304 on normal abnormal B-cells analyzed was 4533 (range 240-17813). 14/45 patients had total absolute hematopoietic cells and acute leukemia has not been well-established. lymphocyte counts greater than 4.0 x 10^9/l, though these lymphocytoses were due to Design: We evaluated the expression of CD304 on blasts in 89 patients with acute the underlying T-LGL. 5 patients met criteria (11%) for a B-cell dyscrasia, with a mean myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), as well as the age of 57 years. Of these 5 patients, 2 patients had a sIg negative population consisting distribution of expression of CD304 in 36 blood and bone marrow specimens by flow of greater than 80% of B-cells, while 3 patients had an abnormal kappa/lambda ratio. cytometry. Chart review of the affected patients showed that 3 had evidence of an M-protein while Results: CD304 was expressed in 32% of acute leukemias and was expressed 1 had hypogammaglobulinemia. Although 3 patients had CD19+ CD5+ populations significantly more frequently in precursor B-cell ALL (14/22, 64%, median level of of B-cells, none of these patients had such a population in a quantity greater than 4.0 expression 70%) than in acute myeloid leukemia (11/56, 20%), p<0.0001. CD304 x 10^9/l (max 3.72 x 10^9/l). expression demonstrated no subtype specificity in AML. CD304 was detected in 1 of 5 Conclusions: 11% of T-LGL patients in this series had a concurrent B-cell dyscrasia by precursor T-cell ALL but was not detected on blasts in the few myelodysplasia samples FC that can be described as an MBL. This incidence appears higher than that published (0/4) or the single juvenile myelomonocytic leukemia specimen examined. All cases for the general population. Altered immune status of T-LGL patients may be an etiologic (3/3) of PDC leukemia evaluated at our institution were positive. CD303 was not detected factor, but this remains speculative. Further evaluation of the clinical significance of in any acute leukemia analyzed (0/50) with the exception of the 3 cases of PDC leukemia. these MBLs is warranted. On normal hematopoietic elements, there was weak to moderate expression of CD304 on erythroid progenitors (CD71bright, CD45-), weak expression on B-cell progenitors (both 1214 Mast Cells as a Part of a Prognostic Panel in Hodgkin early precursor-B cells, CD34+CD20-CD10+CD19+, and late precursor-B cells, CD34- Lymphoma CD20+CD10+CD19+, with median expression level of 17% and 27%, respectively), MT Howard, B Pohlman, P Elson, ED Hsi. Cleveland Clinic, Cleveland. moderate expression on plasma cells (CD38brightCD45dim), and moderate expression Background: Therapy for classical Hodgkin Lymphoma (cHL) therapy results in on PDCs (CD303+CD45+). A small subpopulation of lymphocytes, likely regulatory cure in a most patients, but a subset of cHL patients suffer relapse and die from their T lymphocytes, also expressed CD304. disease. Biologic prognostic markers may assist in risk stratification of cHL patients. Conclusions: Our findings indicate that CD304 is expressed on a wide variety of MAL and BCL2 expression in Reed-Sterberg (RS) cells and T-cell subsets (FOXP3/ hematopoietic cells and its expression is not restricted to PDCs. Its expression on Granzyme B(GzB) ratio) have been shown to be prognostic markers in cHL. Other a variety of leukemias limits its specificity in identifying PDC neoplasms. Finally, microenvironmental constituents such as mast cells (MC) have been suggested as expression of CD304 on precursor-B cell ALL was generally stronger than that observed additional markers. We studied our cohort of patients for MC infiltrates and correlated on normal precursor-B cells, suggesting that it may have utility in the detection of it with outcome. minimal residual disease in ALL. Design: A tissue microarray was constructed using 99 patients with cHL. Immunohistochemistry (IHC) for tryptase was performed on these microarrays. MCs 1212 Expression of Notch Proteins in Diffuse Large B Cell Lymphomas were counted and expressed as MC/hpf (using a 40x objective and 20mm ocular). The (DLBCL) Is Associated with a Favorable Clinical Outcome MCs/hpf (hot spot counting) were compared to the available clinical data, failure free SM Homan, YA Jalil, CE Sheehan, MS Tai, A Hayner-Buchan, JS Ross, T Nazeer. Albany survival (FFS) and overall survival (OS). Medical College, Albany, NY; NY Oncology, Rexford, NY. Results: The 99 patients had a mean age of 30 years, and 50/99 were male. Most of Background: Notch is a family of four transmembrane receptors that regulate cell the patients (62%) had Stage I/II disease, International Prognostic Score (IPS) values differentiation, proliferation and survival. Dysregulation of the Notch signaling pathway ≤2 (80%), and nodular sclerosis histology (82%). The mean MC hotspot count was 5.2 has been implicated in the pathogenesis of many human neoplasms. In hematolymphoid (range 0- 38.6). In 28% of cases, the RS cells expressed Bcl-2, 19% expressed MAL, malignancies, activating mutations resulting in constitutive Notch signaling have been and 75% of patients had FoxP3+:GzB+ ratios >1.0 (i.e. a FoxP3 “bias”). Multivariable clearly shown to be associated with T-cell malignancies. The role of Notch signaling in analysis considered age, stage, IPS, histology, presence of bulky disease, MC count, B-cell malignancies is still less clear; however, recent studies suggest a growth inhibitory FoxP3 bias, and expression of Bcl-2 and MAL. and pro-apoptotic function. This study evaluated Notch 1, 2 and 4 proteins in DLBCLs and correlated their expression with HIV status, tumor location, and overall survival. 268A ANNUAL MEETING ABSTRACTS

Table 1. FFS Conclusions: All patients with t(14:19)(q32;q13)-positive B-cell neoplasms and IgH/ Factor Hazard Ratio P-value BCL3 fusion in this study are small B-cell leukemias with atypical morphologic and MAL (pos./neg.) 5.14 <.001 immunophenotypic features and are frequently associated with additional chromosomal FoxP3 Bias (present/absent) 5.84 .002 changes including trisomy 12. The IgVH genes are usually unmutated. Bcl-2 (pos./neg.) 5.68 .009 MC count (>6/≤6) 3.56 .01 Age (≥45/<45) 3.06 .02 1218 Characterization of Collagen Vascular Disease-Associated Using the hazard ratios from the final models in Table 1, a scoring system was created Bone Marrow Changes by Morphologic and Immunohistochemical that counts the number of poor prognostic factors present. Patients were stratified into Assessment two groups: patients that had 0-1 poor prognostic factors and patients that had two or K Hunt, M Salama, C Sever, K Foucar. U. of New Mexico, Albuquerque, NM; U. of more. Estimated 5-year FFS for patients with 0-1 factors was 89%, vs. 51% for patients Utah, Salt Lake City, UT; Pathology Associates of Albuquerque, Albuquerque, NM. with 2 or more factors (P<.001). Estimated 5-year OS for patients with 0 or 1 factors Background: Collagen vascular diseases (CVD) are frequent in the differential was 100%, vs. 63% for patients with 2 or more factors (P<.001). diagnosis for unexplained cytopenias. Unfortunately, there are a paucity of large studies Conclusions: Increased MCs is associated with a poorer prognosis in patients with cHL. characterizing the bone marrow findings in CVD, making the diagnosis difficult without As part of a battery of immunophenotypic findings evaluating both tumor cells and the substantial clinical history. This study aims to identify associated morphologic and microenvironment, MC counts may be useful in risk stratification for cHL. immunohistochemical (IHC) abnormalities in a large series of CVD cases. Design: 101 cases of CVD and 12 controls were selected. Diagnoses were confirmed 1215 Simultaneous Detection of FLT3/ITD and NPM Mutations in AML with chart review. The following data was collected: CBC, peripheral blood smear with Normal Cytogenetics Using Formalin-Fixed, Paraffin-Embedded Bone morphology, bone marrow aspirate smear, clot and core biopsy morphology, iron stain, Marrow Biopsies and manual quantitation or semi-quantitation of IHC for the following antibodies: CD3, Q Huang, C Chen, W Chen, LM Weiss. City of Hope National Medical Center, Duarte, CD20, CD34, CD42b, MPO, Hgb A and CD68. Specifically, ten high power fields were CA. counted and averaged. All morphologic and IHC review was performed in a blinded Background: Acute myeloid leukemia (AML) with normal cytogenetics represents fashion. The results were compared between patients with CVD and normal controls. approximately 40-50% of de novo AML. Stratified prognostic determinants are required Results: Lymphoid aggregates were identified in 47% (40/85) cases of CVD vs. 42% to predict which patients in this category have increased risk of relapse, resistance to (5/12) of controls. Lipogranulomas (LG) were identified in 11% (11/101) cases of CVD therapy or long term disease outcomes. Recently, we developed a one step multiplex but were in none of the 13 controls. Sea blue histiocytes (SBH) were found in 6% (6/101) PCR assay for simultaneous detection of FLT3/ITD and NPM mutations in AML patients of CVD cases and none of the controls. Bony trabeculae were abnormal in 36% (31/87) in fresh specimens. Now we report that we modify and validated the methodology and of CVD and in 17% (2/12) of controls. Megaloblastic changes (at least mild) were present apply it to formalin-fixed, paraffin embedded bone marrow biopsies, enabling us for in 30% (30/101) of CVD cases versus none of the controls. A pseudo-myeloproliferative retrospective studies by using archived materials. (MPD) appearance was identified in 5% (5/101) CVD cases versus none of the controls. Design: Multiplex PCR was performed on genomic DNA using formalin-fixed, paraffin Quantitation of cell lineages via IHC is listed in figure 1. embedded bone marrow biopsies with specific primer sets designed to detect the presence or absence of FLT3/ITD and NPM gene 4 bp insertion mutations in patients with AML with normal cytogenetics. A specific set of b-globin primers was used simultaneously with FLT3 and NPM primers to ensure DNA integrity and PCR amplification. The results were compared and correlated with that of tests by using fresh materials. Results: Bone marrow trephine biopsy and clot sections from 39 AML patients with normal cytogenetics were included in this study. The expected amplified products for wild type FLT3, wild type NPM and b-globin were at 152 bp, 168 bp and 282 bp, respectively. FLT3/ITD mutations produced larger peaks ranging from 167 to 225 bp, while NPM mutation yield a single peak at 172 bp, 4 bp larger than the wild type. In the study, wild type FLT3, NPM and internal control were successfully amplified in 25/39 cases (64%). Bone marrow clot sections yielded excellent results while trephine biopsy with decalcification process had a marked impact on PCR amplification. Of 25 successfully amplified cases, 14 cases of FLT3/ITD and 12 cases of NPM mutations were identified. Ten of 25 cases were positive for both FLT3/ITD and NPM mutations. A100% correlation in regarding to the mutational status and the size of mutations of FLT3 and NPM genes was obtained by comparing the results from fresh materials and tissue in the cases studied. Conclusions: The multiplex PCR method provides a one step, rapid assay for detection Conclusions: Many of the investigated features showed no difference between of FLT3/ITD and NPM mutation using archived tissue. The test demonstrates excellent CVD and control groups. Lymphoid aggregates were common in both groups and correlation with the results from fresh materials and provides a valuable tool for immunohistochemical stains showed similar quantitation of various cell types. However retrospective studies. However, bone marrow biopsies with decalcification have major LG, bony abnormalities, megaloblastic changes, SBH and pseudo-MPD were more impact on the reliability of the assay. prevalent in the CVD group. These features may be helpful in strengthening suspicion of CVD and triggering further clinical investigation. 1217 The t(14;19)(q32;q13)-Positive B-Cell Leukemia: A Comprehensive Genetic and Clinicopathologic Study of Six Cases 1219 Comparative Analysis of 3q26 (EVI1 MDS1) Breakpoints in CML- YO Huh, RP Ketterling, F Vega, JE Kim, LJ Medeiros, LV Abruzzo. University of Texas BP and AML by Interphase FISH Using Bacterial Artificial Chromosome M. D. Anderson Cancer Center, Houston; Mayo Clinic, Rochester. Probes Background: The t(14;19)(q32;q13), involving the BCL3 locus at chromosome 19q13 KJ Jabbar, P Lennon, LJ Medeiros, J Abraham, P Hu, P Lin. MD Anderson Cancer and the immunoglobulin heavy chain gene locus at 14q32, is a rare recurrent cytogenetic Center, Houston, TX. abnormality in patients with B-cell neoplasms. B-cell neoplasms harboring t(14;19) have Background: Chromosome rearrangements involving 3q26 occurs in acute myeloid been most commonly reported in B-cell chronic lymphocytic leuekmia (CLL). However, leukemia (AML), myelodysplastic syndrome (MDS) and chronic myelogenous a recent study reported that the t(14;19) is not restricted to CLL. We previously reported leukemia in blast phase (CML-BP) resulting in EVI1/MDS1 gene disruption. The 3q26 seven cases of small B-cell leukemia with t(14;19). These cases share many features with rearrangements are heterogeneous with variable breakpoints in different diseases making CLL but differ from CLL in their morphologic and immunophenotypic features. FISH analysis challenging. One study, however, found in 3 of 4 CML-BP carrying the Design: Over the past two years, six additional cases with t(14;19)(q32;q13) were inv(3)(q21q26) that the breakpoint is located within the EVI1 gene detectable by BAC identified at the Cytogenetics Laboratory. Clinical and laboratory findings were clone RP11-82c9 (BJH 2007;136:806-813). Based on this report, we performed a larger reviewed. The morphologic features of peripheral blood and bone marrow smears, clot scale study comparing CML-BP and AML with inv(3)(q21q26) using the same clone. and core biopsy specimens, and lymph nodes were examined. Immunohistochemical Design: Cases of AML, CML-BP with inv(3)(q21q26) from 1999-2007 were searched stains for ZAP-70, cyclin D1 and bcl3 were performed on bone marrow biopsy or clot from our database and those with adequate materials were analyzed. A dual-color section. Flow cytometric immnophenotyping was performed on bone marrow aspirates. interphase fluorescence in situ-hybridization assay using RP11-82c9 (labeled green) Conventional cytogenetic analysis and FISH analysis for detecting IGH/BCL3 fusion and centromere 3 (labeled red) were performed. Presence of split green signals indicates was performed using a homebrew dual-color, dual fusion FISH probe. IgVH mutation EVI1 gene rearrangement. anlysis was performed on the bone marrow specimen. Results: We analyzed 18 cases with inv(3)(q21q26), 11 CML-BP and 7 AML. The RP11- Results: The patient cohort included 4 men and 2 women, with a median age of 62 82c9 clone identified EVI1 rearrangement in 13/18 (72%) cases, including 10 (91%) years (range 51-79). All had absolute lymphocytosis, 4 had lymphadenopathy, and CML-BP compared with 3/7 (43%) of AML cases. The positive signals ranged from 1 had splenomegaly. Lymphocytes in blood and bone marrow aspirate smears were 6.5% to greater than 32 % in the CML-BP cases and 6% to greater than 32% in the AML predominantly small but cytologially atypical and demonstrated significant heterogeneity. cases. One case of CML-BP had 3.5% signals and was considered indeterminate. Flow cytometric immunophenotyping showed an atypical immunophenotype with low Conclusions: The RP11-82c9 clone is preferentially involved in CML-BP and a subset CLL scores and positive CD38 in 5 of 6 cases. Immunohistochemical stain showed of AML with inv(3)(q21q26). This clone is useful for detection of residual disease. overexpression of bcl3 in all cases. ZAP-70 was positive in 3/6 cases. IgVH mutation analysis revealed unmutated gene in 5/6 cases. Conventional cytogenetic studies demonstrated trisomy 12 in 4 patients and additional cytogenetic changes were found in 5 patients. FISH analysis verifiedIGH/BCL3 fusion in all 5 cases tested. ANNUAL MEETING ABSTRACTS 269A

1220 Flow Cytometric Immunophenotyping of Systemic Mastocytosis coexpression of CD23); (2) cyclin D1/IgH negative (by FISH or immunohistochemistry); in the Bone Marrow and (3) available concurrent non-BM tissue biopsy for review. KJ Jabbar, Y Huh, S Wang, LJ Medeiros, MR Johnson, S Verstovsek, JL Jorgensen. UT Results: The median age at diagnosis was 68 yrs (range= 42-91); M:F=45:30. 47 patients MD Anderson Cancer Center, Houston, TX. had a lymph node biopsy following the PB or BM FC study; 22 had splenectomy; the Background: By WHO criteria, systemic mastocytosis (SM) involves at least one remaining tissue biopsies were from lacrimal gland, epiglottis, lung, pleura, small bowel, extracutaneous organ. Detection of aberrant expression of CD2 and/or CD25 on omentum, liver, bone, and skin. Tissue review showed that 33 of 75 patients (44%) had mast cells is a minor criterion for diagnosis, and this is commonly achieved through involvement by CLL/small lymphocytic lymphoma, despite lack of prototypic flow immunohistochemisty (IHC) on bone marrow (BM)biopsies. In this study we assessed cytometric findings in PB or BM, based on typical morphology and immunophenotype; flow cytometry (FC) on BM aspirates as an alternative technique for detection of aberrant no MCL were identified. Marginal zone lymphoma (MZL) was diagnosed in 24 patients mast cells, in initial diagnosis and in follow-up after therapy. (32%); of these 8 had typical histologic features of splenic marginal zone lymphoma, 3 of Design: We studied 50 adults (23-88 years old) who met WHO criteria for SM, including mucosa-associated lymphoid tissue lymphoma, and the remaining 13 were unspecified. 20 men and 30 women by flow cytometry. BM aspirates were stained with CD2 and Lymphoplasmacytic lymphoma (LPL) was detected in 7 patients (9%) and large cell CD25, conjugated to FITC or PE, along with CD45 PerCP-Cy5.5 and CD117 APC for lymphoma in 6 (8%). 1 patient had a high grade B-cell lymphoma, unspecified, with gating. Other antigens included CD11c, CD35, CD59, CD63 and CD69 (all from BD CDK6 translocation. The remaining 4 patients (5%) were diagnosed with a low grade Biosciences, San Diego, CA), and non-specific staining was measured with matched B-cell lymphoma, NOS. isotype controls. Data were acquired on FACS Calibur cytometers (BD), collecting Conclusions: Although CD5 positivity is most commonly associated with CLL and from 2x105 to 106 events in most cases, and analyzed with CELL Quest (BD). Staining MCL, a significant minority of cases do not fall into these 2 categories. Phenotypically patterns were compared to normal mast cells (in staging marrows negative for non- unusual CLL, MZL and LPL were the most common diagnoses in this group of patients. Hodgkin lymphoma). Applying strict FC criteria, using genetic studies, and deferring to a lymph node/tissue Results: By flow cytometry, aberrant over expression could be demonstrated using diagnosis in non-classical cases is critical for accurate diagnosis and classification of CD25-PE in 45/50 (90%) SM cases and using CD2-PE in 41/50 (82 %) cases. In cases CD5+B-LPDs. stained with both CD2 conjugates, 31/40 (77.5 %) cases were positive with CD2-PE while 28/40 (70%) were positive with CD2-FITC. For CD25 staining, cells were brighter 1223 p53 Immunohistochemistry Predicts 17p13(TP53) Deletion in with the CD25-PE conjugate, but the same cases were also positive with CD25-FITC. CLL Among cases studied with both FC and IHC there was 100% concordance for CD25 A Jiang, C Qi, H Chang. University Health Network and University of Toronto, expression, with 25/26 (96%) cases positive. For CD2 staining, FC was more sensitive Toronto, Canada. than IHC, with 14/23 (61%) positive by FC and 9/23 (39%) positive by IHC. The other Background: Chromosome 17p(TP53) deletion is an adverse prognostic factor markers studied by FC were also frequently aberrant, with over expression compared associated with disease progression and chemotherapy resistance in patients with chronic with normal mast cells seen for CD35 in 43/50 (86%) SM cases, CD59 in 46/50 (92%), lymphocytic leukemia (CLL). This abnormality can be detected in 7-15% of CLL by CD63 in 44/49 (90%), and CD69 in 39/48 (81%). Flow cytometry detected far fewer interphase fluorescencein situ hybridization (FISH). Previous studies found that nuclear mast cells (0.002-7%) than estimated by morphology and IHC (2-80%), with 10-fold p53 protein expression detected by immunohistochemistry (IHC) is associated with to 10000-fold lower levels seen by FC. poor overall survival; however, it is not clear whether nuclear p53 protein expression Conclusions: Flow cytometry is a rapid and sensitive technique for characterizing can predict 17p deletion in CLL. aberrant mast cells in BM aspirates. Large numbers of cells must be acquired, and this Design: Paraffin-embedded bone marrow biopsies from 110 CLL patients were method is not suitable for absolute quantitation. The detection rate is comparable to evaluated for nuclear p53 protein expression by IHC with an antibody against p53 IHC for CD25 aberrancies, and superior for CD2. (DO-7 clone). Fifty-three samples were obtained at diagnosis, while 57 were obtained at progressive or relapsed stage of the disease. Interphase FISH was performed on 1221 Light Chain Only Plasma Cell Dyscrasias Demonstrate Increased paired bone marrow aspirate specimens with probes for 17p13(TP53) deletion, and Bone Marrow Microvessel Density and Mast Cells other CLL-associated genetic prognostic factors including 13q deletion, 11q22 (ATM) JR Jacobsen, GJ Stoddard, SR Tripp, SL Perkins, TW Kelley. University of Utah & deletion, and trisomy 12. ARUP Laboratories, Salt Lake City, UT; University of Utah School of Medicine, Salt Results: IHC detected nuclear p53 protein expression in 14 (12.7%) patients while Lake City, UT. interphase FISH detected hemizygous 17p deletion in 15 (13.6%), of whom 10 patients Background: Plasma cell myeloma (PCM) is an aggressive and incurable disease (66.7%) had progressive or relapsed disease. All nuclear p53 protein immunoreactive with poor long-term survival. Prognostic factors for PCM include cytogenetic, clinical, cases had 17p deletion (p<0.001). Thus, the sensitivity of the IHC p53 test was 93%, serologic, and histologic features. Bone marrow microvessel density (MVD) and mast and the specificity was 100% to predict 17p(TP53) deletions. Interphase FISH detected cell (MC) numbers have also been evaluated as predictive markers but have a much 13q deletion, 11q22 (ATM) deletion and trisomy 12 in 48.0%, 14.1% and 16.2% of the less clear relationship to outcome. CLL cases respectively. There was no association between p53 expression and these Design: Initial bone marrow (BM) biopsies with at least 5% involvement by PC genetic risk factors, or other clinical laboratory parameters such as age, gender, leukocyte dyscrasia (PCD) including newly diagnosed patients and those with recurrent disease count, haemoglobin level, platelet count or marrow infiltration pattern. were studied. BM transplant recipients were excluded. Biopsy adequacy was defined Conclusions: IHC, a widely available and inexpensive tool, detects nuclear p53 protein as 10 evaluable high power fields (HPFs). Stains for MC tryptase and CD34 were expression which is predictive for 17p deletion in CLL. p53 IHC can be used to risk- performed to highlight MCs and vasculature, respectively. MCs were counted in ten stratify CLL patients for innovative therapies. HPFs. MVD was quantified by counting the number of small vessels in the three HPFs with the greatest vessel density. BM plasma cell percentage was drawn from the original 1224 Among Alpha Thalassemia Patients (pts), Genotypes Are Closely pathology report. Correlation was assessed with a Pearson correlation coefficient. Associated with the Degree of Microcytosis/Mean Corpuscular Volume Statistical comparisons of continuous variables were made using the Fisher-Pitman (MCV) Values permutation test for independent samples. X Jiang, D Grisak, M Lareau, MTS Rad, A Mansoor. Calgary Laboratory Services Results: 54 patients (22 F, 40.7% and 32 M, 59.3%) with age 40-78 years were included. (CLS)/University of Calgary, Calgary, AB, Canada. 48 cases were classified as PCM, 5 as MGUS, and 1 as amyloidosis. Ig subtypes included Background: α thalassemia is characterized by quantitative defects in globin chain 8 light chain only (LC, 14.8%), 15 IgA (27.8%), 30 IgG (55.6%), and 1 IgM (1.7%) and synthesis due to gene deletions. Minor deletions are associated with silent carrier (-α/ were not correlated with age or percent BM plasma cells. LC-only cases demonstrated αα) or indolent clinical states (-α/-α OR --/αα) . Major deletions result in severe significantly higher MVD (p=0.038) and MCs (p=0.022) than other subtypes. Overall, disease, like Hb H disease (--/-α) or fatal Hydrops fetalis (--/--). It is an important D/D MVD was significantly correlated with percent BM plasma cells (r=0.49, p<0.001) but in hypochromic microcytic anemia with normal iron studies, specially in specific ethnic there was no correlation between BM mast cells and plasma cells or the MVD. groups. Genotype determination is essential in thalassemia minor pts to avoid severe Conclusions: This study demonstrates that PCDs expressing LC-only have significantly disease among offsprings. Identification of subset to be screened for gene deletion increased MVD and BM mast cells compared to the other subtypes. Overall, the MVD based on CBC data alone is difficult. In this study, we determined the association and percentage BM plasma cells also correlated, suggesting that neoplastic plasma cells between MCV & various gene deletions in a cohort of hypo / microcytic anemia pts influence stromal vascularity. The higher MVD in the LC-only subtype may, in part, with normal iron studies. contribute to the more aggressive behavior of this variant. Design: Analysis of 1,714 pts (> 95% non-Caucasian) with normal serum iron studies, referred for Hemoglobinopathy screening to our tertiary care facility was conducted. 1222 CD5+ B-cell Lymphoproliferative Disorders: Beyond Chronic CBC / Iron studies were performed on automated analysers. Determination of Hb A2, Lymphocytic Leukemia (CLL) and Mantle Cell Lymphoma (MCL) HbF / etc was conducted by HPLC methodology (BIO RAD, Germany). Established D Jevremovic, RS Dronca, WG Morice, ED Remstein, PJ Kurtin, CS Zent, CA Hanson. single tube multiplex PCR screening (AJCP 2002), capable of detecting all genotypes Mayo Clinic, Rochester, MN. was utilized in all. Pts were divided into group A: ≥12 yr (MCV ref. 82-100) and group Background: Flow cytometric (FC) analysis of peripheral blood (PB) and bone B: 2-11 yrs (MCV ref. 75-91). Regression analysis was used to model for various marrow (BM) is a common method for evaluating B-cell lymphoproliferative disorders genotypes. Chisquare test determined statistical correlation & p <0.05 was considered (B-LPDs). CD5 positivity is usually considered characteristic of either CLL or MCL, significant. depending on co-expression of CD23, intensity of surface immunoglobulin (sIg) Results: In group A (n=1531, median age 43 yrs, range 12-93, M:F 0.7:1); MCV ≤ 73 and CD20, and the protein or genetic status of cyclin D1. However, other neoplastic was noted in 659(43%); 74-81 in 809 (53%) & ≥ 82 in 63 (4%) pts. In pts with MCV B-LPDs may express CD5, albeit infrequently. In this study we have reviewed the tissue ≤ 73; 551 (84 %) had ≥2 gene deletions ((p <0.0001; PPV 81%; NPV 95%), majority pathology of CD5+ B-LPDs that do not fulfill diagnostic criteria for CLL or MCL on (77%) being cis deletion (--/αα) (SEA: 83%). Pts with MCV 74-81, only 8% had ≥ 2 FC studies of PB or BM. gene deletions, while most (443/809) (55%) had single gene deletion (3.7kb =55%; Design: We identified, using PB or BM FC analysis, 75 patients with a CD5+ B-LPD 4.2 kb= 7.7%). In group B (n=183; median age 6, M:F 1.3:1); MCV ≤ 68 was noted in that were seen at the Mayo Clinic between 1996 and 2008 and met the following 99(54%); MCV 69-74 in 59 (32%) and MCV ≥ 75 in 25 (14%). In pts with MCV < 68 inclusion criteria: (1) lack of typical CLL FC profile (dim sIg, dim CD20, uniform CD5, (n=99), 70% had ≥2 gene deletions (p <0.0001; PPV 70%; NPV 90%); cis (73%) & tran 270A ANNUAL MEETING ABSTRACTS

(27%), (SEA: 65%). In pts with MCV 69-74, single gene deletion was most common (32/59 (54%) while ≥ 2 gene deletions was seen in only 5 pts (3%). Conclusions: In thalassemia minor pts, MCV ≤ 73(adults) OR ≤ 68 (pediatric) are predictive of two α gene deletions; while single gene deletion is most common when MCV is 74- 81 (adults) & 69- 74 (pediatric).

1225 MUM1 and CD138 Immunophenotype as Prognostic Markers in Patients with Multiple Myeloma A Junger, C Fibich, M Voralia, H Neufeld, H Doell, SK Verma, JF DeCoteau, EE Torlakovic. College of Medicine, University of Saskatchewan, Saskatoon, SK, Canada; Saskatchewan Cancer Agency, Saskatoon, SK, Canada. Background: Both CD138 and MUM1 are expressed at the plasma cell stage of B-cell differentiation and their expression is generally widely present in multiple myeloma. The percentage of MUM1+ cells vary from case to case. The percentage of CD138+ cells also varies from case to case, but to a lesser degree than MUM1. High MUM1 mRNA expression was recently reported as unfavorable prognostic marker in MM. We investigated predictive value of CD138 only, MUM1 only, and CD138/MUM1 protein expression and coexpression respectively. Design: Seventy patients, median age 62 years (range 36-92); 38 were treated with high dose chemotherapy and autologous stem cells transplantation (SCT) and 32 with conventional chemotherapy alone (CTX). Median follow up was 807 days (range 20- 3396), mean=1010 days). Bone marrow biopsies at diagnosis were studied by single Conclusions: MNDA is a potentially useful marker in the recognition of normal and double immunohistochemistry for MUM1, CD138, or both. The percentage of marginal zone cells and the diagnosis of NMZL cases, with a limited expression among positive plasma cells in 500 tumor cells was recorded. FL. which can offer potential value in the analysis of normal B-cell subpopulations and Results: Cox regression analyses revealed that higher the percentage of MUM1 or the differential between NMZL and FL. CD138 positive plasma cells, as well cells with MUM1/CD138 coexpression, worse the overall survival (OS) and progression-free survival (PFS) (Table 1). Percent positive 1227 Differential Staining of Phospho-AKT in Normal Marginal Zone cells as continuous values and a cut off point of ≥ 85% positive cells were established Cells Versus Marginal Zone B Cell Lymphoma as significant for PFS, but for OS only continuous values were significant, while≥ 85% S Kemp, KA Rizzo. Indiana University, Indianapolis, IN. positive cells cut off point showed only statistical trend for shorter OS. The results Background: Marginal zone B cell lymphomas (MZL) are subclassified as nodal were independent of the type of the treatment for PFS and OS and independent of ß2- marginal zone B cell lymphoma, extranodal marginal zone B cell lymphoma of MALT microglobulin for PFS, but not OS. type (MALT lymphoma) and splenic marginal zone lymphoma (SML). Due to the non- PFS OS specific immunophenotype in MZL, investigations have focused on new diagnostic and p-value 95% CI p-value 95% CI prognostic markers. AKT(PKB) plays a significant role in cell survival and apoptosis SCT 0.340 0.687 (0.317-1.486) 0.001 0.262 (0.118-0.585) MUM1 (CD138+/-) 0.016 1.047 (1.008-1.086) 0.107 1.026 (0.995-1.058) and is activated via phosphorylation. Few reports show that phospho-AKT is seen in CD138 (MUM1+/-) 0.039 0.687 (0.318-1.487) 0.024 1.037 (1.005-1.069) certain hematopoietic malignancies. We undertook a study to analyze the expression CD138+/MUM1+ 0.010 1.027 (1.006-1.048) 0.016 1.022 (1.004-1.040) of phospho-AKT in normal marginal zone B cells versus B cells in marginal zone B Conclusions: The extent of MUM1 and CD138 protein expression appears to play a cell lymphoma. role in biology of multiple myeloma and cases that show less of either MUM1 or CD138 Design: 21 cases were retrieved and included 5 cases of non-malignant spleens, 1 case or both may represent biologically less aggressive entities with, regardless the type of of SML, 9 cases of MALT lymphoma and 5 cases of MZL in a lymph node. H&E and the treatment, significantly longer PFS and OS. immunostains performed on paraffin sections were reviewed and marginal zone cells were analyzed for phospho-AKT staining. The percent of overall positive cells were measured on a 0-4 scale (0= 0 cells, 1 = 1-25%, 2 = 26-50%, 3 = 51-75%, 4 = 76-100%) 1226 MNDA, a New Marker Useful in the Recognition of Nodal and the intensity of staining was analyzed on a 0-2 scale (0 = no staining, 1 = minimal, Marginal Zone Lymphoma and Its Differential with Follicular Lymphoma 1.5 = moderate, 2 = marked). G Kanellis, G Roncador, S Montes-Moreno, A Arribas, M Mollejo, Y Campos-Martin, Results: The age range was between 1 and 79 years old. The location of the MALT M Piris. CNIO, Madrid, Spain. lymphomas included the stomach (7 cases), breast (1 case) and thyroid (1 case). Two of Background: Specific immunohistochemistry markers for NMZL are not known, and the stomach MALT lymphoma cases were from the same patient, the first biopsy was those of FL (CD10, BCL6) not always conclusive. We tried to clarify this grey area of H.pylori positive; the subsequent one was H. pylori negative. The marginal zone cells in differential diagnosis by using the novel marker Myeloid Nuclear Differentiation Antigen 4 of the 5 non-malignant splenic cases were completely negative, while one case showed (MNDA), developed by the Monoclonal Antibody Unit of our Centre, as suggested by minimal staining (1) in less then 25%(1) of the cells. In the marginal zone lymphoma a previous expression profiling study. cases, 11 of the 15 cases (73%) scored a 4 for overall positive cells, 1 case scored a Design: Gene expression profile studies were performed in a series of small B cell 3 (7%) and 4 cases scored a 2 (27%). Statistical analysis via student t-test showed a lymphoma cases.Differentially expressed genes among small B cell lymphoma subtypes statistical significance in the number of positive cells between the malignant and non- were identified by paired comparison using t-test analysis.A new monoclonal antibody malignant groups (p=1.77 x 10^-8). Analysis of the staining intensity of phospho-AKT against MNDA protein was generated and immunohistochemical characterization of a within the malignant group showed a large majority of the malignant cases contained series of 464 B- cell Non-Hodgkin Lymphoma cases was performed. moderate to marked (1.5-2) staining intensity(87%, 13 of 15 cases). Results: Gene expression profile identifies MNDA as a component of NMZL signature. Conclusions: Our study shows differential positive staining of phospho-AKT between A MoAb recognizing MNDA was prepared and validated through Western Blotting and normal and malignant marginal zone B cells. Analysis of the activation of the AKT transfected cell lines. MNDA expression is nuclear and, in reactive lymph nodes, was pathway in marginal zone lymphomas may lead to diagnostic immunohistochemical seen by mature granulocytes and monocytes with great intensity. With a lesser intensity markers as well as insights into activation/upregulation of signaling pathways. the MNDA expression was found within the B cells of the hyperplastic IgD-negative marginal zone, but not seen by monocytoid B cell, and especially not by germinal centre cells(GC). The analysis of a series of 464 B cell NHL showed a heterogeneous 1228 Immunophenotypic Alterations by Flow Cytometry in Patients expression of MNDA (Table 1). Of greater interest is the expression of MNDA among with Granulocyte Colony Stimulating Factor (G-CSF) Therapy May Mimic NMZLs and FLs. The former showed 76% (41/54) positivity and an expression pattern Changes Seen in Myelodysplastic Syndrome (MDS) that was marginal, or marginal with interfollicular constituent. MNDA positivity in FA Khokhar, LJ Medeiros, JL Jorgensen. UT MD Anderson Cancer Center, Houston, GC was observed in cases of follicular colonization. In contrast, only 5% of FL cases TX. expressed MNDA (10/183) with an intrafollicular staining intensity which appeared Background: G-CSF is often given to neutropenic patients after chemotherapy or less intense than the one seen in the rim of the neoplastic follicules. stem cell transplant (SCT), and occasionally to patients with suspected MDS. Kussick and Wood (Arch Pathol Lab Med. 2003; 1140-1147) described immunophenotypic changes seen in G-CSF-treated patients by flow cytometry (FC). In this study, we assessed FC findings in a series of patients with known G-CSF therapy, or status/post (s/p) chemotherapy for acute myeloid leukemia (AML). Design: Patients were identified from our institutional databases from 2006-2008. All patients had known G-CSF therapy within the last 21 days, or were s/p chemotherapy for AML with no available history of G-CSF therapy. All showed diploid cytogenetics, with morphologic features on bone marrow aspirates (BMA) not diagnostic for dysplasia or residual disease. Patients who received G-CSF included 2 SCT patients; 1 AML patient and 7 patients with other neoplasms, all s/p chemotherapy; and 1 patient with neutropenia of unknown etiology. BMA specimens were studied with a cytopenia screening panel that included CD13, CD16, CD45 and CD56. Side scatter (SSC) was measured on granulocytes, gated using CD45. FC results were compared with ranges established using 20 apparently normal bone marrows, submitted for lymphoma staging but negative for disease. ANNUAL MEETING ABSTRACTS 271A

Results: Patients with known G-CSF therapy most often had an altered expression Conclusions: SHH signaling pathway is activated in DLBCL. Our data show that this pattern of the maturation markers CD13 and CD16 (Table 1). Many also had decreased pathway plays an important role in cell survival and proliferation in DLBCL and that granulocyte SSC (correlating with cytoplasmic hypogranularity). A few had increased inadequate activation of this pathway may confer a poor prognosis in DLBCL. expression of CD56 on granulocytes and/or monocytes. Patients s/p chemotherapy for AML had similar findings, at a lower frequency. The patient with neutropenia of 1231 Insulin-Like Growth Factor II m-RNA Binding Protein 3 (IMP3) Is unknown etiology had a followup BMA 3.5 months later, which showed reversal of all Differentially Expressed in Lymphoid Compartments and Is a Potentially immunophenotypic changes. Novel Marker of Normal and Neoplastic Germinal Center B-Cells Total Altered CD13/ Decreased Increased CD56+ Increased CD56+ RL King, T Pasha, MR Roullet, PJ Zhang, A Bagg. Hospital of the University of patients CD16 pattern SSC Granulocytes Monocytes Known G-CSF 11 7 (64%) 5 (45%) 3 (27%) 1 (9%) Pennsylvania, Philadelphia, PA. AML s/p chemotherapy 9 2 (22%) 3 (33%) 2 (22%) 1 (11%) Background: IMP3 is a member of the insulin-like growth factor II m-RNA binding Conclusions: G-CSF therapy led to immunophenotypic alterations in most patients, protein family of proteins that play a role in RNA trafficking and stabilization, and but the pattern of changes varied from patient to patient. Some patients s/p AML cell growth and migration during embryogenesis, but which are down-regulated in chemotherapy had similar findings, perhaps due to the effects of endogenous growth adult tissue. However, IMP3 has recently been shown to be overexpressed in several factors. All of these FC findings have also been described in patients with MDS. We epithelial malignancies, with increased expression correlating with aggressive believe that a suggested diagnosis of MDS by FC should be made with caution in a behavior. These findings implicate IMP3 as an oncofetal protein and suggest its patient with a history of recent G-CSF therapy. role as a biomarker for adverse outcomes. To the best of our knowledge, there is no published literature evaluating IMP3 in normal or neoplastic lymphoid tissue. In a pilot immunohistochemical (IHC) study of normal lymphoid tissue, we observed that IMP3 1229 Anaplastic Multiple Myeloma Is Associated with High-Risk expression is largely restricted to germinal center (GC) B cells. Molecular Cytogenetic Abnormalities Design: We evaluated the expression of IMP3 in a series of lymphomas, specifically DT Kim, H Chang. University Health Network and University of Toronto, Toronto, to determine its utility as a potentially novel marker of those of GC origin. IHC was Canada. performed on a series of 148 (predominantly B cell) lymphomas in tandem with other Background: The WHO classification recognizes anaplastic multiple myeloma (AMM) pertinent IHC stains and correlated with WHO subtypes. as a rare morphologic variant characterized by pleomorphic multinucleate plasma Results: Heterogeneous expression of IMP3 was noted among different B cell cells with prominent nucleoli. AMM is associated with extramedullary infiltrates and lymphomas. The strongest expression was seen in malignant cells of both classic and an adverse prognosis but little is known about the underlying molecular cytogenetic nodular lymphocyte predominant Hodgkin lymphoma [26/30 (87%) cases positive]. abnormalities. We systematically analyzed 11 cases of AMM and compared their Bright expression was evident in 100% [12/12] of Burkitt lymphoma cases. Follicular molecular cytogenetic features with 180 newly diagnosed non-anaplastic MM. lymphoma showed weak positivity in most cases [14/16 (88%)]. While all cases [27/27 Design: AMM cases were morphologically identified according to WHO criteria, their (100%)] of diffuse large B cell lymphoma (DLBCL) were positive for IMP3, there clonal plasma cells immunophenotyped and clinical features reviewed. The clonal was wide variability in staining intensity, which did not correlate with a proposed plasma cells from bone marrow aspirates were examined by interphase fluorescencein classification into activated B cell vs GC B cell origin, as gauged by IHC. By contrast, situ hybridization (FISH) for myeloma-related genetic abnormalities. The FISH analysis lower proportions (10-30%) of all other lymphoma subtypes studied (mantle cell, included probes for translocations t(11;14) (cyclin D1), t(4;14) (FGFR3), deletions marginal zone, small lymphocytic, B lymphoblastic, and T cell lymphoma) were 13q14 and 17p(p53), and amplifications of 1q21(CKS1B). IMP3 positive. Results: There were 7 males and 4 females with a median age of 61 years (range, Conclusions: This is the first study to evaluate expression of IMP3 in lymphoid 39-70). Four patients had IgA, 3 IgG kappa, and 4 light chain only. The median bone tissues, revealing it to be a potentially novel GC B cell marker, with distinct intensities marrow plasmacytosis was 85% (range, 40-100%). Interphase FISH detected CKS1B of expression between different GC B lymphomas. However, expression of IMP3 in amplifications (3-8 FISH signals) in 10 (91%) of 11 cases, the median percentage of DLBCL is variable and does not appear to have GC B cell phenotypic associations, at myeloma cells with CKS1B amplification was 80% (range, 40-100%). FISH detected least in terms of IHC algorithms. Further studies are needed to elucidate the functional hemizygous p53 deletions in 5/11, 13q deletions in 4/11, t(4;14) in 4/11, and t(11;14) in role of IMP3 in normal and neoplastic B cells. 2/11 cases. One case had 4 coexisting genetic abnormalities, 5 cases had 3 abnormalities, and the remaining cases had 1-2 abnormalities. In comparison, the newly diagnosed MM had CKS1B amplification, p53 deletion, 13q deletion, t(4;14) and t(11;14) in 34%, 1232 CD20-Positive Multiple Myeloma: A Subtype Associated with a 11%, 41%, 13% and 13% respectively. AMM had significantly higher prevalence of Worse Prognostic Profile CKS1B amplification (p=0.0003), p53 deletion (p=0.001) and t(4;14) (p=0.004) than S Kitahara, A Yung, R Alsabeh. Cedars-Sinai Medical Center, Los Angeles, CA. that of non-anaplastic MM. Background: CD20 positivity has been reported in 13-22% of patients with multiple Conclusions: AMM represents a subset of MM frequently harboring high-risk genomic myeloma (MM). The exact clinical significance of CD20 expression in MM is not clear, aberrations, particularly, CKS1B amplification. The high prevalence of CKS1B however, this subset is of interest because these patients may benefit from rituximab amplification, p53 deletion, and t(4:14) may indicate genetic instabilities of AMM cells therapy. Some studies suggest that CD20-expressing MM reflects a more aggressive resulting in a more aggressive behavior of the disease. MM subtype. However, recent studies have demonstrated a correlation between CD20 and a favorable prognostic marker t(11;14). The aim of this study is to describe the relationship between CD20 and genetic abnormalities detected by conventional 1230 Sonic Hedgehog Signaling Pathway as a Therapeutic Target in karyotype analysis and/or fluorescent in situ hybridization (FISH), and to explore the Diffuse Large B-Cell Lymphoma prognostic implication of CD20 expression. JE Kim, RR Singh, C Milito, JH Cho-Vega, M Karanikou, F Kasbidi, LJ Medeiros, R Design: 50 cases of MM from 2002 to 2008 with genetic information (conventional Luthra, F Vega. MD Anderson Cancer Center, Houston, TX. karyotype on 49 cases and/or FISH for deletion (del) of 13q) on 18 cases) were evaluated Background: Sonic hedgehog (SHH) signaling is involved in the regulation of cell for expression of CD20, PAX-5, bcl-1, p53 and CD56 by immunohistochemistry. proliferation and differentiation of embryonic and adult stem cells. Deregulation of this Results: pathway have been reported to result in the initiation and maintenance of a broad range CD20-positive (n=8) CD20-negative (n=42) of cancers including basal cell carcinoma, medulloblastoma, prostate cancer, cancers % CD20 (+) MM cells 20 - 100 - of the digestive tract, lung, breast and liver. However, the role of SHH/GLI signaling % MM cells/biopsy (mean) 84 44 in diffuse large B-cell lymphomas (DLBCL) has not been explored. Plasmablastic morphology 2 (25%) 11 (26%) Design: SHH and GLI-1 were immunohistochemically assessed in a tissue microarray PAX-5 (+) 1 (13%) 1 (2%)* containing 36 DLBCL tumors and by Western blot (WB) analysis of 6 DLBCL cell bcl-1 (+) 7 (88%) 7 (17%) p53 (+) 2 (25%) 8 (19%) lines (Pfeiffer, DOHH2, MS, MCA, Ly3 and Ly10). For comparison, tissue microarrays CD56 (-) 4 (50%) 11 (26%) containing cases of follicular lymphoma as well as mantle cell lymphoma cell lines Deletion of 13q (FISH) 3/5 (60%) 1/13 (8%) (SP-53 and Z-138) were assessed.The biological effect of inhibition of SHH pathway Abnormal karyotype 1/7 (14%) 12 (29%) was assessed using a cell viability (MTT assay) and clonogenicity assays after treatment Translocations involving chromosome 14 1/7 (14%) 3 (7%) with the SMO inhibitors, cyclopamine and SANT-1, and after transfection with siRNA Hyperdiploid 0/7 (0%) 3 (7%) specific for GLI1. The presence of extra copies of GLI1 gene was investigated using * - weak staining real-time quantitative PCR. Conclusions: CD20 expression is seen in 16% (8 of 50) of MM cases and the majority Results: SHH expression (cytoplasmic) was detected in 32 of 36 (89%) DLBCL tumors (7 of 8) are associated with diffuse bcl-1 expression. When expressed, CD20-positivity and was intense in 15 cases (41.7%). GLI1 expression (nuclear) was detected in 35 is seen in 20-100% of myeloma cells, and can potentially be a good target for rituximab of 36 (97.2%) DLBCL and strong nuclear expression was seen in 12 (34.3%) cases. therapy. CD20-positive MM is associated with a higher tumor burden in bone marrow High expression levels of both proteins in DLBCL cell lines were confirmed by WB. In biopsies and more commonly demonstrates adverse prognostic features, such as contrast, in follicular lymphomas we found expression of SHH and GLI1 in only 2 of del(13q) and negative expression of CD56. Hyperdiploid DNA content, indicative of 23 and 8 of 23 cases, respectively. We found a correlation between high expression of a good prognosis, was not present in any of the CD20-positive MM. CD20-expression GLI1 and lower survival in DLBCL patients. Pharmacologic inhibition of SHH signaling assessment may be useful in evaluating MM. induced cell death (35-80%) and decreased clonogenecity of DLBCL cell lines in a dose- dependent manner in comparison with controls (tomatidine and DMSO). Importantly, 1233 Bone Marrow Biopsy in Patients with HCV: Spectrum of Findings cyclopamine treatment showed no effect on colony formation or cell viability in cell and Diagnostic Utility lines with no overexpression of SHH signaling or in the cell viability of non-neoplastic JM Klco, B Geng, M Lisker-Melman, EM Brunt, A Hassan, F Kreisel, JL Frater. B-lymphocytes. Extra copies of GLI1 were detected in 5 of 11 (45.4%) DLBCL tumors Washington University School of Medicine, Saint Louis, MO. analyzed, including 3 cases with 4 or more extra copies of GLI1. Background: Patients with hepatitis C (HCV) develop a number of hematologic disorders including cytopenias, which are felt to stem primarily from hypersplenism 272A ANNUAL MEETING ABSTRACTS and/or antiviral medications. To date, no study has reported on the spectrum of bone and in the KM-H2 human Hodgkin lymphoma cell line. Lastly, KM-H2 cells were marrow morphologic changes in patients with HCV. treated with various concentrations of MIF, ISO-1 (MIF Inhibitor), sphingosine-1- Design: 49 adult patients (28 M, 21 F) with HCV without HIV coinfection that underwent phosphate or complete growth medium (10% FBS) to determine the effects, if any, on a bone marrow biopsy were identified. The bone marrow findings, peripheral cell proliferation and migration. counts and features of chronic hepatitis, including cirrhosis, splenomegaly, treatment, Results: Genotyping of the -173 SNP in 60 Hodgkin patients revealed 60% (36/60) G/G, and MELD score (a system for assessing the severity of chronic liver disease) are 33% (20/60) G/C and 7% (4/60) C/C. While in control cases there were 77% (75/97) G/G, reported. 23% (22/97) G/C and 0% (0/97) C/C. Immunohistochemistry demonstrated staining for Results: 59% (23/39) of the patients presented with cirrhosis, either by radiographic MIF and CD74 in the KM-H2 cell line and in both Hodgkin Reed-Sternberg cells and studies or liver biopsy, 76% (29/38) with splenomegaly and 12% (6/49) had a history background lymphocytes in tissue samples from Hodgkin patients. Treatment of KM-H2 of antiviral treatment with interferon and ribavirin. The average MELD score was 9.5 cells with various concentrations of MIF and/or ISO-1 made no significant difference in (range 2.3-28.2, max. score 40). The patients typically presented with anemia (mean: proliferation, as assessed by cell number, after 2 days of treatment in a variety of culture 10.2 g/dl) and thrombocytopenia (mean: 135 109/L) with a normal WBC count (mean: conditions. Interestingly, in cell migration assays, sphingosine-1-phosphate (5nM) was a 5.11 109/L). 36% (17/47) of patients presented with pancytopenia, which was the most potent chemoattractant for the KM-H2 cells, however, MIF and ISO-1 had no significant frequent indication for bone marrow biopsy. The average marrow cellularity was 50%. effect on migration alone or in combination with sphingosine-1-phosphate. Dyserythropoeisis was the most common finding on biopsy (39%, 19/49). 11 (22% of Conclusions: These results suggest an increase in the -173 C allele frequency in Hodgkin total) of those patients had dysplasia in one or more additional lineages. Other findings lymphoma patients compared to controls. In addition, both MIF and CD74 are expressed included AML (x2), hemophagocytosis (x1), plasma cell myeloma (x1) and polyclonal in tissue from Hodgkin lymphoma patients and in the KM-H2 Hodgkin lymphoma lymphocytosis (x7). The highest diagnostic yield of bone marrow abnormalities was in cells. However, further study is required to elucidate the role, if any, that MIF plays in the pancytopenic patients with two patients with AML, 5 with multilineage dysplasia regulating Hodgkin Reed-Sternberg cell proliferation and migration. (MLD), and 5 with isolated dyserythropoeisis. The five patients with MLD had an average marrow cellularity of 66% with no ringed sideroblasts (n=2). There was no 1236 Hemoglobinopathy Work-Up at Hematology Laboratory of Tufts correlation in bone marrow findings and MELD score, stage of chronic hepatitis or Medical Center: 3-years Experience splenomegaly. S Kondratiev, SM Johnson, H-Y Luo, DHK Chui, IB Rosenwald. Tufts Medical Center, Conclusions: 1. Patients with pancytopenia, even with clinical features of hypersplenism Boston, MA; Boston Medical Center, Boston. and/or documented antiviral medication warrant a bone marrow biopsy for hematologic Background: Hemoglobinopathies are common inherited diseases. No single laboratory evaluation. 2. Degree of bone marrow dysfunction is independent of stage of chronic test has adequate sensitivity and specificity for detection of all hemoglobinopathy hepatitis. 3. Hematologic malignancy is rare in this population yet there is a high syndromes; therefore a group of tests is required. The purpose of this study is to define percentage of morphologic abnormalities. 4. Exercise caution to avoid misdiagnosis a practical approach for the diagnosis of hemoglobinopathies at our Medical Center. of primary myelodysplasia. Design: We retrospectively reviewed all adult cases (18 y and older) submitted for hemoglobinopathy work-up at the Tufts Medical Center between 2005-2007. Initially, 1234 Myelodysplastic Syndromes Following Therapy with Purine cases were stratified based on HPLC and MCV results. Zinc protoporphyrin (ZPP) for Analogues — Therapy Related Myeloid Neoplasms? possible iron deficiency and molecular tests were performed if indicated. M Kluk, C Toomey, E Hochberg, R Hasserjian. Massachusetts General Hospital, Results: A total of 832 cases were referred for hemoglobinopathy work-up during the Boston, MA. 3-y period, initially with CBC, blood smear and HPLC. Among the 619 cases with Background: Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) normal HPLC, 237 had microcytosis (MCV<80). 57 cases had normal ZPP (≤80) may occur as complications of chemotherapy and/or radiation therapy (XRT) and are and were sent for DNA-based molecular testing. 93% (53 cases) had α-thalassemia, classified as therapy-related AML or MDS (t-AML/MDS). These neoplasms typically and only 7% (4 cases) had no globin gene mutation detected. Of the 180 cases with follow alkylating agent or topoisomerase II inhibitor therapy, exhibit characteristic ZPP>80, most were referred for iron studies. 44 cases, however, were sent for molecular cytogenetic findings, and have a very poor prognosis. It is unclear if purine analogues testing, mainly because of target cells found on blood smear. 70% (31 cases) showed (fludarabine, cladrabine, and pentostatin) also can cause AML or MDS, as the features of α-thalassemia (28), α- and β-thalassemia (1), and variant Hb (2). The majority of the myeloid neoplasms occurring after such therapies have not been well-characterized. 382 cases with normal HPLC and MCV≥80 did not require further study, but 3 cases Design: We searched the pathology files from one institution over a four-year period were sent for molecular testing as part of family study. They showed α-thalassemia (2004-2008) for bone marrow samples taken from patients who had been treated with (1), variant Hb (1), and no mutation (1). The group with abnormal HPLC included 213 purine analogues and that were diagnostic of AML or MDS (PA-AML/MDS); cases cases. There were 95 cases with MCV<80 and elevated HbA2 and/or HbF. Molecular treated with any additional chemotherapeutic agents or XRT were excluded. These cases study was done in 35 cases and confirmed abnormalities in 91% (32 cases) including were compared to a control group of 16 cases of AML or MDS arising in patients who β-thalassemia (17), α-thalassemia (6), HbH disease (2), α- and β-thalassemia (2), had been treated with alkylating agents (ALK-AML/MDS). variant Hb (5) and no mutation in 9% (3 cases). Among 22 cases with MCV≥80 and

Results: We identified 5 cases of PA-AML/MDS that occurred in two men and three elevated HbA2 and/or HbF, 12 cases were sent for molecular study and detected HPFH women with a median age of 74; over the same time period, 54 cases of t-AML/MDS (1), HPFH and α-thalassemia (1), β-thalassemia (1), and normal test (9). Furthermore, were diagnosed. All patients had been treated with fludarabine (3 patients) or fludarabine HbS (51 cases), HbC (22), HbS/C (5), HbE (13) were detected by HPLC. Molecular and rituximab (2 patients) for chronic lymphocytic leukemia (4 patients) or marginal study was required in 41 cases based on MCV and morphology and confirmed Hb S, zone lymphoma (1 patient). Compared to the control group of ALK-AML/MDS cases, C, or E (13) frequently in combination with α-thalassemia (26). the PA-AML/MDS cases occurred with a shorter latency, were less likely to show Conclusions: These results confirm the high frequency and complexity of complex karyotypes, and had a longer median survival (although the latter finding did hemoglobinopathies encountered in today’s US populations. The study emphasizes not reach statistical significance). These data are shown in Table 1. the importance of clinical laboratory and molecular testing in making the correct Comparison of AML/MDS cases occurring after purine analogue therapy (PA-AML/MDS) or diagnosis. alkylating agent therapy (ALK-AML/MDS) PA-AML/MDS (n=5) ALK-AML/MDS (n=16) P Presented as AML 0/5 6/16 NS 1237 Expression of Cytotoxic T Cells in Relapsed/Refractory Hodgkin Developed AML 1/5 8/16 NS Lymphoma Complex karyotype 1/5 13/14 0.006 AF Koreishi, DO Persky, H Cui, A Moskowitz, C Moskowitz, J Teruya-Feldstein. Latency in years, median (range) 1 (0.5-3) 3 (0.5-20) 0.015 Memorial Sloan-Kettering Cancer Center, New York, NY; University of Arizona Survival in months, median 16 4 0.13 Cancer Center, Tucson, AZ. Conclusions: AML or MDS occurring after purine analogue therapy is rare, comprising Background: Regulatory T-cells in the inflammatory background of classic Hodgkin <10% of all t-AML/MDS cases diagnosed during the same time period. Although purine lymphoma (cHL) may play a role in inhibition of the antitumor host immune response analogues are included among agents that can cause t-AML/MDS in the 2008 WHO in patients. The presence of cytotoxic and regulatory T-cells such as TIA-1, granzyme Classification of Myeloid Neoplasms, the cytogenetics and short latency of these cases B and FOXP3 in cHL tissues has been shown to correlate with poor overall survival suggest a pathogenesis distinct from t-AML/MDS caused by alkylating agents. PA-AML/ and also shown to be expressed in a handful of relapsed biopsies. We were interested MDS patients also appear to have superior survival to typical t-AML/MDS. in analyzing expression of these markers in a larger series of relapsed/refractory cHL biopsies and determine if there was any correlation with clinical factors and survival 1235 Role of Macrophage Migration Inhibitory Factor (MIF) in Hodgkin outcome. Lymphoma Design: Cases of relapsed/refractory cHL were consecutively ascertained from the MJ Kluk, KM Hoyt, C Bifulco. Massachusetts General Hospital, Boston, MA; Yale New files of MSKCC and use of tissues was approved by HBUC and an IRB waiver. Haven Hospital, New Haven, CT. A TMA of the reactive inflammatory background was constructed using a fully Background: Macrophage migration inhibitory factor (MIF), a 12.5kDa protein automated arrayer Beecher instrument, ATA-27. TMAs were stained in the Pathology discovered in 1966, is a soluble factor secreted by lymphocytes that inhibits macrophage Core Immunohistochemistry Laboratory using established conditions, evaluated by migration. Subsequent studies revealed that various human cell types express MIF and 2 pathologists (AFK and JTF) and scored using the following criteria: 0=negative; its cell surface receptor, CD74. In addition, MIF promoter polymorphisms are associated 1+=<25%; 2+=25-50%; 3+=50-75%; 4+>75%. A total of 64 evaluable cases of relapsed/ with increased MIF expression and correlate with susceptibility and severity of human refractory cHL with tissue core availability and stain results for TIA-1, granzyme B autoimmune disorders and prostate cancer. We investigated the prevalence of MIF and FOXP3 was obtained. Results were linked with clinical factors and statistically promoter polymorphisms in patients with Hodgkin lymphoma and tested the effects of analysed using SPSS and SAS software. MIF on the proliferation and migration of a Hodgkin lymphoma cell line. Results: The cohort was comprised of 22 females and 42 males. Median age was 29 yrs Design: The-173 G to C single nucleotide polymorphism (SNP) in the MIF promoter (range from 17-62 yrs). High expression of TIA-1 (>75%) correlated with extranodal was characterized by sequencing genomic DNA from 60 cases of Hodgkin lymphoma disease (p<0.001), high risk factors (p=0), > 5 and 10 cm bulky disease (p<0.015; p=0 and 97 control cases. Expression of MIF and CD74 was tested by immunohistochemistry respectively), prior radiation (p<0.02), CD20 positivity (p<0.02), and B symptoms of formalin fixed, paraffin embedded tissue samples from Hodgkin lymphoma patients (p=0). TIA1 and granzyme B showed significant inverse correlation with FOXP3 in ANNUAL MEETING ABSTRACTS 273A relapsed/refractory biopsies (p=0). No significant correlation was seen with high LDH, was accomplished by using PCR primers to amplify exon 12 of JAK2 followed by and relapse in prior radiation site. High expression of TIA1 (>75%) correlated with DNA sequencing. poor overall survival (p<0.007), and event free survival (p<0.05). Results: Three previously recognized point mutations of exon 12 were found in 6 Conclusions: This study confirms the observation of increased TIA-1 and decreased patients; 1 patient had a novel exon 12 mutation. All patients had low Epo. CBC showed FOXP3 in a larger series of relapsed/refractory cHL biopsies initially reported in an erythrocytosis in all patients; the Hgb ranged from 18.3-22 g/dl (med=19.4 g/dl). a handful of cases. Cytotoxic and regulatory T-cells play an important role in the Neutrophilia and/or basophilia were seen in 4 cases; 2 patients had thrombocytosis. The antitumor host immune response in relapsed/refractory cHL and shows clinical BMs were hypercellular (45-100%; med=90%). An erythroid hyperplasia was present in prognostic significance. all cases with M:E ratios ranging from 1:1 to 1:5 (med=1:2). Erythroid maturation was left-shifted in 5 cases, and granulocytic hyperplasia was present in 4 cases. Reticulin 1238 Clonal T-Cell Receptor Gene Rearrangements in Traumatic fibrosis was not increased except in 1 patient who evolved into post-polycythemic Ulcerative Granuloma with Stromal Eosinophilia myelofibrosis. No prominent clusters of large, bizarre megakaryocytes that characterize JAK2V617F positive PV were seen in the exon 12 cases. A spectrum of megakaryocytes CL Kossover, S Budnick, S Li. Emory University, Atlanta, GA. was identified; small to medium-sized forms predominated over larger forms. Atypical Background: T-cell monoclonality is not confined to lymphoproliferative disorders megakaryocytic lobulation and abnormal megakaryocytic chromatin distribution were but additionally has been described in benign reactive conditions. Traumatic ulcerative identified in all cases. Loose clusters of megakaryocytes could be found but were subtle granuloma with stromal eosinophilia (TUGSE) is a typically self-limited lesion of in all cases. Immunohistochemical stain for CD61 confirmed the above findings. the oral mucosa with unclear pathogenesis. Morphologically these lesions consist of Conclusions: The BMs from patients with exon 12 mutations show an erythroid dense and deeply infiltrative mixed inflammatory cells with a prominent eosinophilic hyperplasia and lack the prominent clusters of large, bizarre megakaryocytes that component as well as occasional large atypical mononuclear cells. Although largely characterize classic JAK2V617F positive PV. However, subtle megakaryocytic atypia regarded as clinically benign lesions, recently a few case reports have demonstrated can be identified. Because these cases lack the classic myeloproliferative morphology, molecular evidence of T-cell clonality within these lesions, suggesting that a subset of the BMs from patients with exon 12 mutations may be initially difficult to diagnose. TUGSE might be a low-grade T-cell lymphoproliferative disorder. However, to date Clinically suspected PV with low serum Epo and absent JAK2V617F together with a large study examining the prevalence and clinical significance of T-cell clonality in the BM finding of erythroid hyperplasia and subtle megakaryocytic atypia/clustering TUGSE’s has not been conducted. should prompt an evaluation for an exon 12 mutation. Design: To evaluate by molecular methods the presence of T-cell clonality in TUGSE’s and to investigate the clinical significance of these findings. Results: We performed polymerase chain reaction (PCR) analysis for T-cell receptor 1241 Chromosomal Aberrations in Thymoma: Review of the Literature (TCR) gene rearrangements on 37 cases of TUGSE selected from the 2002-2008 files and Report of Five Additional Cases of the Department of Pathology at Emory University. Clonal TCR gene rearrangements AR Laury, V Nose, P Dal Cin. Brigham & Women’s Hospital, Boston. were demonstrated in 7 of the 37 cases. After blind review of the morphology of all Background: Thymic tumors are rare, composing 0.1% of all adult neoplasms, and 37 cases, two of the clonal cases were diagnosed as atypical lymphoid proliferations among them thymomas, a benign lesion, are the most common. Although cytogenetics based on the findings of cohesive clusters and sheets of atypical large mononuclear cells of thymoma have been reported only as infrequent case reports, chromosome 6 seems with abundant mitoses. The remaining 5 clonal cases were morphologically benign. to be the most frequently involved chromosome. Clinical follow-up was available for 5 of the 7 clonal cases for an average period of 1 Design: We reviewed our electronic reporting system (PowerPath) for in-house cases year and 9 months after the initial biopsy/excision, with no evidence of local recurrence of thymoma with cytogenetic reports between December 2004 and June 2008. Consult or development of systemic T cell lymphoma. cases, tissue submitted from outside hospitals, and thymic carcinomas were excluded, Conclusions: The majority of TUGSE’s demonstrating a clonal TCR gene rearrangement but biopsies, recurrent and metastatic thymomas were included. 61 cases of thymoma are morphologically and clinically benign. Without morphologic and/or clinical were identified; 31 were submitted for cytogenetic analysis, and 18 were successfully evidence of a lymphoproliferative process, T cell clonality in these lesions is not an karyotyped. indication of malignancy. More experience is needed with these lesions for a conclusive Results: Of the eighteen karyotyped thymomas, abnormal karyotypes were identified in statement regarding their long-term biologic behavior and the best overall clinical five. Three of the five cases had chromosome 6 aberrations, e.g. r(6) in two cases and an management. unbalanced rearrangement, der(1;6)(q10:q10), resulting in loss of the short arm (p) of chromosome 6. No rearrangements of chromosome 6 were detected by GTG-banding 1239 Basal Activation of NFkB Protein Signaling Pathway in Follicular in the remaining two karyotypically abnormal thymomas. Lymphoma In Vivo Conclusions: Chromosome aberrations have been so far observed in 14 thymomas, including our 5 cases reported here, with chromosome 6 aberrations being relatively AE Kovach, MK Humsi, ES Jaffe, KR Calvo. National Cancer Institute, NIH, Bethesda, frequent (9 of 14 cases). Ring of one chromosome 6, r(6), is the most common MD. abnormality encountered (4 of 8 cases) , followed by deletion of either the short Background: Follicular lymphoma (FL) is an indolent tumor characterized by the arm (3 cases) or of the long arm (2 cases) . In summary, loss of 6p and 6q are the constitutive expression of the Bcl-2 oncoprotein. We previously reported significantly commonest findings in all subgroups of thymoma, and there is no correlation, thus far, elevated levels of the Th2 associated cytokine IL-4 and basal activation of the with histologic subtype. downstream mitogen activated protein (MAP) kinase Erk in vivo in FL (Calvo K, Blood, 2008). In the current study we sought to investigate the activation state of the Cytogenetic Aberrations in Thymoma Age Gender Thymoma Tybe Karotype NFkB protein signaling pathway which is thought to play an important role in B-cell 1 77 M B2 44,XY,+X,inv(2)(p25q13),del(6)(q15),-8,-16,-17 activation, survival and lymphomagenesis. Here we report post-translational basal 48-49,XX,+del(X)(q24), +i(5p), +?del(7) (q22), der(11) 2 76 F AB activation of several key NFkB signaling proteins in FL in vivo. t(1;11) (q23;25), t(11;?) (p15;?),-18,+r Design: Whole tissue lysates of FL (N=36) and FH (N=16) from fresh-frozen lymph node 3 71 M NOS 46,XY, r(6) 4 70 F NOS 46,XX,t(15;22)(p11;q11) specimens were analyzed by Western blot. FL samples included 12 cases each of grade 1 57,XY,+i(1)(q10),+add(4)(q12),+7,+8,+9,+14,+14,+15, 5 61 M AB (G1), grade 2 (G2), and grade 3a (G3). Blots were probed for the active phosphorylated +16,der(17)t(9;17)(q12;p13),+20,+22 form of NFkB p65 Ser536 and NFkB p105 Ser933. Expression of total c-Rel was also 6 63 F A 46,XX,del(6)(p22p25) determined by Western in a random subset of samples (18 FL, 8 FH). 7 56 F AB 45,XX,pseudic(16;12)(q11;p11.2) Results: Thirty two of 36 FL cases (88.9%) showed prominent phosphorylation of 8 74 M AB 46,XY, r(6) 9 62 M AB 46,XX,del(6)(p22p25) NFkB p65 Ser536 with stong bands on Western blot, in contrast to only 8 of 16 FH 10∗ 69 M AB/B2-B3 46,XY,t(2;16)(q3?1;q24),r(6)(p2?2q2?5) (50%) with overall weaker bands. A striking difference between the groups was also 11∗ 55 F B1 45,X,-X,r(6)(p2?1q2?5) observed in phosphorylation of NFkB p105; 25 of 36 FL cases (69.4%) demonstrated 12∗ 51 M AB 44,XY,del(15)(q2?4),-21,-22 phosphorylation of NFkB p105 Ser 933 versus 4 of 16 FH cases (25%). Total c-Rel 13∗ 42 M B2-B3 50,XY,+1,+1,der(1;6)(q10;q10),i(1)(q10),+7,+8,+14 45-52,-X,Y,psu dic(11;1) (p15;p11),complex 14∗ 46 M B3 protein expression was similar between FL and FH, with most FL cases (34 of 36, 94.4%) karyotype[cp15] and all FH cases (16 of 16, 100%) yielding strong detectable bands. ∗ Current case Conclusions: These findings demonstrate that NFkB protein signalling networks are basally activated in FL in vivo. Combined with our previous studies showing increased IL-4 protein concentrations and basal activation of Erk in FL, these findings suggest that 1242 Dual Color Chromogenic In Situ Hybridization (CISH) Is a complex crosstalk exists between multiple activated pro-survival signaling networks in Rapid, Specific and Sensitive Method for IdentifyingMYC Translocations FL, which may have implications for molecularly targeted therapies. in Paraffin-Embedded Tissue ME Law, A Dogan, ED Remstein. Mayo Clinic, Rochester, MN. Background: The identification ofMYC translocations is important for the classification 1240 A Bone Marrow Morphologic and Immunohistochemical Study of and management of lymphomas. Time is often of the essence as lymphomas that possess Polycythemia Vera (PV) with Exon 12 Mutations MYC translocations proliferate rapidly and often require urgent therapy. Fluorescent MA Lakey, JD Hoyer, A Tefferi, A Pardanani, PL Nguyen, TL Lasho, CA Hanson. Mayo in situ hybridization (FISH) using breakapart probes is widely used to determine the Clinic, Rochester, MN. presence of this translocation. However, as FISH requires the use of a microscope fitted Background: The diagnosis of PV requires the integration of clinical features, laboratory with epifluorescence, samples must be sent to specially-equipped laboratories, which findings (including serum erythropoietin (Epo) levels), bone marrow (BM) morphology, can be time-consuming and cumbersome. Also, it is difficult to visualize morphology and JAK2 analysis. The JAK2V617F (exon 14) is found in 95% of PV. Mutations in exon using fluorescence microscopy. A viable alternative is chromogenic in situ hybridization 12 have also been described in PV. These 2 mutations account for virtually all cases of (CISH), which resembles FISH except chromogen/enzyme reaction products that are PV. A thorough BM study of PV patients with exon 12 mutations has not been done. visible using light microscopy instead of fluorescent tags highlight the appropriate DNA Design: We identified 7 PV patients with exon 12 mutations. JAK2V617F studies sequences. Our aim was to compare the utility of CISH to FISH in identifying MYC were done using an allele-specific PCR analysis. JAK2 exon 12 mutation screening translocations in paraffin-embedded tissue. 274A ANNUAL MEETING ABSTRACTS

Design: Sections from 10 formalin fixed paraffin embedded (FFPE) samples including (M:F-1:1.8 median age 73; range 55-97); seven(20%) had complex and 28(80%) had 6 high-grade B-cell lymphoma samples, 3 cell line cell blocks containing 90%, 10%, simple karyotypes. Structural abnormalities were noted in 7 cases, numerical in 19, and and 0% Raji (t(8;14)/IGH-MYC-positive) cells and 1 FFPE normal tonsil were cut at a combination of both in 9 cases. Multiple related (n=4) and unrelated (n=1) clones were a thickness of 3-5uM and placed onto SuperFrost Plus microscope slides. FISH using identified at diagnosis in 11% of MZL, the former representing clonal evolution. Overall, a dual color breakapart Myc DNA probe (Dako) was performed and results tabulated. the most common aberrations were those of: chromosome (chr) 3(n=16,36%), including Slides were then de-identified and, using Dako DuoCISH kit, the FISH signals were trisomy 3 (n=13,29%), in 3(30%) NMZL, 7(29%) EMZL, and 6(55%) SMZL; chr 1 tagged with anti-FITC-HRP (blue chromogen) and anti-Texas Red-AP (red chromogen). (n=12,27%) in 3(30%) NMZL, 5(21%) EMZL, and 4(36%) SMZL; chr X (n=7,16%) Slides were visually screened to determine the presence or absence of split signals and in 3(30%) NMZL and 4(17%) EMZL, and IgH rearrangements (n=7,16%) in 1(10%) after the results were recorded the slides were re-identified. NMZL, 3(13%) EMZL, and 3(27%) SMZL. However, variability in the chr regions and Results: loci involved was noted in some cases. The IgH partner loci included 3q12, 6p21.1, 8q24, Specimen FISH CISH 9p13, and 17p11.2. BCL6 rearrangements were noted in 2 cases. Recurrent deletions Lymphoma (Cmyc translocation-) Normal (1/1) Normal (1/1) at 7q22-31 and 14q21-2 were only seen in 3 and 2 cases of SMZL, respectively. FISH Lymphoma (Cmyc translocation+) Abnormal (4/4) Abnormal (4/4) analysis in 41 cases identified additional aberrations in 4 MZL with normal and 3 with Raji (90% and 10%) Abnormal (2/2) Abnormal (2/2) abnormal karyotypes. MALT1 translocations were restricted to EMZL (n=5, 21%). Raji (0%) Normal (1/1) Normal (1/1) Normal tonsil Normal (1/1) Normal (1/1) Conclusions: We observed both shared and distinct karyotypic aberrations amongst MZL subtypes. Trisomy 3 and chr 1 abnormalities were the most common and a novel Conclusions: In this small cohort, dual color CISH and dual color FISH were both IgH partner locus was identified (17p11.2). Multiple clones were not infrequent in our 100% specific and 100% sensitive in determining the presence ofMYC translocations series of MZLs. FISH analysis aids in correct classification by identifying subtype using breakapart probes. CISH can also be performed rapidly on FFPE tissue and associated chromosomal aberrations. can be screened and compared to morphology by the pathologist on a standard light microscope. Thus, in addition to its previously demonstrated utility in determining gene region amplification, FFPE CISH is useful in identifying translocations in a rapid, 1245 Inhibition of HDM4 Induces p21 Expression in Mantle Cell specific and sensitive manner on routine FFPE specimens. Lymphoma M Liang, X Han, M Fernandez, M Nguyen, GZ Rassidakis, I Drakos, LJ Medeiros, CE Bueso-Ramos. University of Texas, M.D. Anderson Cancer Center, Houston, TX. 1243 Insulin-Like Growth Factor-II mRNA-Binding Protein Expression Background: Alterations of the p53-murine double minute 2 (MDM2)/MDM4-pathway in Leukemias and Lymphomas are thought to contribute to the pathogenesis of a variety of malignant neoplasms. MP LeGolvan, S Hao, BA Woda. UMassMemorial Healthcare, Worcester, MA. Haploinsufficiency of MDM4 reduced lymphomagenesis in Eµ-myc transgenic mice, Background: Leukemias and lymphomas represent a heterogeneous group of neoplasms. a model of non-Hodgkin’s lymphoma. p53 expression leads to increased expression of Diagnostic markers are already well established in most of the individual neoplasms; p21, a negative cell cycle regulatory protein that inhibits cyclin D1 activity. In mouse however, prognostic markers, while growing in number, are still being elucidated. The model and cell line studies, MDM2 and MDM4 have been shown to synergistically family of Insulin-like growth factor II mRNA-binding protiens (IMP), which includes promote proteosomal mediated degradation of p21 and p53. MDM4 also inhibits IMP1, IMP2, and IMP3, are oncofetal proteins expressed in embryogenesis. While p53-mediated transcriptional activation of p21. The aim of this study was to assess for absent or present in very low levels in adult tissue, they have now been found to be Human homolog of MDM4 (HDM4) expression and its effect on p21 in mantle cell present in a number of cancers. IMP3 has been the most widely studied and recently lymphoma (MCL). has been shown to hold prognostic significance. Yet, IMP3 has had limited study in Design: We assessed HDM4 expression using immunohistochemical methods in reactive leukemias and lymphomas, and neither IMP1 nor IMP2, to our knowledge, has been lymph nodes and MCL. To characterize the differential upregulation of HDM4 in MCL, addressed in these neoplasms. We focused on a wide range of hematopoeitic neoplasms HDM4 mRNA expression was assessed by quantitative real-time polymerase chain to evaluate the staining patterns and possible utility of these newly emerging markers reaction (RT-PCR) in MCL cell lines (Granta 519, Z-138, SP-53, and Mino) and leukemia for diagnostic or, more likely, prognostic use. patient peripheral blood samples. After using small interfering RNA (siRNA) to inhibit Design: 86 cases of representative leukemias and lymphomas from 2004-2008 were HDM4 in MCL cell lines, p21 protein levels were measured by immunocytochemical collected from the archives of UMassMemorial Medical Center and stained with and Western blot analysis. Activation of the p53-apoptotic pathway was assessed by IMP1(Aviva systems BIO), IMP2 (Affinity Bioreagents), and IMP3 (DAKO) from caspase-3 expression by immunocytochemical staining. a previously published protocol using a DAKO autostainer. 9 acute lymphoblastic Results: HDM4 nuclear expression was observed in 18 of 19 MCL, but was negative in leukemias (ALL), 9 acute myeloid leukemias (AML), 8 Hodgkin lymphomas, classic mantle zone B-cells of reactive lymph nodes (n=19) (p<0.01). HDM4 overexpression (CHL), 9 chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL), 7 was confirmed by RT-PCR in all 4 MCL cell lines and 6 MCL patient specimens. Both diffuse large B-cell lymphomas (DLBCL), 10 follicular lymphomas (FL), 8 mantle splicing variants, HDM4-S (containing only p53 binding domain) and full length cell lymphomas (MCL), 7 multiple myelomas (MM), 10 marginal zone lymphomas (FL)-HDM4, were increased compared with normal CD19+ B-cells. The HDM4-S to (MZL), and 9 T-cell lymphomas (PTCL) were utilized. HDM4-FL ratio was significantly increased in MCL compared with normal CD19+ Results: B-cells (p<0.05). After introducing siRNA inhibition of HDM4 in two MCL cell lines, Percentage of Positive Staining of IMP1,2,3 SP53 and Mino, p21 protein levels were induced as shown by Western blot analysis. IMP1 (%) IMP2 (%) IMP3 (%) ALL 33 12.5 66 Immunocytochemical analysis also showed increased p21 expression and active AML 0 100 0 caspase-3, the latter indicating increased apoptosis. CHL 100 50 100 Conclusions: HDM4 is overexpressed in MCL and, at least in part, may exert its effect CLL 0 0 11.1 by suppressing p21 expression thereby enhancing cell cycle progression. Inhibition of DLBCL 100 100 100 HDM4 may serve as a potential therapeutic remedy in MCL by inhibiting cell cycling FL 20 10 100 MCL 0 12.5 37.5 and inducing apoptosis. MM 0 0 0 MZL 0 0 0 1246 Aberrant CD56 Expression in Myelomonocytic Cells Correlates PTCL 0 77.8 25 with Chromosomal Aberrations in Chronic Idiopathic Myelofibrosis (CIMF/ Conclusions: 1. IMPs are expressed in 100% of DLBCL. 2.IMP1 and IMP3 are PMF) useful surrogate markers for CHL when CD30 expression is inconclusive. 3. IMPs are P Lin, B Feng, JL Jorgensen, S Verstovsek. UT MD Anderson Cancer Center, Houston, variably expressed in ALL, PTCL and MCL. Further studies are needed to investigate TX. the possible prognostic significance in these tumors. 4. IMPs are not expressed in MM, Background: CIMF/PMF often exhibits granulocytic and megakaryocytic hyperplasia. and rarely expressed in low grade B-cell lymphomas with the exception of FL. 5. IMP3 Flow cytometry (FC) immunophenotypic features of myeloid cells in CIMF are not well is expressed in all FL and DLBCL. Further study is needed to investigate the possible documented. The aim of the study is to assess the frequency of the aberrant phenotypes in role in diagnosis of lymphomas of follicular center cell origin. 6. IMP2 is expressed in myelomonocytic cells in CIMF and correlate the results with JAK2 and cytogenetics. all AML but IMP1 and IMP3 were not detected. Design: 79 CIMF cases with JAK2 status and karyotypes characterized were identified from 2000 to 2007. Bone marrow aspirates of 35 cases were analyzed by FC with a 1244 Cytogenetic Analysis of Marginal Zone Lymphoma Reveals panel that included CD56. The percentage of myelomonocytes positive for CD56 and Shared and Distinct Aberrations among Different Subtypes their mean fluorescence intensity (MFI) were assessed and compared with the values M Levin, V Murty, B Alobeid, G Bhagat. Columbia University, NY, NY. for 17 cases of MDS (RCMD or RAEB-1) with myelofibrosis, and 20 non-CMPD Background: Marginal Zone Lymphomas (MZLs) are subdivided into three distinct controls. The results were correlated with hematologic parameters, prior therapy, JAK2 categories: nodal (NMZL), extranodal (EMZL), and splenic (SMZL) according to the mutation and cytogenetics. WHO classification, which share similar histologic and phenotypic features, but differ Results: Aberrant phenotypes were observed in 26 of the 35 (80%) CIMF cases analyzed, with regard to genetic alterations. To gain further insights into the relationship between including CD56 overexpression in granulocytes and/or monocytes, and/or decreased these subtypes, we retrospectively reviewed cytogenetic data of all MZLs evaluated at side scatter in granulocytes. The MFIs of the CD56 expression by both granulocytes and our institute over a 10-year period. monocytes in CIMF cases are significantly higher than those of the controls (p<0.001) Design: G-band karyotypes of all MZLs submitted for cytogenetic analysis were and are comparable to or higher than that of MDS with myelofibrosis.JAK2V617F and examined. In addition, FISH with a panel of probes for CEP3, CEP18, MALT1 and IgH chromosomal aberrations were found in 18 (51.4%) and 15 (42.9%) cases, respectively. was performed. Morphology was assessed using H&E stained sections and phenotype The JAK2V617F alleles burdens range from 19%-93% (Median 45%). Aberrant CD56 was determined by flow cytometry and immunohistochemical staining. Cases were expression statistically correlates with cytogenetic aberrations. categorized according to the current WHO classification. Results: Forty-five of 55 MZLs (from 53 patients) had informative karyotypes (10 normal and 35 abnormal). These MZL subtypes comprised 10 NMZL (M:F-1:1.5 median age 72; range 50-84), 24 EMZL (M:F-1:1.3 median age 66; range 36-83) and 11 SMZL ANNUAL MEETING ABSTRACTS 275A

Table 1. CD56 Mean Fluorescent Intensity (MFI) of Granulocytes and Monocytes 1249 CD81 Protein Is Expressed in Normal Germinal Center B-Cells Correlates Positively with Chromosomal Abnormalities in CIMF/PMF and in Subtypes of Human Non-Hodgkin Lymphomas Gran CD56 MFI Mono CD56 MFI SSC MH (Mean+SD) (Mean+SD) (Mean+SD) RF Luo, S Zhao, R Tibshirani, IS Lossos, R Advani, D Gratzinger, A Wong, N Talrega, CIMF: Abnormal karyotype (n=15) 28.01+27.45 28.35+32.01 596.7+175.5 R Levy, S Levy, Y Natkunam. Stanford University, Stanford, CA; University of Miami, CIMF: Normal karyotype (n=20) 12.00+9.73 36.65+27.97 697.2+119.7 Miami, FL. CIMF: JAK2v617+ (n=18) 27.12+27.83 27.81+32.22 693.0+113.0 Background: CD81 is a tetraspanin cell surface protein that regulates CD19 expression CIMF: JAK2v617F- (n=17) 11.23+7.88 38.04+26.37 672.9+158.5 in B lymphocytes and enables hepatitis C virus infection of human cells. Using a novel MDS/MF (n=17) 10.60+8.96 38.04+26.37 775.1+195.7 Control Group (n=20) 4.42+1.14 9.15+4.91 920.3+78.4 statistical methodology, the CD81 gene and germinal center (GC) molecules LMO2 and SSC MH--Side scatter median height BCL6 were found to be highly associated with clinical outcome in patients with diffuse Conclusions: As seen in MDS with myelofibrosis, myelomonocytic cells in more than large B-cell lymphoma (DLBCL) in gene expression profiling studies. Since no data was half of CIMF cases exhibit aberrantly increased CD56 expression. CD56 overexpression available on the expression of its cognate protein, we undertook the characterization of correlates strongly with the presence of cytogenetic aberrations but not with JAK2 CD81 protein in normal and neoplastic hematolymphoid tissues. mutation. Design: Immunohistochemistry for CD81 was performed using a monoclonal anti- CD81 antibody (clone 1D6) on tissue microarrays of over 1000 normal and neoplastic tissues. Expression patterns of GC and non-GC markers in DLBCL were compared 1247 Cytoplasmic Expression of Nucleophosmin Detected by using hierarchical clustering algorithms. RCHOP-treated DLBCL patient biopsies (106) Paraffin Immunohistochemistry Predicts Nucleophosmin Gene Mutations were interrogated with CD81 and the results were correlated with overall survival (OS) in Patients with Acute Myeloid Leukemia and Normal Karyotype and progression free survival (PFS). J Lou, C Qi, W Xu, J Brandwein, S Kamel-Reid, H Chang. University Health Network Results: CD81 protein was found in normal GC B-cells and in subtypes of non-Hodgkin and University of Toronto, Toronto, Canada. lymphomas, including those occurring more frequently in patients with hepatitis C Background: Mutations in nucleophosmin (NPM1) exon 12 and the resulting infection. Staining was infrequent in myeloma, Hodgkin lymphoma, myeloid leukemia, delocalization of NPM into the cytoplasm are the most frequent cellular events occurring hematopoietic precursors in normal bone marrow, and most non-hematolymphoid in 50-60% of acute myeloid leukemia patients (AML) with normal karyotype. Recent tissues. There was no significant association between CD81 expression and OS or PFS studies have suggested that AML patients with NPM1 mutations make up a subgroup (p>0.05). However, in hierarchical cluster analyses of DLBCL, CD81 protein expression with a better prognosis. However, there have been discrepancies about the ability of aligned most closely with that of LMO2, HGAL, BCL6 and CD10. immunohistochemical detection of cytoplasmic NPM (NPMc+) to predict NPM1 CD81 Expression in Selected Lymphomas gene mutations. Here we investigated the correlation between cytoplasmic NPM Lymphoma Subtype Total Positive % Positive immunopositivity and NPM1 gene mutations and their prognostic significance. Follicular 136/164 85% Design: Paraffin-embedded bone marrow specimens from AML patients with normal Diffuse Large B-cell 123/196 63% karyotype were immunostained with a monoclonal mouse anti-human NPM antibody Marginal Zone 23/27 85% (clone 376) that detects both mutant and wild-type NPM1. A case was classified as Mantle Cell 12/18 67% SLL/CLL 6/33 18% NPMc+ if the majority of the leukemic cells were positive for cytoplasmic NPM. NPM1 Lymphoplasmacytic 4/5 80% mutation and FLT3-ITD (internal tandem duplication) status were evaluated by multiplex Precursor B-Lymphoblastic 5/9 56% RT-PCR. Clinical and laboratory features were obtained by chart review. Precursor T-Lymphoblastic 4/10 40% Results: Of the 77 patients, NPM1 mutations were detected in 43 (55.8%) and FLT3- Peripheral T-cell 11/14 79% ITD in 18 (23%) cases. NPM1 mutation was correlated with FLT3 –ITD (p=0.006), Anaplastic Large Cell 4/8 50% NK Cell 9/94 10% CD33 (p=0.01) and lack of CD34 expression (p=0.0001), but not with age, gender, high Multiple Myeloma 13/101 13% WBC count, or FAB subtype. Of the 59 specimens immunostained for NPM, all 31 Hodgkin 5/98 5% cases (53%) that were shown to be NPMc+ by IHC carried NPM mutations; none of Conclusions: CD81 protein is expressed in a GC-associated manner and is present in the 28 cases (47%) with nucleus-restricted NPM1 (NPMc-) carried NPM1 mutations a subset of non-Hodgkin lymphomas including DLBCL. Although its expression as a (p<0.0001). The complete remission (CR) rate was similar between patients with or single marker was not associated with outcome in RCHOP-treated DLBCL patients, it without NPM1 mutations (79% vs 70%, p=0.36). However, a NPM1 mutation positive, is an additional novel GC-associated marker that warrants further study in multi-gene FLT3-ITD negative subset had a favorable event free survival (EFS) (median not models used to stratify DLBCL patients into prognostic subcategories. reached vs. 8.1 months, p=0.05) and a trend to a longer overall survival (OS) (median not reached vs 14.8 months, p=0.13) than patients without this genetic profile. FLT3- ITD positive patients had a worse EFS (5 months vs not reached, p<0.0001) and OS 1250 Decreased Surface Immunoglobulin Light Chain Expression by (8 months vs not reached, p<0.0001) than those without this genetic lesion regardless Mature B-Lymphocytes in Serous Fluids: An Assessment of Frequency of NPM1 mutations status. and Clinical Significance Conclusions: In the absence of FLT3-ITD, NPM1 mutation confers a favorable clinical MS Madsen, JH Lunde, R Bryant, MR Lewis. Univ. of Vermont, Burlington. outcome in normal karyotype AML. NPM IHC accurately predicts NPM1 mutations and Background: The absence of surface immunoglobulin light chain (sLC) expression warrants inclusion in the routine diagnostic and prognostic work-up of AML. by mature B-cells has been found in some cases to be associated with B-cell lymphoproliferative disorders (B-LPD), although this finding is not entirely specific. While the studies addressing this topic to date have largely focused on lymph nodes, 1248 Assessment of Bone Marrow Plasma Cells by Flow Cytometry, we have observed decreased or absent sLC expression in a subset of serous fluid Fluorescence In Situ Hybridization, and Bone Marrow Morphology in 224 specimens. As the clinical implications of such a finding are uncertain, our experience Cases for Diagnosis of Plasma Cell Neoplasms was reviewed. G Lu, WR Stull, W Chen, XX Zhang. UT MD Anderson Cancer Center, Houston, TX; Design: Flow cytometric analyses of serous fluid specimens received over a 4.5 year Medical Laboratory Services, Murrieta, CA; USLabs, Irvine, CA. period (1/1/04-6/30/08) were reviewed, yielding 112 specimens from 98 patients Background: Bone marrow (BM) plasmacytosis is an important criterion for the (M:F = 52:46; median age 70 y, range 16-99) for which sLC data were available. The diagnosis of plasma cell neoplasms (PCN). Although assessment of plasma cell (PC) specimens were predominantly pleural fluids (90/112 = 80%), with smaller numbers of percentages by BM morphologic examination (BMME) and flow cytometry (FC) are peritoneal (15) and pericardial (7) fluids. sLC expression was classified as monotypic both important diagnostic tools, the results are often discrepant. Fluorescence in situ or not monotypic, and level (brightness) of sLC expression was categorized as absent, hybridization (FISH) assay using a multiple color panel is a sensitive method to detect minimal/dim, or typical. genomic abnormalities in malignant plasma cells, and has important prognostic value. Results: Nineteen of 112 specimens (17%) showed monotypic sLC, 20 (18%) contained The aims of this study: (1) to assess the concordance of plasma cell percentages in bone too few B-cells to permit definitive characterization, and 56 (50%) showed polytypic marrow aspirates analyzed by BMME and by FC in cases of PCN; and (2) to determine sLC expression with typical brightness, while the remaining 17 (15%) contained B-cells if there is an optimal cut-off value for percentage of plasma cells assessed by FC in exhibiting dim or absent sLC expression (6 minimal/dim, 11 absent). 15/17 were pleural order to select cases for further analysis by FISH. fluids, and two were pericardial. Two of these 17 cases showed involvement by lymphoid Design: Two hundred and twenty-four bone marrow aspirates with myeloma as indication neoplasms; one showed involvement by a CD5+ B-LPD in a 57 y.o. man who had been were included in this study, and demonstrated monoclonal plasma cell population by previously diagnosed. The second case, involving pleural fluid from an 80 y.o. man 4-color flow cytometry analysis. They were also evaluated by FISH with a probe panel with no prior B-LPD diagnosis, contained B-cells that were large by forward scatter to detect +3, +5, +7, +11, del(13)(q14), del(17)(p13), and IgH locus rearrangements. and cytologically abnormal, fitting with involvement by DLBCL. Four other cases Of the 224 cases, 157 were further studied by BMME. Plasmacytosis was defined as involved patients with prior or concurrent diagnoses (CLL, DLBCL, B-ALL, classical >3% PC on morphologic examination of Wright-Giemsa-stained aspirate smears, with Hodgkin’s) but no evidence of fluid involvement. In the other 11 cases exhibiting 3-9% PC defined as mild, 10-30% as intermediate, and >30% as severe. dim or absent sLC, no evidence of prior, concurrent, or subsequent involvement by a Results: Of the 157 cases, 40 showed mild plasmacytosis, 49 intermediate, and B-LPD was identified. 68 severe; whereas 99 (63.1%) were FISH-positive including 6 cases with mild Conclusions: 15% of serous fluids contained B-cells that lacked monotypic sLC plasmacytosis (15%), 29 intermediate (59.2%), and 64 severe (94.1%). Plasma cells expression but showed decreased or absent sLC. Among this subset, six cases (35%) less than 2% by FC were found in 52 cases that were also evaluated by BMME. Thirty- were seen in patients with prior or concurrent diagnoses of B-LPD, while eleven (65%) three of the 52 showed morphologic plasmacytosis, including 21 that were mild, 11 lacked evidence of prior or subsequent involvement by a B-LPD. In our series, absent intermediate, and 1 severe. Eight of the 33 cases were FISH-positive, accounting for a positive history or concurrent B-LPD diagnosis, the finding of decreased or absent 8.1% of the total FISH-detectable cases. Three of the 8 cases demonstrated adverse sLC without monotypia was not associated with subsequent B-LPD. genomic abnormalities including del(13q) and non-t(11;14) IgH rearrangements. Conclusions: Our results suggest that flow cytometry significantly underestimates plasma cell percentages in bone marrow. Using a PC cutoff of as low as 2% assessed by flow cytometry as an indication for further FISH analysis, at least 8% of FISH- detectable cases would have be missed. 276A ANNUAL MEETING ABSTRACTS

1251 Multiple Myeloma Patients with 13q14 Deletion Show a (complex methodology & high cost); we evaluated expression of cyclin D2 and ITβ7 Significant Downregulation of MicroRNA Gene Mir16a but Not of Mir15a by immunohisto-chemistry (IHC) in a cohort of MM pts and correlated our results A Malik, SU Bozkurt, AA Toor, S Alkan. Loyola Univ Med Ctr, Maywood, IL. with clinical progression. Background: Multiple myeloma (MM) is an incurable malignancy characterized Design: Diagnostic BM biopsy tissue (FFPE) of MM pts (treated with a dexamethasone by a proliferation of clonal plasma cells. The plasma cells often harbor karyotypic based regimen & ASCT) were stained with CD138; CD45, CD20; κ,λ, cyclin D2 and abnormalities such as deletion of chromosome 13q14 (13q14-del), commonly detected ITβ 7 antibodies; using standardized techniques & appropriate controls. Two pathologists by cytogenetics and FISH studies in of patients with MM. Despite well known (blinded to clinical outcome), scored staining as positive (>50% MM cells, irrespective association of 13q14-del with poor prognosis in MM, the exact role of genes associated of intensity) or negative. FISH studies for del 13; t(4;14) & del 17p were performed. with this locus in MM is unknown. MicroRNAs (miRNAs) are naturally occurring, The clinical parameters, response criteria and survival outcomes (PFS and OS) were highly conserved families of transcripts (18–25 nt in length) that are processed from defined according to the international uniform response criteria. OS [amp time to larger hairpin precursors and play an important role in cellular growth in normal and progress (TTP) was determined by Kaplan-Meier method. Cox regression method was neoplastic cells. Recent evidence indicates that miRNAs can also function both as a used for multivariate analysis. tumor suppressor and oncogene. Results: 79 pts between 27-72 yrs of age ((median 54.4) were included. 17.7% had ISS Design: A total of 23 newly diagnosed MM patients with standard karyotyping and stage III, median β 2-microglobulin was 3.29 mg/L (1.16–37.05). Del13q, t(4;14) and FISH assays with viable frozen material for miRNA analysis was available. 7 patients del17p13 were detected in 35.9%, 13.6% and 17.3%, respectively. Post ASCT, 68.8% had 13q14-del with standard karyotyping.All 7 and additional 1 more patients showed achieved a CR or VGPR with mTTP of 2.3 yrs (C.I. 1.8-2.7) and mOS of 8.1 yrs (C.I. 13q14 del by FISH using a probe D13S319 were selected for miRNA analysis. Following 5.3-10.7 ). Expression of cyclin D2 was seen in 76.6% cases while integrin-β7 was positive selection of plasma cells by CD138 purification, miRNA were isolated and noted among 17.7% of samples. In univariate analysis integrin-β7 predicted for a shorter subjected to Taqman reverse transcpitase real time PCR (RT-PCR). Changes in miRNA TTP(mTTP 0.9 vs 2.4 yrs, P=0.008) but not OS (P=0.570), whereas the expression of mir16a and mir15a expression in paired sample were assessed in triplicate ABI 7500 cyclin D2, did not affect TTP or OS. In multivariate analysis (age, β-2 microglobulin, instrument. del13q, integrin β7 and cyclin D2) presence of del13 or integrin β 7 were the only Results: The real- time PCR analysis of miRNA showed that the average number of independent predictors of TTP with HR of 3.892 and 4.029 respectively. miR16a copies was 428 copies per cell in 13q14-del MM samples (range 14 to 1176 Conclusions: Detection of ITβ7 expression by a routinely applicable methodology, copies per cell), while it averaged 7790 copies per cell in 13q14 non-deleted patients identify a high risk group in MM pts. These pts can be spared of toxicities of ASCT (831 to 30573 copies per cell , P=0.0003). In contrast to the miR16a, the miR15a levels through offering of upfront novel therapeutic agents. in general were very low in all myeloma cases. The miR15a averaged 11 copies per cell in the 13q14-del samples while it averaged 45 copies. 1254 Distinct Patterns of Marrow Fibrosis in Patients with Inherited Conclusions: miR16a was downregulated in of the majority of MM samples with Bone Marrow Failure Syndromes the 13q14 deletion as compared to those MM cells that did not harbour this deletion. I Maric, N Giri, D Arthur, P Noel, BP Alter. CC/NIH, Bethesda, MD; NCI/NIH, Therefore, miR16a may have a critical role in the pathogenetic effect of 13q14 deletion Bethesda, MD. in MM particularly considering a recent evidence demonstrating miR16 G0/G1 arrest Background: There are no data on the incidence or significance of bone marrow and decreased cell growth. fibrosis in patients with the inherited bone marrow failure syndromes (IBMFS), genetic disorders characterized by cytopenias, distinctive clinical features, varied molecular 1252 Expression of B Cell Signaling Proteins (PLC-γ and Grb2) pathways and high risk of myelodysplastic syndrome/acute myeloid leukemia (MDS/ and EBV Status Correlate with Expression of CD20 in Classical Hodgkin AML). We studied marrow fibrosis in the four most common IBMFS: Fanconi anemia Lymphoma (FA), Diamond-Blackfan anemia (DBA), Shwachman-Diamond syndrome (SDS) and CC Mankey, S Tripp, SL Perkins, KSJ Elenitoba-Johnson, MS Lim. University of dyskeratosis congenita (DC). Michigan, Ann Arbor, MI; University of Utah, Salt Lake City, UT. Design: We performed a retrospective study of 42 bone marrow biopsies from patients Background: The neoplastic Hodgkin/Reed-Sternberg cells of classical Hodgkin with established diagnoses of IBMFS. Blinded biopsies from 13 DBA, 13 DC, 12 FA, lymphoma (cHL) are B cells which typically show down-regulation of B-cell genes and 4 SDS patients were analyzed. Reticulin fibrosis was graded on a scale of 1-4 including CD20, the B-cell receptor (BCR) and its signaling components. CD20 according to the quantity and pattern of distribution of reticulin. The frequencies of expression is seen in a minority of cHL, but expression of BCR signaling proteins in abnormalities in marrow fibrosis, cellularity, MDS, cytogenetic clones, blood counts, this subset has not been studied. Previous studies have shown that latent membrane and need for treatment were compared between the disorders. protein-1, the EBV oncoprotein, downregulates expression of B cell-associated proteins Results: See Table. Significantly more patients with DC or FA had increased reticulin in HRS cells, but the correlation between EBV status and CD20/BCR signaling proteins fibrosis (grade 2 or 3) compared with DBA or SDS. No patient had grade 4 fibrosis, has not been studied. and grade 3 was present only in 2 FA patients. Marrow hypocellularity was present in Design: Using tissue microarrays constructed from 123 cases of cHL we analyzed all DC and FA and less frequent in SDS or DBA. MDS and cytogenetic clones were expression of two BCR signaling proteins, PLC-γ and Grb2, and CD20 by not seen in DBA. Cytogenetic clones were more frequent in SDS and FA then in DC, immunohistochemistry and EBV status by in situ hybridization. 19 cases of nodular while the frequency of cytopenias and hematologic treatment was consistent with the lymphocyte predominant HL were used as controls. Cases with reactivity in at least underlying diagnoses. 25% of tumor cells were considered positive. BM BM Morphologic Thrombo- On Results: All cases of NLPHL expressed CD20 and PLC-γ, and nearly all cases (17/18) Hypo- Neutropenia Anemia IBMFS fibrosis MDS/Cytogenetic cytopenia Treatment cellularity N(%) N(%) expressed Grb2. In cHL, 17% of cases (21/123) expressed CD20. CD20-positive cases N(%) Clone N(%) N(%) N(%) expressed PLC-γ more often, expressed Grb2 more often and were more often EBER N(%) DBA n=13 2 (15) 5 (38) 0 (0)/0 (0) 0 (0) 3 (23) 9 (69) 9 (69) positive than CD20-negative cases (p<0.01, p<0.01 and p=0.02, respectively). There DC n=13 10 (77) 13 (100) 1 (8)/2 (15) 11 (85) 7 (54) 7 (54) 7 (54) was no correlation between EBER status and expression of PLC-γ or Grb2 (p=0.45 FA n=12 9 (75) 12 (100) 3 (25)/6 (50) 8 (67) 8 (67) 4 (33) 5 (42) and p=0.14, respectively). There was a positive correlation between expression of SDS n=4 1 (25) 2 (50%) 2 (50)/3 (75) 3 (75) 4 (100) 0 (0) 0 (0) PLC-γ and Grb2 (p<0.01). P value (DC/FA/ 0.002 <0.001 NS/0.01 <0.001 0.02 NS NS CD20- CD20- CD20- CD20+ CD20+ CD20+ SDS)vsDBA PLC-γ Grb2 EBER PLC-γ Grb2 EBER NS 3.9% 2/51 1.7% 1/58 22.9% 14/61 40% 2/5 16.7% 1/6 42.8% 3/7 Conclusions: The incidence of grade 2 or 3 bone marrow fibrosis is as high as 75% in MC 9.1% 1/11 16.7% 2/12 57.1% 8/14 20% 1/5 20% 1/5 60% 3/5 DC and FA, and much less common in DBA and SDS. Marrow hypocellularity, MDS, LR 0% 0/3 33.3% 1/3 0% 0/3 33.3% 1/3 66.7% 2/3 66.7% 2/3 cytogenetic abnormalities and pancytopenia are rare in DBA but common in the other LD 0% 0/2 50% 1/2 0% 0/2 IBMFS. Longitudinal studies are required to determine the prognostic significance of NOS 0% 0/17 11.8% 2/17 52.9% 9/17 33.3% 1/3 66.7% 2/3 66.7% 2/3 these findings. all 3.6% 3/84 7.5% 7/93 32% 31/97 33.3% 5/15 38.9% 7/18 60% 12/20 Conclusions: Our study indicates an association between expression of CD20 and the 1255 CD71 (Transferrin Receptor) – An Excellent Marker for Erythroid BCR signaling proteins PLC-γ and Grb2 in cHL suggesting the presence of an intact Precursors in Bone Marrow Biopsies BCR signaling pathway. The association of EBER-1 with CD20 expression does not DK Marsee, GS Pinkus, H Yu. Brigham and Women’s Hospital, Boston, MA. support the notion that EBV leads to loss of B-cell identity. The results of our study Background: Accurate analysis of the erythroid lineage is essential in evaluating bone have implications for potential therapeutic agents that target CD20 and components of marrow biopsies and may be particularly challenging in settings of dyserythropoiesis. the BCR signaling pathway for patients with cHL. The transferrin receptor (CD71), an integral membrane protein that mediates the uptake of transferrin-iron complexes, is highly expressed on the surface of cells of the erythroid 1253 Expression of Intergrin Beta 7 (ITβ7) Identify a “ High Risk lineage. Although the use of CD71 for flow cytometry has been reported, its utility in Group” among Multiple Myeloma (MM) Patients (Pts) Treated with High paraffin-fixed bone marrow biopsies has not been examined. Dose Chemotherapy Followed by Autologus Stem Cell Transplantation Design: To determine whether CD71 represented an effective marker to analyze (ASCT) erythropoiesis in bone marrow biopsies, we compared immunohistochemical studies A Mansoor, SMT Rad, A Klimowicz, A Magliocco, X Jiang, D Stewart, N Bahlis. for several erythroid markers using antibodies to CD71 (clone H68.4), glycophorin A University of Calgary/CLS, Calgary, AB, Canada; Tom Baker Cancer Ctr., Calgary, (clone JC159), and hemoglobin (rabbit polyclonal) and an Envision+ detection system AB, Canada. (Dako). Studies for CD71 were performed following heat-induced epitope retrieval. Background: Gene expression profile (GEP) identified various prognostic sub-groups Paraffin sections of 56 bone marrow biopsies fixed in Zenker’s solution were analyzed, (e.g. c-MAF, MAFB-, MMSET etc) in MM pts. c-MAF translocated in (10%) MM including 10 normal marrows, 10 cases of myelodysplastic syndrome (MDS), 12 cases pts, stimulate cell cycle progression and promote stromal interactions through Cyclin of acute myeloid leukemia (AML; 2 each of FAB subtypes M0-M5), 7 cases of acute D2, ITβ7, & CCR1 as target genes. In MM, ITβ7 is also up-regulated through other erythroid/myeloid leukemia (FAB M6a), 4 cases of acute pure erythroid leukemia (FAB pathways and enhances production of VEGF. Since, GEP has limited applicability ANNUAL MEETING ABSTRACTS 277A

M6b), 5 cases of acute lymphoblastic leukemia (ALL), 3 cases of multiple myeloma, Hierarchical cluster analysis with GC and non-GC markers in DLBCL showed that and 5 cases of metastatic carcinoma. expression of cyclin D2 and D3 were most similar to each other and distinct from GC Results: In normal marrows and in the various disorders, reactivity for CD71 was and non-GC markers. specific and restricted to the erythroid lineage, with the highest intensity of membrane Conclusions: Our results show that cyclin D2 and D3 proteins, in contrast to cyclin staining seen in early forms and the lowest level in late normoblasts. Mature erythrocytes D1, are expressed in a wide variety of hematolymphoid neoplasia. The differential were CD71 negative, greatly facilitating interpretation. In cases of MDS, AML, expression of D-cyclins in DLBCL suggests their relationship to prognosis in this multiple myeloma, and metastatic carcinoma, CD71 served as the most effective disease may be related to cell cycle regulation or another unknown mechanism rather marker for detection of normal or abnormal erythroid precursors due to their distinct than the cell of origin. Whether cyclin D2 is predictive of DLBCL patient outcome in membranous staining pattern and the lack of reactivity for mature erythrocytes. the immunochemotherapy era is under investigation. The importance of D-cyclins and Myeloblasts, lymphoblasts and non-erythroid marrow elements, as well as myeloma CDK in cell cycle regulation and lymphomagenesis render them attractive choices as and carcinoma cells, were CD71 negative. In studies for hemoglobin and glycophorin potential therapeutic targets for further study. A, though erythroid elements were also detected, interpretation was complicated by strong reactivity of the background erythrocytes. Staining pattern for Glycophorin A 1258 LMO2 Expression and the Hans Algorithm in Predicting (membrane) was more distinct than that for hemoglobin (cytoplasmic). In addition, Germinal Center Phenotype and Survival in Diffuse Large B-Cell hemoglobin studies exhibited the most background staining. Lymphoma Treated with Rituximab Conclusions: This study demonstrates that CD71 (transferrin receptor) is an excellent PN Meyer, DD Weisenburger, WL Choi, LM Smith, TC Greiner, P Aoun, J Delabie, RM marker for detection of cells of erythroid lineage in bone marrow biopsies. Braziel, JM Vose, G Lenz, LM Staudt, WC Chan, K Fu. University of Nebraska Medical Center, Omaha, NE; University of Hong Kong Faculty of Medicine, Hong Kong, 1256 Myelomastocytic Leukemia: A Clinical, Pathologic, and China; University of Oslo Medical Center, Oslo, Norway; Oregon Health and Science Molecular Genetic Study University, Portland, OR; National Cancer Institute, Bethesda, MD. A McGuire, LR Shier, J Gotlib, B Medeiros, K Wong, C Corless, DA Arber, TI George. Background: Diffuse large B-cell lymphoma (DLBCL) has wide variation in survival. Stanford University, Stanford, CA; University of Alberta, Edmonton, AB, Canada; Gene expression profiling (GEP) shows DLBCL derived from germinal center B cells Stanford, Stanford, CA; Oregon State Health Sciences, Portland, OR. (GCB) have better prognosis. Immunohistochemical techniques to predict the cell of Background: Myelomastocytic leukemia (MML) is a rare and recently recognized origin, such as the Hans algorithm (Blood 103:275, 2004), have been developed. Recent neoplasm described as involving 2 distinct, but related populations, myeloid and reports show that LMO2 also predicts outcome in DLBCL. Our goal was to study mastocytic. The rare and unusual nature of MML makes diagnosis difficult, with little LMO2 expression and the Hans algorithm in predicting GCB phenotype and survival data to direct its management. This case series is presented to improve diagnosis of in DLBCL treated with rituximab. this rare leukemia. Design: Tissue microarrays (TMA) were created from 175 cases of DLBCL treated with Design: We describe 3 cases of MML retrospectively collected from the Pathology rituximab in combination with CHOP-like therapies. LMO2 expression was determined Dept at Stanford which did not meet diagnostic criteria for other mast cell disorders. by immunohistochemistry. Analysis of CD10, BCL6, and MUM1 expression divided the Immunohistochemistry, flow cytometry and cytogenetics were performed using standard cases into GCB or non-GCB types (Hans algorithm). GEP by microarray analysis was techniques. Allele specific PCR was performed forKIT mutations. performed on 57 cases. The relationships between LMO2 expression, GCB phenotype, Results: Case 1: 64 yo man with AML t(8;21). After induction, a diffuse mast cell GEP, as well as overall and event-free survival, were analyzed. infiltrate (20%) was found expressing CD117+, tryptase+, CD25-. PCR found the Results: LMO2 expression showed good agreement with the Hans algorithm (p<0.0001, imatinib sensitive N822K KIT mutation. The patient relapsed with AML 9 mo after kappa 0.54) or GEP (p=0.0001, kappa 0.50). Using GEP as the gold standard, the complete remission with cytarabine consolidation + imatinib. Case 2: 61 yo woman Hans algorithm showed greater sensitivity (90% vs. 84%), specificity (73% vs. 65%), with pancytopenia. She was refractory to multiple regimens (standard chemotherapy, positive predictive value (80% vs. 74%), negative predictive value (86% vs. 77%), cladribine, and imatinib + prednisone). Her marrow was diffusely infiltrated by tryptase+, and concordance (82% vs. 75%) when compared to LMO2; however, the differences CD117+, CD25- immature mast cells (90%) and bizarre multinucleated mast cells. were not statistically significant. LMO2 expression and the Hans algorithm were both Karyotype was normal. Initial sample could not be amplified; a second sample 1 mo predictive of overall (p=0.0023 and p=0.026, respectively) and event-free survival later was D816V KIT mutation negative. The patient expired 5 mo after diagnosis. (p=0.0038 and p=0.027, respectively). Case 3: 35 yo woman with urticaria treated with antihistamines/prednisone, who later Conclusions: Our results demonstrate that the Hans algorithm and LMO2 are similar presented with pancytopenia. Marrow was diffusely infiltrated by immature mast cells in predicting the GEP, and confirm recent reports that LMO2 is predictive of survival (50%) expressing tryptase+, CD117+, CD25+. A second blast population expressed dim in DLBCL. Unlike some reports, we found that the Hans algorithm is also predictive CD117+, MPO+, CD34-. D816V KIT mutation was negative with normal cytogenetics. of survival in patients treated with rituximab. The patient received cladribine with progressive disease and expired. Conclusions: All patients showed diffuse marrow infiltrates by immature mast cells 1259 Extra Copies of GLI1 Gene at 12q13.12 Are Detected in a Subset lacking the D816V KIT mutation, with an aggressive course refractory to chemotherapy. of Cases of Trisomy 12 Negative Chronic Lymphocytic Leukemia/Small Findings not previously reported included CD25 expression in mast cells, bizarre Lymphocytic Lymphomas multinucleated mast cells, lack of a myeloid neoplasm and the N822K KIT mutation. C Milito, FA Khokhar, K Newton, RR Singh, F Kasbidi, E Schlette, H Yao, I Biasoli, LJ Previous reports have shown favorable responses to polychemotherapy and marrow Medeiros, D Jones, R Luthra, F Vega. MD Anderson Cancer Center, Houston, TX. transplant suggesting MML should be treated similarly to AML rather than as a systemic Background: Trisomy 12 is detected in approximately 20-30% of chronic lymphocytic mast cell disease. Further study of MML is needed. leukemia/small lymphocytic lymphoma (CLL/SLL). Previously, a structural analysis of chromosome 12 in CLL identified a minimally gained region limited to bands 12q13- 1257 Characterization of D-Cyclin Proteins in Hematolymphoid 15. Glioma-associated oncogene homologue-1 (GLI1) gene, at 12q13, encodes one of Neoplasms the effectors that mediate sonic hedgedog (SHH) signalling. Abnormal expression of RA Metcalf, S Zhao, R Levy, IS Lossos, Y Natkunam. Stanford University, Stanford, GLI1 is observed in several cancers. Here, we report frequent extra copies of GLI1 in CA; University of Miami, Miami, FL. trisomy 12 negative CLL. Background: D-cyclins (D1, D2, D3) play a central role in the cell cycle and lymphoma Design: We analyzed GLI1 (12q13) and WNT1 gene (12q14.3) copy number using pathogenesis. t(11;14) in mantle cell lymphoma (MCL) causes overexpression of real-time qPCR in a series of 71 BM or PB specimens involved by CLL. This set of cyclin D1 by deregulating cyclin-dependent kinases (CDK) and their targets. Gene cases included 53 cases without trisomy 12 and 18 cases with trisomy 12. Expression expression profiling studies uncovered cyclin D1-negative MCL that overexpress of SHH and GLI1 was assessed immunohistochemically (IHC) in 22 cases as well as in cyclin D2 or D3. Cyclin D2 was also identified as a poor prognostic marker in diffuse additional 11 LN involved by SLL. The biological effect of inhibition of SHH signaling large B-cell lymphoma (DLBCL) patients treated with CHOP chemotherapy. Our aim pathway on CLL cells was assessed using cell viability assays after treatment with SHH in this study was to characterize the expression of D-cyclins in hematopoietic tumors inhibitors, cyclopamine and SANT1. CD19+ non-neoplastic B-lymphocytes collected at the protein level. from the peripheral blood of 4 healthy donors were used as controls. Design: Over 600 tumors were stained using immunohistochemistry for cyclins D1, D2, Results: Extra copies of the GLI1 gene were detected in 21 (39.6%) of 54 CLL cases and D3 on tissue microarrays. Hierarchical cluster algorithms were used to compare the without trisomy 12; 15 (71.4%) cases had 1 extra copy and 6 (28.5%) had at least 2 extra staining of the three D-cyclins with other germinal center (GC: CD10, LMO2, BCL6, copies of GLI1. In contrast, 1 extra copy of WNT1, a more telomeric gene than GLI1, HGAL) and non-GC (BCL2, MUM1) markers in 143 DLBCL. was noted only in trisomy 12 cases. The presence of either trisomy 12 or extra copies Results: Of the 3 D-cyclins, cyclin D2 was most widely expressed in hematolymphoid of the GLI1 gene correlated with strong nuclear expression of GLI1 as detected by IHC as well as non-hematopoietic tissue types. (p<.001). In cases with extra copies of GLI1, most of the CLL cells exhibited moderate Immunohistochemistry of D-Cyclins in Hematolymphoid Neoplasms to strong nuclear expression of GLI1; however, in cases without trisomy 12 or without Diagnosis Cyclin-D1 Cyclin-D2 Cyclin-D3 extra copies of GLI1 gene, expression of GLI1 was restricted to prolymphocytes and Diffuse large B-cell lymphoma 3/110 95/194 44/220 paraimmunoblasts. Pharmacologic inhibition of SHH signaling decreased cell viability Follicular lymphoma, grade 1 0/39 15/38 1/37 of CLL cells, but not that of non-neoplastic B-lymphoid cells. Extra copies of GLI1 Follicular lymphoma, grade 2 0/48 9/41 5/56 Follicular lymphoma, grade 3 0/69 21/61 10/71 correlated with high RAI stages (p=.0067). Marginal zone lymphoma 0/27 10/28 2/35 Conclusions: Extra copy numbers of GLI1 gene are frequent in trisomy 12 negative CLL Mantle cell lymphoma 15/18 3/18 1/18 cases; they can be predicted by IHC and their presence correlate with a high RAI stage. CLL/SLL 0/36 11/36 2/38 This data also suggests that SHH signaling has a role in the survival of CLL cells. Precursor B lymphoblastic lymphoma 0/7 4/7 3/14 Precursor T lymphoblastic lymphoma 0/12 10/11 3/12 Peripheral T-cell lymphoma 2/18 10/18 1/20 Anaplastic large cell lymphoma 0/7 3/5 2/8 Hodgkin lymphoma 0/0 76/101 21/125 Total 20/391 267/558 95/654 278A ANNUAL MEETING ABSTRACTS

1260 Follicular Lymphoma Involving the Small Intestine: Is There an in the GCB type are miR-331-3p, 151-5p, 28-5p, 454, 210, 183 and miR-138 and Association with Gastric Disease or Helicobacter Pylori? uregulated in the ABC subtype are miR-222, 144, 451, 221, 148a). RN Miranda, T Muzzafar, LJ Medeiros. M.D. Anderson Cancer Center, Houston, TX. Background: Follicular lymphoma (FL) involving the small intestine is uncommon and its etiology is unknown. Rare case reports of FL of small intestine have shown an association with gastric Helicobacter pylori infection, and it has been suggested a pathogenic role (Gastrointest Endosc 2001; 54:92). In this study we evaluated the hypothesis that associated gastric disease or Helicobacter pylori infection might play a role in pathogenesis of FL of small intestine. Design: We identified 23 patients who had FL involving the small intestine who also had simultaneously obtained stomach biopsy specimens at our institution over the past 10 years. Their clinicopathologic features are presented. Results: The study group included 11 women and 12 men, with a median age of 60 years (range, 39 - 81). Three distinct groups were identified: Localized and presumably primary (n=7), disseminated (n=9) and patients with a history of FL (n=7). Lymphoma involved the duodenum (n=15), ileum (n=5), and jejunum (n=3). Histologically, the FL was low-grade in 22/23 cases; one patient with a history of FL had a grade 3 tumor. FL was simultaneously identified in the stomach of 7 (30%) patients: 1 of 7 (14%) localized, 2 of 9 (22%) disseminated, and 4 of 7 (57%) with a history of FL. Chronic active gastritis with Helicobacter organisms was identified in 4 patients: 2 (29%) localized, 1 (11%) disseminated, and 1 (14%) with a history of FL. Conclusions: A unique miRNA signature is identified to associate with poor clinical Conclusions: Patients with FL involving the small intestine who undergo biopsy are a outcome in R-CHOP treated DLBCL patients. A model based on the expression of 4 heterogeneous group. There is no apparent association between the presence of chronic miRNAs is able to predict for OS and EFS, which is independent of IPI score. MiRNA active gastritis or Helicobacter infection with FL involving the small intestine. expression profiling is able to identify particular miRNA species that are differentially expressed between DLBCL subtypes, providing a valuable technology to identify 1261 Mitotic Index and Cell Cycle Regulatory Proteins as Predictors of differentiation stage-related miRNAs and its potential gene targets. Behavior in Typical, Non-Blastoid Mantle Cell Lymphoma A Mogal, A Cashen, J Frater, A Hassan, F Kreisel. Washington University, St. Louis, 1263 Comparison of Non-Hodgkin Lymphoma Subtypes in Guatemala MO. and North America/Western Europe Background: Mantle cell lymphoma (MCL) is clinically more aggressive with a median A Morovic, H Molina-Kirsch, J Diebold, K Maclennan, K Muller-Hermelink, B survival of < 4 y. However, a small subset shows an indolent course with an overall Nathwani, J Armitage, D Weisenburger, for the International Non-Hodgkin Lymphoma survival of up to 10 y. Routinely applicable prognostic markers are needed to identify Classification Project. University of Nebraska Medical Center, Omaha, NE. the different prognostic patient subgroups. While mitotic index (MI), Ki-67, loss of Background: Comparisons of the relative frequencies of subtypes of non-Hodgkin p16INK , and p53 overexpression have all been reported as markers of poor clinical lymphoma (NHL) between different geographic regions are rarely documented in the outcome in B-cell lymphomas, no study addresses the significance of these markers in literature. In this study, we contrast the frequencies of NHL subtypes between developed distinguishing the indolent cases of MCL from the more aggressive forms. countries and a developing country, Guatemala. We used a new immunostain algorithm Design: MI, Ki-67, p16INK, and p53 expression were analyzed in 2 groups of pts. with for the classification of diffuse large B-cell lymphoma (DLBCL) into germinal center typical MCL (total 10 cases). Blastoid variants were excluded. One group included B-cell-like (GCB) and non-GCB subtypes, and compared the frequencies of these chemoresistant pts. (chemorefractory or initially chemoresponsive, but progressed within subtypes between Guatemala and the developed countries. 1 y of treatment), the other group was chemoresponsive pts. with no progression within Design: We reviewed 194 consecutive cases of NHL from Guatemala and contrasted 1 y of treatment. Sections were examined by 2 pathologists. MI and p53 expression were the frequency of the different subtypes to those in North America and Western Europe quantitated by counting the # of positive nuclei per 20 100x fields in areas of highest (Ann Oncol. 9:717,1998). Also, we subclassified 83 cases of DLBCL from Guatemala activity. Ki-67 was quantitated by counting 1000 cells using a 100x objective in the areas to GCB and non-GCB subtypes using a new algorithm (Mod Pathol. 21:250A, #1144, of highest expression. p16INK was quantitated by % of staining malignant cells. 2008) and the following antibodies: GCET1, CD10, BCL6, MUM1 and FOXP1. We Results: 3/9 pts. failed chemotherapy and treatment resistant, while 6/9 responded to compared the frequencies of the GCB and non-GCB subtypes in the Guatemalan series chemotherapy. All treatment resistant pts. presented with significantly higher serum with two series from developed countries, one representing North America and Western lactate dehydrogenase (LDH) levels, compared to treatment responders (p<0.007). While Europe (PNAS.100:9991, 2003) and the another representing North America alone no significant differences were found between the treatment resistant and treatment (Mod Pathol. 21:250A, #1144, 2008). responsive MCL for MI (mean:1.4vs.1.7,p= 0.33), Ki-67 (mean:30.3vs.15.7,p=0.1), Results: DLBCL is the most common NHL in Guatemala (45%) and its frequency and p53 (mean:17.5vs.4.5,p=0.26), higher p16 expression was noted for the 2/3 is higher when compared to North America or Europe (28-31%). The frequency of treatment resistant MCL (10-15%) compared to treatment responsive MCL where all follicular lymphoma is markedly decreased in Guatemala (10.3%) compared to North cases were negative. America (31-32%) and Europe (17-28%). Nasal NK/T cell lymphoma is rare in North Conclusions: Mitotic index, Ki-67, and p53 were not useful in predicting response to America and Europe (0-2%), but it made up 7.7% of all NHL in Guatemala. When the chemotherapy, and their expressions showed no correlation with serum LDH levels. new algorithm was applied to 83 DLBCL cases from Guatemala, 44 were classified These markers may not be useful in distinguishing the subset of MCL patients with as GCB and 39 as non-GCB subtype. This group was compared to the DLBCL series an indolent course. Despite to earlier reports that loss of p16INK protein expression in from North America and Europe which included 217 cases (134 GCB, 83 non-GCB), non-Hodgkin lymphomas is a frequent finding associated with tumor progression, all of and the series from North America which included 147 cases (84 GCB, 63 non-GCB), treatment responsive MCL lacked expression of this marker, suggesting that p16INK may but there were no significant differences between these groups, with p values of 0.17 not be of significance in the negative cell cycle regulation of this lymphoma. and 0.57, respectively. Conclusions: DLBCL is the most common NHL in Guatemala and its relative 1262 MicroRNA Expression in DLBCL: Identification of Unique miRNAs frequency is increased compared to North America and Western Europe, whereas the Signatures Related to Clinical Outcome Prediction in R-CHOP Treated relative frequency of follicular lymphoma is decreased in Guatemala. There were no Patients significant differences in the frequency of DLBCL subtypes in Guatemala compared to North America and Europe. S Montes-Moreno, N Martinez, A Saez, C Montalban, MH Rodriguez, B Sanchez- Espiridion, SM Rodriguez Pinilla, GB Lemarbre, WM Rehrauer, M Waknitz, KH Young, MA Piris. Spanish National Cancer Center, CNIO, Madrid, Spain; UW Paul P. 1264 Early-Onset, EBV-Negative PTLD in Pediatric Solid Organ Carbone Comprehensive Cancer Center, University of Wisconsin School of Medicine Transplant Recipients: Spectrum of Plasma Cell Neoplasms with Favorable and Public Health, Madison, WI. Prognosis Background: MicroRNAs (miRNAs) can constitute markers of malignant A Morovic, DW Coulter, W Sanger, PF Coccia, P Aoun. University of Nebraska, Medical transformation, outcome or sensitivity to specific therapies. In this study we analyzed Center, Omaha, NE. the miRNA profile of a large series of R-CHOP treated DLBCL patients and correlated Background: Epstein-Barr virus (EBV)-negative post-transplantation with clinical outcome variables. lymphoproliferative disorders (PTLD) are rare in comparison to EBV-positive PTLD, Design: First, a set of 36 cases was studied by miRNA and Gene Expression profiles by usually occur more than five years after transplantation, and have a poor response array tech. Both outcome-related candidate miRNAs and Cell of Origin (Wright et al. to decreased immunosuppression. Few studies have examined EBV-negative PTLD PNAS USA. 2003) related miRNA candidates were identified. After this, the expression in pediatric patients. The purpose of this study was to characterize the clinical and of 14 miRNA candidates was studied by RT-PCR in a large series of 90 samples and pathological features of EBV-negative PTLD arising in pediatric solid organ transplant results were correlated with IPI score and outcome data (OS, EFS). (SOT) recipients at our center. Results: Two microRNA expression based models were defined to correlate with Design: 66 pediatric (age < 19 years) SOT recipients who developed PTLD from 1991- outcome. These models summarize the expression of 4 miRNAs each and correlate 2008 were identified. The clinical, histological, molecular and cytogenetic features of with OS (Hazard ratio 1.2, 95%CI=1.05-1.38, p=0.004) and EFS (Hazard ratio the EBV-negative cases were further characterized, including immunostains for CMV 1.14, 95%CI=0.99-1.3, p=0.055) in the univariable Cox regression analysis. In the and HHV8, and in situ hybridization for kappa, lambda and EBERs. multivariable Cox regression analysis, the miRNA-based models remain independent of Results: EBV-negative PTLD was identified in 6/66 patients (9%), and material for study IPI, being both variables significant as outcome predictors (figure 1). 12 miRNAs were was available for 5/6 patients. There were 3 females and 2 males, ranging in age from 10 found to be differentially expressed between DLBCL subtypes (FDR<0.05) (upregulated months to 7 years (median, 3 years) at the time of PTLD. All 5 patients had received a combined liver and small bowel transplant, and were on an immunosuppressive regimen ANNUAL MEETING ABSTRACTS 279A consisting of tacrolimus and prednisone. EBV-negative PTLD developed 9 to 22 months All 19 cases of T-ALL/LBL were negative for CD20, CD22, CD79a, PAX-5, MUM-1, post-transplantation (median, 15 months).The morphology included atypical plasma cell and OCT.2. However, of the 19 T-ALL/LBLs, 15 (79%) and 12 (63%) demonstrated hyperplasia (1/5), plasmacytoma-like (3/5), and multiple myeloma (1/5). Bone marrow PU.1 and BOB.1 expression, respectively. involvement was identified in 3/4 cases. Serum monoclonal or oligoclonal paraproteins Conclusions: We have found that PAX-5 is an excellent pan-B and pan-pre-B-cell were identified 4/5 patients. EBV, CMV and HHV-8 were negative by serum PCR, marker and should be used as the first-line B lineage-specific marker for paraffin EBERs and immunohistochemistry in all cases. Molecular studies demonstrated clonal immunophenotyping B-ALL/LBL and BL. Pax-5 demonstrated better specificity than IgH gene rearrangements in all cases. Karyotyping demonstrated trisomy 3 in one case, BOB.1 and PU.1 and better sensitivity than CD79a, CD22 and CD20. Our results and +X in another case; FISH cytogenetic studies are in progress. One patient died of suggest that the expression of Pax-5 antigen precedes the expression of CD22, CD79a pneumonitis before treatment. The remaining four patients were treated with high-dose and CD20. Although it is reported that OCT2 is an early B-cell differentiation antigen, corticosteroids, and one patient also received thalidomide. All remain in remission 31-48 only 6% of our B-ALL/B-LBL cases were positive. months (median, 42 months) after diagnosis. Conclusions: We describe a group of EBV-negative PTLD arising in pediatric SOT 1267 DNA Methylation Biomarkers in Myelodysplastic Syndromes recipients, characterized by a histological spectrum ranging from atypical plasma cell VT Nguyen, S Nalluri, MX Wang. University of Missouri School of Medicine, Columbia, hyperplasia to plasmacytoma-like or myeloma-like. In comparison to other reported MO. EBV-negative PTLD cases, these occur early after transplantation, and can be treated Background: Myelodysplastic syndrome (MDS) is defined as a clonal stem cell disorder with high-dose steroids with good response. characterized by single/multilineage dysplasia and ineffective hematopoiesis. Thirty percent of patients with MDS eventually progress to acute myeloid leukemia (AML), 1265 The Disruption of the ER Stress Response Pathway by R-CHOP and but the majority of patients die from marrow failure. Although half of all patients Proteasome Inhibition in DLBCL Converges in GRP78/Bip and GADD153/ have clonal chromosomal abnormalities, yet none are specific for MDS. Emerging Chop clinical and experimental evidence indicate the importance of epigenetic alterations, A Mozos, G Roue, O Balague, A Lopez-Guillermo, D Colomer, E Campo, A Martinez. especially aberrant DNA hypermethylation, in the pathogenesis of MDS. Currently, Hospital Clinic, IDIBAPS, U. Barcelona, Barcelona, Spain; Netherlands Cancer Institute, there are no diagnostic molecular biomarkers available for MDS. In this study, we Amsterdam, Netherlands. identified several hypermethylated loci in MDS patients, which have a potential use Background: The ER stress response controls the homeostasis of the ER. This pathway as diagnostic biomarkers. involves Ire1-Xbp1, Perk-Chop and Atf6. The chaperone Bip and the transcription Design: Genomic DNA was extracted from 21 MDS, 48 AML and 8 non-tumor patient factor Xbp1 are key players in this response. We have shown that Xbp1 activation in bone marrow aspirates as well as 3 AML cell lines (KG1, KG1a and Kasumi-1). The DLBCL is associated with aggressive disease. Proteasome inhibition (PI) disrupts the DNA was subjected to digestion with 4 methylation sensitive restriction enzymes. The ER stress response in myeloma but the role of this response in DLBCL treated with PCR target regions were carefully selected to ensure a complete digestion. Digested R-CHOP or PI is unknown. normal cell DNA will not be amplified by PCR. Conversely, hypermethylated regions in Design: We analyze the expression of Bip, Chop, Xbp-1, Ire-1 and Noxa by western tumor cells, being resistant to methylation sensitive restriction enzymes, remain intact blot and rtPCR in 9 lymphoma cell lines. DLBCL cell lines were treated either with and therefore can be differentially amplified. Thus, the presence of PCR products is R-CHOP or PI. Cell viability was analyzed by annexin V and MTT assays. We also indicative of DNA methylation at those particular loci in the tumor cells. analyze Bip and Chop expression in 138 DLBCL by immunohistochemistry. Results: We analyzed 18 CGI DNA methylation “hot spots”, which have been implicated Results: All cell lines expressed Bip, Ire1 and active Xbp1 and 6 also expressed in leukemogenesis. The selected CGIs are contained within the genes involved in tumor Chop. All DLBCL cell lines were sensitive to R-CHOP and MG-132 but resistant suppression, cell cycle regulation, cellular proliferation and migration, DNA repair and to Bortezomib. After treatment, Bip expression was altered in all cell lines studied, apoptosis. Nine of 18 genes were found to be methylated in at least one AML cell line, induced by PI and reduced by R-CHOP. SUDHL16 was the most sensitive to PI (30% while only 2 of 18 genes (SOCS-1 and PCDHGA12) were methylated in a subset of MDS vs 50% viability) and showed high Bip expression unaltered by treatment but lacked patients’ bone marrows. A strong methylation in PCDHGA12 gene was observed in 41 Chop that was highly induced by treatment. Both Ire1 and Xbp1 were reduced by of 48 (85%) bone marrows from AML patients, while a weak PCDHGA12 methylation MG-132. The proapototic molecules Chop and Noxa were induced only in SUDHL16. was also seen in 3 of 8 non-tumor bone marrow samples. None was observed in normal R-CHOP reduced Bip expression more than 60% without altering Ire1 and Xbp-1. Only blood cell DNAs. in SUDHL-16 cells Chop was induced, although at lower levels than PI treatment. Conclusions: Using a novel PCR method, we were able to demonstrate aberrant DNA We studied Bip and Chop expression in reactive tissues and in 138 DLBCL. Bip was methylations in both AML and MDS patients’ bone marrow samples. A greater number expressed in the light zone of the germinal centers (GC) in association with the plasma of methylated genes were detected in AML than in MDS samples. Both SOCS-1 and cell transcription factors. Chop was upregulated in GC. Bip was expressed in 96 out PCDHGA12 were methylated in MDS and AML. Epigenetic evolution with increased of 138 (69.5%) and Chop in 59 out of 101 (58.4%) DLBCL. Neither Bip or Chop had level of methylation may reflect the biological nature of the progression from MDS impact on complete response rate, disease-free or overall-survival. to AML. Conclusions: In summary R-CHOP and MG-132 treatments disrupt ER stress response in DLBCL and target Bip expression. In cell lines, high Bip expression and lack of 1268 Precursor T-Lymphoblastic Lymphoma: An Immunophenotypic Chop was correlated with an impaired response to R-CHOP but a better response Analysis of 186 Cases to PI. Although Bip expression alone had not clinical impact in DLBCL patients, JL Patel, MA Lones, M Abromowitch, MS Cairo, SL Perkins. University of Utah, Salt the combination of Bip and Chop expression may help to identify patients that may Lake City, UT; St. Joseph’s Medical Center, Orange, CA; Children’s Memorial Hospital respond to PI. of Omaha, Omaha, NE; Columbia University, New York, NY. Background: Although ample data regarding the immunophenotypic characterization of 1266 Expression Profiling of Transcription Factors in Precursor precursor T-acute lymphoblastic leukemia (T-ALL) is available, the equivalent is not true Lymphoblastic Leukemia (ALL/LBL) and Burkitt Lymphoma (BL): Utility for patients who present with precursor T-acute lymphoblastic lymphoma (T-LBL). of PAX-5 Immunostaining as Pan-Pre-B-Cell Marker Design: We present a review of flow cytometric data from 186 patients with a diagnosis MR Nasr, N Rosenthal, S Syrbu. University of Iowa Hospitals and Clinics, Iowa City, of T-LBL derived from the Children’s Oncology Group study database (protocol# IA. A5971), which included lymphoblastic lymphomas and required less than 25% bone Background: The optimal use of transcription factors available to determine B-lineage marrow lymphoblasts as a study criterion. Multiple T-cell, B-cell, myeloid, and other specificity in B-ALL/LBL has not been determined. We undertook an extensive markers were evaluated. immunohistochemistry study of a panel of B-cell transcription factors in B-ALL/LBL, Results: T-ALL/LBL and BL seeking to find those with the best specificity and sensitivity. Immunoprofile of T-LBL (n=186) Design: Tissue microarrays were constructed with duplicate paraffin-embedded tissue Marker Number of Cases (n) Positive (%) cores from 34 B-ALL/ABLs, 19 T-ALL/LBLs and 30 BLs samples. Immunostaining CD1a 67 72.8 was performed on all specimens using antibodies against PU.1, OCT.2, BOB.1, MUM- CD2 127 91.4 cCD3 109 84.5 1, PAX-5, CD20, CD79a and CD22. Each core was evaluated for the percentage of sCD3 64 46.0 tumor cells staining and recorded in 4 grades (G) [G (0): 0%, G (1): 1-25%, G (2): CD4 110 71.9 26-50% and G (3): >50%]. CD5 145 95.4 Results: The number of positive cases and the grade of staining for each of the CD7 143 96.0 transcription factors are shown in the table. CD8 117 78.0 CD4+/CD8+ 129 79.1 B-ALL/LBL (n=34) BL (N=30) T-ALL/LBL (n=19) CD4-/CD8- 129 18.6 PAX-5 (Grade) 34 (3) 25 (3); 5 (2) 0 TdT 174 89.7 BOB.1 (G) 31 (3); 1 (2) 25 (3); 2 (2) 11 (3); 1 (2) CD10 131 56.5 PU.1 (G) 33 (3) 11 (3); 6 (2); 6 (1) 13 (3); 2 (2) CD34 109 20.2 CD79a (G) 25 (3); 4 (2) 25 (3); 4 (2) 0 Any B-cell marker 138 8.7 CD22 (G) 15 (3); 12 (2) 13 (3); 1 (2) 0 Any myeloid marker 73 15.1 CD20 (G) 2 (2) 26 (3); 4 (2) 0 Multiple myeloid markers 73 4.1 OCT.2 (G) 2 (3) 12 (3); 5 (2); 6 (1) 0 cCD3: Cytoplasmic CD3 expression, sCD3: Surface CD3 expression MUM-1 (G) 0 1 (3); 4 (2) 0 All B-ALL/LBLs expressed PAX-5 (34/34 [100%]), 32 (94%) of 34 expressed BOB.1, 33 (97%) of 34 expressed PU.1, 29 (85%) of 34 expressed CD79a, 27 (79%) of 34 expressed CD22, 2 (6%) of 34 expressed CD20 and OCT.2, and none (0%) expressed MUM-1. The percentages of cases expressing PAX-5, BOB.1, PU.1, CD79a, CD22, CD20, OCT.2, and MUM-1 in BLs were 100% (30/30), 90% (27/30), 77% (23/30), 97% (29/30), 47% (14/30), 100% (30/30), 77% (23/30), and 17% (5/30), respectively. 280A ANNUAL MEETING ABSTRACTS

Immunoprofile of TdT-Negative T-LBL (n=17) cases were scored for intensity (weak, moderate, or strong) and localization within the Marker Number of Cases (n) Positive (%) nucleus and/or cytoplasm. BCL-10 (Dako; 1:4000) was also examined and scored in CD1a 11 72.7 a similar manner. CD2 13 76.9 Results: Moderate to strong nuclear and cytoplasmic staining was identified for p65 in cCD3 13 100.0 sCD3 14 57.1 4 out of 10 cases (40%), with the remainder showing only weak cytoplasmic staining. CD4 15 60.0 P50 showed only weak cytoplasmic staining, without nuclear staining in any case. All CD5 14 92.9 cases showed weak cytoplasmic staining for BCL-10, with no nuclear positivity. CD7 15 100.0 Conclusions: Our study identified a significant proportion (40%) of PCMZL, with CD8 15 80.0 nuclear positivity for p65 indicative of classical NF-kB pathway activation. However, CD4+/CD8+ 15 53.3 CD4-/CD8- 15 13.3 this did not correlate with p50 or BCL-10 expression in our study. It is unclear whether CD10 12 16.7 this is related to different antibody clones. Alternative pathways of NF-kB activation CD34 9 11.1 may also be present in cases negative for nuclear expression of p65 or p50. The lack of Any B-Cell Marker 14 7.1 significant BCL-10 expression as demonstrated in this study suggests a diminshed role Any Myeloid Marker 9 11.1 of this protein in the NF-kB pathway and supports a lower frequency of (1;14)(p22;q32) Multiple Myeloid Markers 9 0.0 or t(11;18)(q21;q21) in PCMZL. These results are indicative of a role of NF-kB in a cCD3: Cytoplasmic CD3 expression, sCD3: Surface CD3 expression subset of PCMZL, warranting further evaluation in a larger series. Conclusions: Our study provides additional immunophenotypic data on T-LBL which may be diagnostically useful. Although it has been reported that myeloid antigens are often present in T-ALL/T-LBL, this was a distinctly uncommon occurrence in our 1271 Strong Expression of D2-40 in Lymphoma-Associated Stroma Is study group. Of the myeloid markers studied, CD33 is the most likely to be aberrantly Associated with Superior Outcome in Diffuse Large B-Cell Lymphoma expressed. The TdT-negative subset is interesting, as these often represent diagnostically D Peker, CC Chang, F Hagemeister, Y Zu, A Ewton, C Curry, D Jones. The Methodist challenging cases. Although the absolute number of these cases is small (n=17 or 9.1% Hospital, Houston, TX; MD Anderson Cancer Center, Houston, TX; Mount Sinai of total cases), it appears that TdT-negative T-LBLs are less likely to express aberrant Medical Center, Miami Beach, FL. B-cell markers, myeloid markers, CD10, and CD34. They are also less likely to co- Background: Stroma in lymphoid tissue include follicular dendritic cells (FDC), express CD4/CD8 but are likely to retain expression other T-cell markers, particularly fibroblastic reticulum (FRC), endothelium, and sinus lining cells (SLC), which can cytoplasmic CD3, CD5, and CD7. secrete growth-promoting cytokines and regulate cell adhesion. Degree of stromal activation likely reflects bidirectional interactions between stroma and lymphoma, and be readily assessable markers for classification and outcome prediction.We investigate 1269 Utility of Disease-Specific FISH Panels in the Diagnosis of how the degree of stromal activation correlates with subtype and outcome in a cohort Hematologic Malignancies of untreated diffuse large B cell lymphoma (DLBCL). KP Patel, KH Ramesh, R Conte, D Wei, H Ratech, L Cannizzaro. Montefiore Medical Design: Tissue microarrays (TMA) were constructed from 62 de novo DLBCLs, with Center, Bronx, NY. 3 1.2 mm cores per case. Immunohistochemical staining was done for dendritic cell Background: Identification of cytogenetic abnormalities is integreal to diagnostic differentiation (caldesmon, CD1a, CD21, CD23, D2-40, low-affinity nerve growth work-up of hematologic malignancies. Chromosomal analysis (CA) by conventional factor receptor (LNGFR), S100), (myo)fibroblast differentiation (smooth muscle actin GTG-band karyotyping has multiple known limitations. Fluorescent in situ hybridization (SMA), transglutaminase (TG) and Factor XIIIa), and SLC activation (D2-40, TG). (FISH) based probes can detect abnormalities not evident on GTG-banding. The use Cases were classified into germinal center (GC) and activated B-cell (ABC) types of multiple disease-specific probes as FISH panels (PF) provides information about using BCL6, CD10, MUM1/IRF4 and CD138, as previously described. Distribution multiple specific targets allowing simultaneous evaluation of diagnostic, therapeutic and intensity of stromal staining was scored semiquantitatively on a 3-tier scale, and and prognostic markers. We report highly sensitive FISH panels for routine diagnostic comparison made with tissue arrays containing a variety of other lymphoma types (n testing of different categories of hematologic malignancies. = 61), and reactive lymphoid tissues (n = 20). Design: A total of 315 consecutive Bone marrow or unstimulated blood samples were Results: CD21, CD23, D2-40, LNGFR and SMA were variably expressed in the FRC/ analyzed by conventional GTG-banding and simultaneous staining of interphase nuclei FDC of DLBCL stroma; CD1a, Factor XIIIa, S100 and caldesmon were not expressed with disease-specific FISH panels. The disease categories with respective number of to any significant degree. There was coordinate upregulation of D2-40 in SLC and probes for each panel were as follows: MDS (7), AML (12), CLL (5), ALL (9), MPD stroma in 38/62 cases (61%) that correlated to a degree with presence of CD21 in FDC (8), MM (4) and NHL (6). A detailed clinicopathologic analysis of all the cases was consistent with a common mode of regulation. D2-40 was also strongly expressed in performed. the SLC and FDC/FRC of reactive lymphadenitis and Castleman’s disease but largely Results: PF identified abnormalities in 27.3% cases with normal/failed CA. PF analysis absent in stroma of low-grade B-cell lymphomas. The degree and intensity of D2-40 was most promising in detecting abnormalities in ALL (58.1%), CLL (44.8%), AML expression in FDC/SLC in DLBCL was correlated with superior outcome (p = 0.005, (29.3%) and MM (24.1%) where the detection of abnormalities by CA was normal, Kaplan-Meier). Expression of D2-40 or other stromal markers were not correlated with had failed or was inconclusive (see table). GC versus ABC status, or extranodal versus nodal localization in DLBCL. MDS (%) AML (%) ALL (%) CLL (%) NHL (%) MM (%) MPD (%) Conclusions: Stromal activation state is highly regulated in DLBCL. Upregulation of Only PF 12 (10.5) 12 (29.3) 36 (58.1) 13 (44.8) 5 (18.5) 7 (24.1) 1 (7.7) Positive D2-40 in SLC and stroma represents a distinct phenotype in DLBCL that correlates with PF and CA 9 (7.9) 7 (17.1) 7 (11.3) 3 (10.3) 5 (18.5) 2 (6.9) 2 (15.4) good outcome but does not correlate with molecular subtype (i.e. GC vs ABC). Positive PF and CA 93 (81.6) 21 (51.2) 17 (27.4) 13 (44.8) 16 (59.3) 18 (62.1) 10 (76.9) Negative 1272 PRAME Expression in Follicular Lymphoma (FL) and Diffuse Large Only CA 0 (0) 1 (2.4) 2 (3.2) 0 (0) 1 (3.7) 2 (6.9) 0 (0) B Cell Lymphoma (DLBCL) Positive TOTAL (315) 114 (100) 41 (100) 62 (100) 29 (100) 27 (100) 29 (100) 13 (100) M Perez, JA Shafer, C Curry, Y Zu, A Ewton, G Dotti, C Chang. The Methodist Hospital, Houston, TX; Baylor College of Medicine, Houston, TX. Only 1.9% cases showed positive results with CA where PF was normal/failed. The Background: Preferentially expressed antigen of Melanoma (PRAME) is a cancer-testis clinicopathologic correlation shows improved turnaround time, better sensitivity and antigen over expressed in many types of solid tumors and leukemia. The importance of identification of low level abnormalities in treated cases with PF. this antigen is its immunogenic properties and sparse expression in normal tissues. It Conclusions: This study showed that a FISH panel, when used alone or in conjunction therefore has the potential use as a marker of minimal residual disease and as a target for with conventional karyotyping, significantly improves diagnostic yield of a variety of immunotherapy. We have recently demonstrated our ability to generate PRAME specific hematologic malignancies. After an initial screening with a complete panel, selected cytotoxic T lymphocytes that can kill PRAME positive blasts in chronic myelogenous probes can be used for the follow up analysis to identify the response to treatment, leukemia (Quintarelli et al Blood 2008). To further evaluate the potential use of this residual disease or relapse. novel therapeutic approach to treat lymphomas, we evaluated PRAME expression in FL and DLBCL, the two most common types of non-Hodgkin’s lymphoma. 1270 The Nuclear Factor-Kappa B Pathway: A Role in Primary Cutaneous Design: Tissue microarray (TMA) blocks were constructed from 66 archived cases of Marginal Zone B-Cell Lymphomas? de novo DLBCL and 31 de novo cases of FL, each case consisting of three 1.2 mm MC Patel, S Bhagavathi, S Warren, M Amin, RK Malhotra, AM Blenc. William Beaumont core sections. IHC staining with commercially acquired PRAME antibody (Abcam) Hospital, Royal Oak, MI; Indiana University, Indianapolis, IN. was performed on TMAs. Cases were classified as positive or negative for cytoplasmic Background: Nuclear factor-kappaB (NF-kB) is a family of transcription factors that and/or membrane staining based on >20% of cells showing expression. are required for T- and B-cell development, proliferation, and survival. Constitutive Results: Normal lymphocytes did not express the PRAME antigen based on the expression of the NF-kB signaling pathway has been recognized as a critical pathogenetic negative control on the DLBCL TMA. DLBCL cases negative for the PRAME antigen factor in lymphoma, including Hodgkin lymphoma and a variety of T- and B-cell non- demonstrated a positive background control in endothelial cells. Thirty seven cases Hodgkin lymphomas. Current experimental and preclinical data has shown the NF-kB (56%) of DLBCL stained positive for the PRAME antigen. However, the expression pathway to be a promising therapeutic target. However, little is known about the role of of PRAME in DLBCL did not correlate with germinal-center or activated B-cell type NF-kB in primary cutaneous marginal zone B-cell lymphomas (PCMZL). The aim of of DLCBL (based on the expression of immunohistochemical markers of BCL-6, this study is to examine the role of NF-kB activation in previously classified PCMZL CD10 and MUM1), with high or low IPI scores, or with nodal or extranodal disease. and also evaluate the expression of BCL-10, an NF-kB protein activator which is Furthermore, no significant correlation with overall survival was identified. For FL, aberrantly expressed in extranodal marginal zone lymphomas, particularly those with 24 cases (77%) of FL showed PRAME expression: Grade I (10/12), Grade II (7/10), t(1;14)(p22;q32) or t(11;18)(q21;q21). and Grade III (7/9). Design: We evaluated ten cases of formalin fixed-paraffin embedded PCMZL, from Conclusions: IHC, with PRAME antibody, demonstrated that a substantial percentage the archives of two institutions (2000-2008). The activation of the NF-kB pathway of FL and DLBCL cases expressed this cancer-testis antigen. This suggests that the was evaluated using an immunostain for p65 (Cell Signaling Technology, polyclonal; PRAME antigen may be a possible target for immunotherapy with our recently developed 1:50) and p50 (Cell Signaling Technology, 178F3; 1:300) subunits of NF-kB. All technology for subsets of FL and DLBCL cases expressing PRAME. Additionally, ANNUAL MEETING ABSTRACTS 281A

PRAME may serve as a marker for minimal residual disease using real-time quantitative 1274 The Sensitivity and Specificity of Granulocyte and Monocyte RT-PCR for these lymphoma cases. However, PRAME expression cases did not provide Immunophenotype Abnormalities in the Detection of Myeloid significant risk stratification data for clinical outcome in our cohort of DLBCL cases. Neoplasms JM Polski. University of South Alabama, Mobile, AL. 1273 Novel 5 Probe FISH Panel Predicts Complex Karyotype in MDS Background: Flow cytometric immunophenotyping (FCI) of peripheral blood and with Inadequate Cytogenetic Evaluation bone marrow is a useful and widely utilized technique for diagnosis and classification AD Pierce, CL Schmitt, BJ Skipper, KK Reichard. Univ of New Mexico, Albuquerque, of hematopoietic neoplasms. However, routine FCI is usually limited to analysis of NM. lymphocytes, blasts, and plasma cells. Granulocytes and monocytes are less commonly Background: A complex karyotype (≥3 cytogenetic abnormalities) in myelodysplastic studied, but compelling evidence exists that detection of granulocyte and monocyte syndrome (MDS) confers an unfavorable prognosis. Karyotype, along with blast abnormalities can be useful in diagnosis of myelodysplastic syndrome (MDS) and percentage and number of cytopenias, predicts MDS outcome according to the IPSS. chronic myeloproliferative disease (CMD). Such outcome prediction is hampered when metaphase karytoping is inadequate. Design: Consecutive cases of peripheral blood (n=47) and bone marrow (n=56) We aimed to identify a targeted FISH panel to predict a complex karyotype in such specimens submitted for FCI due to various clinical indications were included in this situations. prospective study. A four-color flow cytometry data was collected. The granulocyte and Design: We identified 309 cases of MDS with adequate cytogenetics (≥16 metaphases). monocyte immunophenotype was compared to normal controls. Age, sex, diagnosis and karyotypes were catalogued. Chromosome 7 or ≥3 abnormalities Results: Granulocyte and monocyte abnormalities were common in peripheral blood were defined as an unfavorable karyotype, per convention. Karyotypic abnormalities were (46.8%) and bone marrow aspirate (26.8%) specimens. The abnormalities included the correlated with cytogenetic complexity, co-occuring abnormalities and diagnosis. following. Monocytes (number of cases): increased expression of CD56 (20), CD2 (5), Results: Of 309 cases, the M:F ratio was 1.8. 72 (23%) cases had a normal karyotype, CD23 (5), CD7 (3), CD22 (1), HLA-DR (1), and lack of expression of HLA-DR (1). while 237 (77%) were abnormal. Of abnormals, 88 (28%) had unfavorable karyotypes, Granulocytes (number of cases): lack of significant granulocyte maturation (7), decreased including 22 cases of non-complex chromosome 7 abnormalities. Exclusively complex side light scatter (4), lack of expression of CD61 (2), expression of CD25 (3), and CD56 abnormalities included -5, followed by -13, -12/12q, -20 and -19/19q (Fig. 1). Monosomy (1). The number of abnormalities ranged from 0 to 4 per case. Only 19 of 38 abnormal 5 co-segregated with -13 (p=0.04): no other significant correlation occurred. FISH cases had evidence of myeloid neoplasia such as acute myeloid leukemia (AML), for -5, -13, -12/12q, -20 and -19/19q would detect 58% of complex cases with 100% MDS, or CMD (specificity of 50%). The overall sensitivity was 73%. The specificity specificity. FISH for -18/18q, -5, -16/16q, -17/17q, -13, and -12/12q would increase of individual abnormalities ranged from 20-100%. Three or more abnormalities had detection to 65% with minimal decrease in specificity (99%) (Fig. 2). specificity of 100%, but were present in only 2 cases. Conclusions: The results confirmed the published data showing high prevalence of granulocyte and monocyte abnormalities in MDS and CMD. Similar abnormalities were present in many cases of AML. This study also documented additional, not previously reported, granulocyte and monocyte abnormalities such as expression of CD25 in granulocytes, lack of expression of CD61 in granulocytes, and expression of CD22 in monocytes. These findings can be useful in clinical flow cytometry.

1275 High-Risk Genetic Prognostic Factors Including 17p(p53) Deletion, 1q21 Gains and 1p21 Loss Do Not Affect Bortezomib Therapy Responsiveness and Survival of Relapsed/Refractory Myeloma Patients C Qi, Y Trieu, D Reece, H Chang. University Health Network and University of Toronto, Toronto, Canada. Background: Multiple Myeloma (MM) patients with unfavorable molecular cytogenetics have a poor prognosis irrespective of treatment with conventional chemotherapy or autologous stem cell transplant. We and others have reported the proteasome inhibitor bortezomib is active in relapsed/refractory MM patients with genetic risk factors including chromosome 13q deletions and t(4;14). However, it is unclear whether chromosome 1 abnormalities (1p loss or 1q gains) and 17p(p53) deletion, which are adverse prognostic factors in MM, influence the outcome of bortezomib treated relapsed/refractory MM patients. Design: We evaluated 85 bortezomib treated patients with relapsed/refractory MM and correlated response, survival and genomic status detected by interphase cytoplasmic fluorescence in situ hybridization (cIg-FISH). CIg-FISH was performed on clonal plasma cells from MM bone marrow aspirates with probes to detect del (17p)(p53), del(1p21), and amp (1q21)(CKS1B). Results: Forty-eight (56%) patients had an objective response to bortezomib with median time to treatment failure (TTF) and overall survival (OS) of 5.4 and 15.1 months, respectively. cIg-FISH detected hemizygous 17p(p53) deletion in 15/74 (20%), 1p21 deletions in 17/69 (25%) and 1q21 amplification in 30/76 (39%) cases. There was no significant difference in response to bortezomib for patients with or without 17p(p53) deletions (69% versus 52%, p=0.26). The median TTF (5.4 vs. 5.0 months, p=0.92) or OS (11.5 vs 12.8 months, p=0.34) was similar in patients with and without 17p(p53) deletions. Likewise, the CR rare, TTF and OS were not significantly different in patients with or without 1p deletions or 1q amplifications. Conclusions: Our data suggest that bortezomib is an effective salvage therapy for refractory/relapsed myeloma irrespective of high-risk genetic factors including 17p(p53) deletion, chromosome 1p deletion or 1q amplification.

1276 Detection of Mono/Oligo Clonal IgH Gene Rearrangements and Characterization of Associated Clinicopathologic Features in Acute Myeloid Leukemia X Qiu, P Lin, X Sun, J You, LJ Medeiros, CC Yin. MD Anderson Cancer Center, Houston, TX. Background: It has been shown that immunoglobulin heavy chain (IgH) gene rearrangements, a function of B-cells and plasma cells, can be detected in non-B-cell malignancies including acute myeloid leukemia (AML). However, the clinicopathologic features of this subset of AML has not been studied. Design: We assessed IgH gene rearrangements by PCR using V and J consensus primers Conclusions: In MDS with suboptimal karyotyping, a targeted FISH panel for -5, -13, followed by genescan in 105 AML cases. We also evaluated IgH and Ig kappa light -12/12q, -20 and -19/19q will predict a complex karyotype in 58% of complex cases. chain (IgK) gene expression in six AML cell lines (HEL, HL-60, KG-1, NB-4, THP-1, The standard FISH panel (-5/5q, -7/7q, +8 and del20q) yields minimal information on OCI-AML3) by RT-PCR, Western blot and immunohistochemistry. karyotypic complexity except -5. These findings are clinically relevant, obviating an Results: Monoclonal and oligoclonal IgH gene rearrangements each was detected in additional bone marrow aspirate for karyotyping (given ample interphase tumor cells four cases, accounting for 7.6% of total cases. There were five men and three woman are present for FISH). (age, 33-82 years, median, 68). The leukemias were classified as acute myelomonocytic leukemia (M4, n=5), AML with t(8;21) (n=1), AML arising from myelodysplastic syndrome (AML/MDS, n=1), and therapy-related AML (n=1). Multilineage dysplasia was observed in all cases. Immunophenotyping showed that the blasts were uniformly positive for CD13, CD33, CD34, CD38, CD117, HLA-DR, and myeloperoxidase; CD14 and CD64 were also expressed by the M4 cases. No lymphoid marker was expressed 282A ANNUAL MEETING ABSTRACTS except for CD19 by the case with t(8;21). Six cases had diploid karyotype. The AML/ Conclusions: By using a high-resolution approach to identify chromosomal abnormalities MDS showed del(7q) and del(20q). The other showed t(8;21)(q22;q22). Five cases were in these closely related post-follicular B-cell lymphomas, we demonstrated recurrent positive for Flt-3 internal tandem duplication (ITD). One was positive for Ras mutation. and disease-specific patterns of abnormalities and redefined minimal deleted/amplified All patients received chemotherapy. With a median follow-up of 8 months (range, regions. TNFAIP3, a negative regulator of the NF-kB pathway, was commonly deleted 1-22 months), five died of disease, one had persistent AML, the one with t(8;21) was in npMALT, LPL, SMZL and NMZL, highlighting its potential pathogenetic role. in clinical remission with low-level disease detected by RT-PCR. One patient was lost to follow-up. Studies with six AML cell lines revealed that IgH gene transcripts were 1279 A Novel Translocation in MALT Lymphoma Involving a G-Protein expressed in HEL, KG-1, THP-1, OCI-AML3, and IgK gene transcripts were detected Coupled Receptor (GPR34) and Immunoglobulin Heavy Chain Gene in all. This was further confirmed by Western blot and immunocytochemistry. Activates NFκB Pathway Conclusions: Mono/oligo clonal IgH gene rearrangements is present in a subset of ED Remstein, AJ Novak, T Akaska, M Manske, M Gupta, TE Witzig, MJS Dyer, SM AML. These cases often demonstrate myelomonocytic differentiation, multilineage Ansell, A Dogan. Mayo Clinic, Rochester, MN; University of Leicester, Leicester, dysplasia, diploid karyotype, higher frequency of Flt-3 mutation, poor clinical outcome, United Kingdom. and is not associated with lymphoid marker expression. Further study to evaluate the Background: In this study we present a new translocation in MALT lymphoma which characteristics and function of the rearranged IgH gene is warranted to explore its targets an orphan G-protein coupled receptor (GPR34) and provide evidence that this implications in the pathogenesis, detection of minimal residual disease, as well as may be a unique oncogenic mechanism leading to NFκB mediated oncogenesis in targeted therapy of AML. MALT lymphoma. Design: We used a long-distance inverse polymerase chain reaction (LDI-PCR) 1277 Gene Expression Profiling of Post-Follicular B-Cell Lymphomas technique to clone the breakpoint involving IGH and an unknown partner in a case of Identifies New Biological Insights with Potential Diagnostic and Therapeutic parotid MALT lymphoma. The LDI-PCR results were confirmed by FISH and array- Applications based comparative genomic hybridization (aCGH) analysis (Agilent 244A platform). ED Remstein, WJ Chng, G Huang, D Sun, PJ Kurtin, A Dogan. Mayo Clinic, Rochester, The expression of genes at the breakpoint was investigated by quantitative RT-PCR, MN; National University Hospital, Singapore. global gene expression profiling (GEP) (Affymetrix U133 Plus 2.0 platform). The ability Background: Splenic marginal zone lymphoma (SMZL), mucosa-associated of the genes identified as the target of the translocation to induce NFκB activation was lymphoid tissue (MALT) lymphoma, nodal marginal zone lymphoma (NMZL) studied using a transient transfection system in HeLa cells. Genetic and GEP data from and lymphoplasmacytic lymphoma (LPL) are all low-grade post-follicular B-cell previously characterized MALT lymphoma cases and normal splenic B-ells were used lymphomas that show overlapping morphologic and phenotypic features and whose for comparison purposes. molecular relationship has not been clarified. Our aim was to use gene expression Results: LDI-PCR identified a novel breakpoint involving IGHSA2 on chromosome profiling to identify the molecular relationship between these entities using unsupervised 14 and Xp11.4. The breakpoint on chromosome Xp11.4 was adjacent to two genes, methods, identify disease-specific signatures and markers using supervised methods, GPR82 and GPR34, both of which code for orphan G-protein coupled receptors. The and identify functional implications of these signatures using a modified gene-set breakpoint also fell within and disrupted a larger gene called CASK. FISH and aCGH enrichment analysis. studies confirmed LDI-PCR findings on the original tumor specimen. 64 other MALT Design: Gene expression profiling was performed on frozen tissue from 35 SMZL, 25 lymphomas lacked Xp11.4 region abnormalities by FISH and/or aGCH. Quantitative MALT lymphomas (15 salivary gland, 3 stomach, 3 bowel, 3 lung, 1 lacrimal gland), 24 RT-PCR and GEP found that GPR34 RNA but not GPR82 or CASK was markedly NMZL, 24 LPL (10 Waldenstrom’s macroglobulinemia (WM), 14 non-WM), 4 normal overexpressed by the tumor, indicating that GPR34 gene is deregulated upon its lymph nodes and 4 normal spleens using the Affymetrix U133plus 2.0 chip. translocation to the IGHSA2 switch region. Interestingly, to a lesser extent GPR34 Results: Using hierarchical clustering, MALT lymphoma formed a tight cluster mRNA was overexpressed in most MALT lymphomas compared to normal B-cells regardless of tumor site. In addition, SMZL and NMZL formed another large cluster. suggesting mechanisms other than the translocations may upregulate GPR34. Transient LPLs segregated into 2 main clusters corresponding almost uniformly to WM/LPL and expression of GPR34 in HeLa cells resulted in increased phosphorylation of Ik-Ba non-WM/LPL types, with occasional samples interspersed among the SMZL and NMZL. indicating that GPR34 could activate NFκB pathway. Using supervised analysis with ANOVA across the different disease groups, we identified Conclusions: 1. We identified a novel IGH translocation partner in MALT lymphoma, clusters of genes that were specifically overexpressed in MALT lymphoma (MMP7, LTF GPR34. 2. GPR34 is expressed by MALT lymphomas irrespective of the translocation. and SFRP2) and LPL (PRDM1 (BLIMP1), XBP1 and TNFRSF17 (BCMA)). Consistent 3. In vitro, overexpression of GPR34 results in activation of the NFκB pathway and with the unsupervised analysis, SMZL and NMZL had little difference and shared the may be a novel mechanism MALT lymphoma oncogenesis. 4. Targeting GPR34 may over-expression of genes such as CD22 and WNT3. Clustering of these samples based be a therapeutic option in MALT lymphoma. on the pathways and genesets that were enriched in the individual tumors as compared to their normal tissue counterparts showed a mutually exclusive pattern with significant 1280 Selective Expression of the Immunoregulatory Lectin, enrichment of NFKB-related genesets and genes in LPL and those of B-cell receptor Galectin-1, Identifies Precursor B Cell Acute Lymphoblastic Leukemias signaling in SMZL and NMZL. (ALL) with MLL Rearrangements Conclusions: In describing the molecular relationship between these closely related SJ Rodig, P Juszczynski, K Takeyama, J Ouyang, J Faber, S Armstrong, MA Shipp, JL post-follicular B-cell lymphomas (SMZL, NMZL, MALT and LPL), we identified Kutok. Brrigham & Women’s Hospital, Boston, MA; Dana-Farber Cancer Institute, novel diagnostic markers that may help distinguish these lymphomas from one another, Boston, MA; Children’s Hospital, Boston, MA. and also identified molecular pathways that are differentially activated in the different Background: Translocations of the mixed lineage leukemia (MLL) gene on chromosome lymphomas. These may represent potential therapeutic targets. 11q23 are found in over 70% of infant leukemia and associated with a poor prognosis. MLL translocations generate a new chimeric gene, in which N-terminal portion of MLL 1278 Post-Follicular B-Cell Lymphomas: A High Resolution Array- is fused to C-terminal sequence from multiple different partners. Given the variety Based Analysis Identifies Disease-Specific Patterns of Chromosome of MLL fusion partners, molecular techniques such as FISH or Southern blotting are Abnormalities not able to detect all genetic abnormalities involving MLL. In addition, these time- ED Remstein, E Braggio, WJ Chng, G Huang, M Barrett, D Sun, R Fonseca, A Feldman, consuming techniques are not always available at initial diagnosis. A Dogan. Mayo Clinic, Rochester, MN; Mayo Clinic, Scottsdale, AZ; National Design: To identify molecular markers of MLL translocation that might be used in University Hospital, Singapore; Translational Genomics, Phoenix, AZ. rapid diagnostic assays, we compared the transcriptional profiles of primary ALLs with Background: Low-grade post-follicular B-cell lymphomas including splenic marginal and without MLL translocations. Galectin-1 (Gal1) transcripts were significantly more zone lymphoma (SMZL), mucosa-associated lymphoid tissue lymphoma (MALT), nodal abundant in MLL-rearranged ALLs (MLL-ALL) from two extensive and independent marginal zone lymphoma (NMZL) and lymphoplasmacytic lymphoma (LPL) show datasets. overlapping morphologic and phenotypic features and their molecular relationship is Results: Consistent with the differences in transcript abundance, Gal1 protein was unclear. We performed a comprehensive high-resolution study to identify distinctive significantly more abundant in MLL+ ALL cell lines than in MLL- ALL lines by molecular markers to define these lymphomas genomically. western blotting. To assess the diagnostic utility of Gal1 expression in ALL subtypes, Design: We analyzed 119 frozen specimens (22 pulmonary MALT (pMALT), 20 non- we performed Gal1 immunostaining of a large series of primary ALLs with known MLL pulmonary MALT (npMALT), 34 SMZL, 21 NMZL and 22 LPL (8 Waldenström’s status. All MLL-rearranged pre-B ALLs had abundant Gal1 expression (10/10 cases, Macroglobulinemia (WM) and 14 non-WM)), using human Agilent 244A platform by 100%); in marked contrast, only 1 of 38 pre-B ALLs without an MLL translocation array-based comparative genomic hybridization (aCGH) analysis. expressed Gal1 (3%), p<0.001. We also evaluated Gal1 expression in ALL subtypes by Results: 93% of patients had an altered genome. LPL had the most copy number flow immunophenotyping and found its expression to be specific to MLL+ cells. The abnormalities (median 7.5/patient; range: 0-44), followed by npMALT (6.5; 0-33), deregulated gene expression in MLL+ leukemias may be related to the altered histone NMZL (5; 0-31), SMZL (4; 0-56) and pMALT (3.5; 1-11). LPL had the largest overall methyltransferase activity. Thus, we analyzed histone 3 lysine 79 (H3K79) dimethylation size of copy number abnormalities (average 245.15Mb/patient), followed by NMZL in the Gal1 promoter region and found that it was 5 fold higher in MLL+ pre-B ALL (188.6Mb), npMALT (187.2Mb), SMZL (126.4Mb) and pMALT (109Mb). Partial/total cells than in control cells, suggesting that this epigenetic modification may be a means gains of chromosome 3 and 18 were ubiquitous and were most common in npMALT for Gal1 overexpression in MLL. (60% and 50%, respectively) and pMALT (27% each). Trisomy 12 was also ubiquitous. Conclusions: We conclude that: 1) Gal1 is a novel, highly specific marker of ALLs 6q deletions were identified in LPL (55%), npMALT (35%), SMZL (12%) and NMZL harboring an MLL translocation. 2) Detection of Gal1 by immunohistochemical staining (5%), encompassing a minimal deleted region (MDR) at 6q23 that included TNFAIP3. or flow immunophenotyping is amenable to routine diagnostic pathology, and 3) Gal1 SMZL and npMALT had 7q deletions, delineating two MDR. LPL, NMZL and SMZL overexpression is likely driven by the altered histone methyltransferase activity of the had interstitial 11q deletions, NMZL had monosomy 13, and LPL and SMZL had MLL fusion protein complex. interstitial 13q14 deletions encompassing MIRN15A and MIRN16-1. Some recurrent abnormalities were exclusive to specific entities: 1p deletions and 1q gains in NMZL; a focal 2p gain including BCL11 and REL in npMALT, partial 8q gains in LPL, and trisomy 4 in LPL/WM. ANNUAL MEETING ABSTRACTS 283A

1281 Angioimmunoblastic T-Cell Lymphoma with Hyperplastic CD25, partially expressing PD1, negative for FoxP3. - B-cells showing high expression Germinal Centres (Pattern I): Clinicopathological and Immunohistochemical of markers reporting for; proliferation (Ki67), apoptosis( Low Bcl2, High MCL1 and Study of 10 Cases BIRC5), NF-kB activation (IkBα-p, MUM1, TRAF1 and nuclear NFKB1) and BCR M Rodriguez-Justo, A Attygalle, P Munson, G Roncador, MA Piris. University College signalling (CD40, ZAP70). London Hospital, London, United Kingdom; Royal Marsden Hospital, London, United Conclusions: PCs seem to constitute a distinctive tissue compartment, where NF-kB Kingdom; Spanish National Cancer Centre, Madrid, Spain. activation could take place in response to signals from specific dendritic and T-cell Background: AITL is an aggressive T-cell lymphoma which natural history is not fully subpopulations. Interestingly, PC B-cells exhibit a strong expression of cytoplasmic understood. Up to 16% of cases show a pattern of hyperplastic germinal centres (pattern and nuclear TRAF1, a molecule described to be induced in response to CD40 signalling I) and the majority of these cases are initially misdiagnosed as reactive hyperplasia. by B-cells. MCL1 expression by PCs could inhibit pro-apoptotic signals from B-cell The correct identification of AITL - pattern I remains a challenge and the aim of this receptor mediated through NF-kB. study is to characterize phenotypically this variant with the use of follicular T-cell (TFH) markers PD1 (antibody NAT-105) and CXCL13. 1284 MUM1/IRF4 Is Expressed in a Subset of Follicular Lymphoma Design: AITL cases from files of the Spanish National Cancer Centre and University with BCL6 Translocation and Short Survival College London were reviewed and 10 cases with pattern I were identified. Cases were HJ Rogers, J Bodo, T Jin, TE O’Brien, ED Hsi. Cleveland Clinic, Cleveland, OH; stained for CD3, CD4, CD8, CD30, CD21/CD23, CD10, CXCL13 and PD1. EBV MetroHealth Medical Center, Cleveland, OH. status was assessed by LMP1 and/or EBER and PCR for IgH and TCR was performed Background: There is a lack of prognostic markers that predict the highly variable to evaluate clonality. Clinical and demographic data were obtained from referring clinical outcomes of follicular lymphoma(FL). MUM1/IRF4 is a transcription factor physicians. 21 lymph node biopsies with reactive lymphoid hyperplasia (including of post-germinal center(GC) B lymphocytes which plays a role in terminal phases of HIV lymphadenopathy) were used to compare PD1 and CXCL13 expression in reactive differentiation. Normally, there is an inverse relationship between BCL6 and MUM1 vs. neoplastic cases. expression in the GC. However, we have observed MUM1 expression in a subset of Results: 10/10 (100%) cases showed strong positive PD1 staining in the “outer zone” FL cases, which may have a more aggressive clinical course in terms of requirement of the lymphoid follicle, perifollicular area and around high endothelial veins. CXCL13 for treatment and outcome in other data sets. We evaluated MUM1 expression in a new showed variable staining in neoplastic cells in 8/10 cases. In 10/10 cases CD21/CD23 cohort of FL cases to further characterize their pathologic features. highlighted only a subtle expansion of follicular dendritic cells. In reactive hyperplasias, Design: Tissue microarray(TMA) blocks were constructed in 44 newly diagnosed PD1+ve cells were found in the germinal centre with a few T-cells in the interfollicular FL cases. We performed immunohistochemistry(IHC) for MUM1, CD10, BCL2 and T-cell zone. EBV was found in 9/10 cases. Clinically 6/7 cases presented with stage BCL6, and interphase FISH for BCL2 and BCL6 rearrangements. IHC stains were IIIB/IV B and in 2/10 cases consecutive biopsies showed “progression” to classical considered positive if >20% of cells expressed the marker. Cytologic grade, clinical patterns II and III (diffuse). stage, FLIPI score and overall survival(OS) were obtained. Quantum-dot(Qdot) dual Conclusions: PD1 has useful diagnostic utility in the distinction and identification of immunofluorescence(IF) for MUM1 and BCL6 was performed on a subset of MUM1 AITL with hyperplastic germinal centres in conjunction with the appropriate clinical positive cases to examine the coexpression of the markers. setting. CXCL13 is variable expressed and its diagnostic utility is limited. PD1 identifies Results: MUM1 was expressed in 13 cases(29.5%). BCL2, CD10, and BCL6 were neoplastic cells in the outer zone of the lymphoid follicle, suggesting that AITL – pattern expressed in 100%, 89% and 91%, respectively. BCL2 and BCL6 translocations were I may originate from T cells in the outer zone, in harmony with previous immunological FH seen in 90% and 18%, respectively. MUM1 positive cases showed higher cytologic grade studies. As the majority of cases in our series presented clinically with stages IIIB/ than MUM1 negative cases(P=.037, Fisher’s Exact). Interestingly, MUM1 expression IVB, progression from pattern I to patterns II and III may represent “histological was associated with BCL6 translocation(46.2%, P=.003, Fisher’s Exact) but not with transformation” rather than clinical progression. other markers. Univariate Cox regression analysis of pathologic and clinical features showed that MUM1 expression, BCL6 translocation and lack of BCL2 translocation 1282 Cutaneous CD4-Positive Small/Medium-Sized Pleomorphic were associated with shorter OS(hazard ratio 4.06, 7.86 and 8.05, respectively; P<.02 T-Cell Lymphoma Expresses Follicular T-Cell Markers for each). In multivariate analysis, BCL6 and BCL2 translocations retained significance. SM Rodriguez-Pinilla, G Roncador, JL Rodriguez-Peralto, M Mollejo, JF Garcia, S Qdot IF stain confirmed bright coexpression of MUM1 and BCL6 in 49.2% of MUM1 Montes-Moreno, FI Camacho, P Ortiz, MA Limeres-Gonzalez, A Torres, E Campo, P positive FL cells. Navarro-Conde, MA Piris. CNIO, Madrid, Spain; Hospital 12 de Octubre, Madrid, Spain; Conclusions: MUM1 expression in FL was associated with higher cytologic grade, Hospital de Gran Canaria, Gran Canaria, Spain; Hospital Rio Hortega, Valladolid, Spain; BCL6 translocation and shorter OS. Aberrant coexpression of MUM1 and BCL6 was Hospital Clinic, Barcelona, Spain; Hospital Arnau de Vilanova, Valencia, Spain. confirmed in MUM1 positive FL cells. Although some caution may be necessary because Background: Cutaneous CD4+ small/medium-sized- pleomorphic T-cell lymphoma of limited study size and heterogeneous treatment, MUM1 expression in FL may be a (CSTCL) is a cutaneous T- cell- lymphoma defined by a predominance of small-to- biologic predictor of aggressive clinical outcome in FL. medium-sized- CD4+- pleomorphic- T- cells, with a favorable clinical course. Cases are also characterized by the presence of a rich infiltrate of reactive B- cells. Recently, 1285 Arsenic Trioxide/IFN-α Combination Eradicated ATLL in SCID it has been reported that follicular helper- T- cells (TFH- cells) display a distinct gene Mice: A Focus on Morphology expression profile, positive for PD-1, CXCL13, and BCL-6. We investigate here the ST Saab, H El Hajj, A Bazarbachi, G Zaatari. American University of Beirut, Beirut, expression of PD-1 and other T - cells markers in CSTCLs and discuss its biological FH Lebanon. significance. Background: We reproduced a previously described Adult T-cell leukemia/lymphoma Design: Sixteen CSTCLs were included in this study, 20 reactive inflammatory (ATLL) mouse model to evaluate systemic involvement, disease progression and survival conditions, 10 primary cutaneous marginal zone, 10 cutaneous follicular center and 5 effects of arsenic trioxide (As), interferon-α (IFN) and their combination (As/IFN). primary CD30+ cutaneous lymphomas. They were immunohistochemically analyzed Design: Splenic lymphoma cells were transfered from Tax transgenic mice to SCID for a large panel of markers. Double immunoperoxidase labeling of paraffin sections mice by IP injection. After inoculation (d0), SCID mice were randomized to receive As, was performed for PD-1, OCT-2, and BCL-6. Clonal Ig and TCR rearrangements and IFN, or As/IFN(d6-30);10 mice per group. Another As/IFN group (N=10) underwent EBV (EBER) expression were also evaluated. Morphological and clinical data were identical treatment protocol. Mice were sacrificed at d21, 26, and 30. Finally, As/IFN reviewed. in two treatment intervals (d6-30 and 42-54)was tested on ten mice. The spleen, liver, Results: There was a dense polymorphic lymphoid infiltrate throughout the dermis. lung, bone marrow and kidney were harvested for gross examination and paraffin Atypical- large CD4-positive cells were positive for PD1, CXCL13, and BCL6 in all embedding for all mice. cases, and were attached in small clusters, or formed rosettes around CD30/OCT2- Results: ATLL was established with untreated mice showing extensive organ positive B-cell blasts. EBV was not detected in any of the cases. A dominant T-cell involvement(26d average survival). As and IFN showed significant survival advantages clone was identified in 14 cases, while all cases were polyclonal for IgH. None of the over untreated mice and similar systemic involvement. All died of disease(50 and 45d, patients had lymphadenopathy, showed any evidence of systemic disease, nor did have respectively). any previous history of mycosis fungoides or drug reactions.

Conclusions: FTH-cell markers are not exclusive to angioimmunoblastic T-cell lymphoma, but may also be seen in neoplastic cells of CSTCLs and rarely in mycosis fungoides. Moreover, these findings suggest that B cell stimulation by THF could also take place in some cutaneous T -cell lymphomas, thus offering an explanation for the increased number of reactive B-cells regularly present in this lymphoma type.

1283 “Pseudfollicles”, Survival Niches for B-CLL Cells Due to NF-K B Activation Pathway SM Rodriguez-Pinilla, B Herreros, I Castillo, P Algara, S Montes-Moreno, R Diaz de Otazu, M Perez Guillermo, MJ Mestre, C Bellas, MA Piris. CNIO, Madrid, Spain. Background: Proliferation centres (PCs) are a distinctive tissue finding in Chronic Lymphocytic Leukaemia, of unknown significance. Cell composition analysis of proliferation centres may clarify the functional relevance of this tissue compartment. Design: A series of 75 lymph nodes in CLL cases using both tissue microarrays (TMA) and whole tissue sections have been analyzed immnuhistochemically for a large panel of markers. Results: Specific cell subpopulations present in PCs have been identified, such as: - Dendritic cells, positive for SDF1(CXCL12), STAT1 and actin, negative for other markers of interdigitating and follicular dendritic cells. - T-cells coexpressing CD4 and 284A ANNUAL MEETING ABSTRACTS

related to fetal type erythropoiesis (hemoglobin F expression) in MDS patients. Conclusions: CA-I was a superior marker to other erythroid markers for the identification of erythroid dysplasia in paraffin-embedded bone marrow trephine biopsy sections. In combination with CD34, myeloperoxidase, and CD42b, CA-1 immunohistochemistry may be a useful adjunct for histological diagnosis of MDS.

1287 Nutlin-3 and Velcade Synergistically Induce Cell Cycle Arrest and Apoptosis in Multiple Myeloma through Activation of p53 Pathway MN Saha, J Jayakar, H Chang. University Health Network and University of Toronto, Toronto, Canada. Background: p53 is rarely mutated in multiple myeloma (MM). p53 protein levels are regulated by murine double minute 2 (MDM2) through a well-established autoregulatory feedback loop. Modulation of the wild-type-p53 (wt-p53) levels in multiple myeloma (MM) has been reported by a recently developed small molecule, Nutlin-3 that antagonizes MDM2 and disrupts the p53-MDM2 interaction. Velcade, a proteasome inhibitor, has been shown to trigger apoptosis in MM cells in vitro. In this study, we investigated synergistic activation of the p53 by these two drugs and explored their effect on cell cycle arrest and apoptosis in MM cells. Design: Two human myeloma cell lines MM1.S (shown to carry wt-p53) and LP1 (harboring mutant p53 gene) were used in this study. MM cells were exposed to velcade or nutlin-3 either individually or in combination, after which cell viability, cell cycle pathways and apoptosis was monitored. The degree of induction of p53 As/IFN cured 40% of mice. At 6mo sacrifice, organs showed near normal morphology. function was assessed by Western blot (WB) analysis of the samples immunostained The drug combination tripled survival in the rest (57d). Survival was further enhanced with antibodies against p53 and its two immediately downstream transcriptional targets, with two courses of As/IFN (80% still alive at 14 mo) and tripled survival (59d) in those p21 and MDM2. that died of disease; slightly improved from the single treatment interval group. Results: Both velcade and nutlin-3 up-regulated p53 expression in MM1.S cell lines. Combined treatment of 1 µM of nutlin for 24 hr complemented with 5 nm of velcade for last 6 hr could further increase the up-regulation of expression of p53 along with p21 and MDM2 indicating a synergistic activation of p53. This synergistic effect was also manifested by 30-40% reduction in the number of viable cells. These events were associated with p53-dependent cell cycle arrest at G1-S check point as observed by a reduction of S phase fraction with an increase of the G1 phase fraction. Cell cycle arrest was followed by induction of apoptosis as evaluated by Annexin-V and propidium iodide (PI) binding and flow cytometry analysis and caspase-3 activation by WB analysis. Either velcade or nutlin-3 could lead to apoptosis individually. However, their combined treatment could lead to a significantly higher percentage of the cells into apoptosis. These events leading to specific p53 activation were observed only in the cells carrying wt-p53 (MM1.S) but not in the cells with mutant p53 (LP1). Conclusions: Our data suggest that velcade and nutlin-3 can synergistically activate p53 pathway to induce cell cycle inhibition and apoptosis in vitro. The combination of these two drugs provides a potential novel therapeutic approach for MM patients.

1288 Analyses of Micro-RNA Expression in Classical Hodgkin Lymphoma Tumors B Sanchez-Espiridion, J Alves-ferreira, M Canales, ME Rodriguez, MM Morente, MA Piris, JF Garcia. Spanish National Cancer Centre, Madrid, Spain; Hospital La Paz, Despite persistent organ involvement at d21, 26 and 30, splenic weights gradually Madrid, Spain; MD Anderson International, Madrid, Spain. declined with significant tumor regression at d30, signifying a delayed tumor response Background: Micro-RNAs (miRNAs) are small non-coding RNAs which regulate the to As/IFN. expression of multiple target mRNAs, playing a key role in the control of the different Conclusions: This in vivo model is useful in the morphological evaluation of tumor biological processes implicated in cancer pathogenesis. Initial studies are showing behavior under chemotherapy effect. As/IFN significantly increased survival and cured that miRNA losses and gains may explain some of the biological and clinical features a subset of mice. Addition of a second treatment course further enhanced survival and of different lymphoma types, and recent analyses of miRNA expression in classical the proportion of cured mice. Hodgkin Lymphoma (cHL) cells and tumors have also demonstrated deregulated expression of some miRNAs. 1286 Carbonic Anhydrase I Immunohistochemistry Is Useful for Design: To further analyze the implication of miRNAs in the pathogenesis of cHL, we Detecting Erythroid Dysplasia in Paraffin-Embedded Bone Marrow have profiled miRNA in a series of advanced cHL patients and cell lines, including 29 Sections fresh frozen lymph samples of cHL whose clinical data were availble. As controls, 4 Y Sadahira, J Tsuchiyama, H Nishimura, T Akiyama, I Irei, S Hamazaki. Kawasaki fresh frozen lymph nodes and 4 fresh frozen tonsils were used. We also analyzed miRNA Medical School, Kurashiki, Japan. profiles from five cHL-derived cell lines (L-540,L-1236,L-428,HDLM-2 and KMH-2) Background: In paraffin-embedded tissue erythroid precursors (EP) are not easily allowing us to characterize microRNA signatures corresponding to tumor HRS cells differentiated from other type of precursors. Moreover, it is difficult to recognize and their reactive cell microenvironment. Hybridization was done using total RNA with erythroid dysplasia. To date, antibodies against hemoglobin, glycophorin, spectrin Agilent 8x15K Human miRNA Microarray Kit v.2 for detection of 723 human and 76 (Sadahira et al. J Clin Pathol 1999;52:919-21), and E-cadherin have been used to identify human viral microRNAs (Agilent Technologies). erythroid cells in paraffin sections. However, the ability of these antibodies to identify Results: We identified 123 miRNAs differentially expressed between cHL tumors and erythroid dysplasia has not been reported. controls (37 upregulated and 86 downregulated miRNAs in tumor samples). Some of the Design: Bouin’s fluid-fixed paraffin-embedded bone marrow trephine biopsy sections most downregulated (miR-31, miR-5905p, miR-285p,) and upregulated (hsa-miR-220c, (300 cases of hematological disease including 52 cases of MDS and MPD/MDS from hsa-miR-526b, hsa-miR-495) miRNAs have been previously related to other cancer 2005 to 2008) were stained with rabbit anti-human carbonic anhydrase (CA-I) antibody types. Targets of these miRNAs included some of the genes identified as deregulated (Nordic Immunology) (1:200 dilution) using antigen-retrieval technique and automatic in cHL, like CHEK1, BUB1, CDK9 (involved in mitotic chekpoint regulation) and immunostainner. The specificity of the antibody was determined by Western blotting some transcription factors such us E2F. Interestingly, the miRNA signature in cHL of erythrocytes. The bone marrow sections were also stained with CD34 for blasts, also included some EBV-encoded miRNAs (upregulation of miR-BART12 and myeloperoxidase for myeloid cells, CD42b for megakaryocytes, and eryhtroid markers downregulation of miR-BHRF1-3) potentially associated with the viral latency status (hemoglobin A and F, glycophorin C, and E-cadherin). and that may serve as an immunomodulatory mechanism in the tumors. In addition, Results: Anti-CA-I antibody resulted in diffuse cytoplasmic and nuclear staining of EP the availability of clinical data allowed us to identify a set of miRNA with prognostic but not that of other hematopoietic cell types. The late EP showed a slightly stronger and predictive capacity in advanced cHL. stainining than early EP. In contrast to anti-glycophorin C (Ret49f), immature erythroid Conclusions: Our results suggest possible roles of human miR-31, miR-590, miR- cell clusters were clearly identified in hematopathologic samples because erythrocytes 220c, hsa-miR-526b, and viral (EBV-encoded) miR-BART12; miR-BHRF1-3 in the showed faint staining for CA-I. The anti-CA-I also stained giant proerythroblasts in pathogenesis of cHL. These results will be validated in an additional series of patients human parvovirus B19 infection and UT7/EPO (an erythropoietin-dependent cell line using RT-PCR paraffin embedded samples. possessing characteristics of CFU-E), suggesting that this antigen is expressed from the early erythroid differentiation stage. However, EP of fetal liver showed weaker staining than EP of adult bone marrow. In MDS and MPD/MDS, megaloblastoid change, binucleated EP, and impaired EI formation could be pointed out using CA-I immunohistochemistry because loosely arranged megaloblastoid EP showed fairly weaker staining for CA-I. This abnormal expression of CA-I in erythroid cells may be ANNUAL MEETING ABSTRACTS 285A

1289 Structural Alterations of the 14q32/IGH Locus in CLL Revealed by Cases were analyzed for histological features, immunophenotype (CD20, CD79a, Fluorescence In Situ Hybridization and Array-Based Comparative Genomic cIg, MUM1, CD30, CD15), EBER in situ, and TCR and IgH gene rearrangement by Hybridization PCR. Cases were classified as 1) hyperplasia with inc. EBV+ cells (RH); 2) nodal or RL Sargent, LV Abruzzo, MJ Keating, WG Wierda, LJ Medeiros, R Luthra, D Jones. 3) extranodal polymorphic EBV+ B-cell lymphoma (N-polylym/E-polylym); 4) nodal UT-MD Anderson Cancer Center, Houston, TX. EBV+ DLBCL including plasmablastic lymphoma (PBL). Background: In chronic lymphocytic leukemia (CLL) alterations of the immunoglobulin Results: RH was diagnosed in 28; median age 67(45-93); M=F. All cases tested were heavy chain (IGH) locus on chromosome 14q32 have prognostic significance; however, polyclonal by IgH PCR. T-cell clonality or a restricted TCR pattern was seen in 3. their structure and frequency are poorly characterized. In our study we used fluorescence Features included preserved architecture, spectrum in cell size of EBV+ cells with in situ hybridization (FISH) and array-based comparative genomic hybridization (CGH) frequent localization to germinal centers. Most patients had self-limited disease. 1 patient to better characterize these alterations. progressed to N-polylym in 1 year. There were 38 N-polylym; median age 73(30-94); Design: Within our database we identified 66/2759 CLL cases (2.4%) with 14q32 18M:20F. Most patients had generalized LNs. IgH PCR was clonal in 22/33(66%); TCR alterations by conventional karyotypic analysis (CC), of which 85% were of IGH- was clonal in 22%; 33% showed a restricted pattern and TCR was polyclonal in 44%. unmutated type. We performed FISH and CGH in a subset of these cases (9 bone EBV+ cells exhibited a range in cell size. Cells were positive for CD79a, MUM1, and marrow), and compared them to cases without a history of 14q32 alterations by CC (7 CD30 but often negative for CD20. Clinical progression was common. There were 35 bone marrow and 2 peripheral blood) and to 5 normal controls. FISH was performed E-polylym; median age 76 (55-101); 21M:14F. Most common sites were oral cavity, using a break-apart probe to the IGH locus (Vysis) on either interphase cells or formalin- including gingiva, tongue, and lips (13); tonsil or nasopharynx (7); adrenal gland (4); GI fixed, paraffin-embedded tissue sections. CGH was performed using a custom-designed tract (3). 58% of cases tested were monoclonal by IgH PCR. A clonal or restricted TCR CGH array that contained 44,000 60-mer oligonucleotide probes with an average spatial pattern was seen in 17%. Hodgkin-like (HRS) cells were common. Immunotype showed resolution of 75 kilobases augmented with additional probes spanning the IGH gene CD79a+, CD20 variable, MUM1+, CD30+. HRS-like cells expressing CD30 and CD15 locus (Agilent Technologies) and labeled genomic DNA hybridized against labeled were seen in 5 cases in sites unusual for CHL (oral cavity, skin, adrenal). There were 11 pooled normal reference DNA. nodal DLBCL and 4 PBL in nodal or extranodal sites; median age 77; M=F). Results: 5/9 cases (55%) with 14q32 alterations by CC showed IGH rearrangements by Conclusions: RH with increased EBV+ cells is frequent in the elderly, but is rarely FISH, of which 4/5 (80%) appeared non-balanced by CGH, with 0.1-0.3 megabase (mb) a precursor to EBV+ B-cell lymphoma. Most EBV+ B-cell LPDs are extranodal, deletions in the IGH locus. Of the 4/9 remaining cases with 14q32 alterations by CC, 2 resemble those seen in the post-transplant setting, and may contain CD30+/CD15+ cells showed deletions of IGH/14q32 by FISH/CGH and 2 showed no detectable alterations mimicking CHL. IgH PCR is usually clonal, but clonal T-cell populations are often seen by FISH/CGH. 2/9 cases without 14q32 abnormalities by CC showed deletions of and may reflect an antigen-driven response in an immunosenescent host. IGH/14q32 by FISH/CGH. The remaining 7/9 cases without 14q32 abnormalities by CC did not demonstrate abnormalities by FISH but showed 0.1-0.5 mb deletions within 1292 Uniform Expression of Notch1, Suppressor of B-Cell Specific Gene the IGH loci by CGH. No significant 14q32/IGH alterations were noted by CGH in Expression, in Plasmablastic Lymphoma 4 normal samples. AC Seegmiller, A Maleki, W Kabbani, P Shang, HY Wang, W Chen. UT Southwestern Conclusions: Although CC identifies alterations of 14q32 in a small percentage of Medical Center, Dallas. CLL cases (largely restricted to the unmutated subtype), FISH and CGH identify Background: While the loss of B-lineage specific gene expression is a distinctive additional 14q32 alterations, including apparent cryptic translocations and deletions feature of plasmablastic lymphoma (PL), the underlying mechanism remains poorly of the IGH locus. Further, a customized array design provides additional accuracy understood. Notch1 signaling is central for T vs. B-lineage decision in that it promotes in delineating the IGH/14q32 aberrations compared to FISH. The high incidence of T-cell development, while inhibiting B-cell development by interfering with the activity small deletions within the IGH locus identified by CGH may reflect normal lineage- of transcription factors E2A and EBF. In addition, Notch1 signaling also positively associated VDJ recombination, or tumor-associated genetic instability or promiscuous regulates the mTOR pathway. In this study, we explore the mechanism of loss of recombination. B-cell phenotype by correlating expression of B-cell markers with that of Notch1 and downstream targets of the mTOR pathway. 1290 A Novel Flow Cytometric Antibody Panel for Distinguishing Burkitt Design: Institutional database searches yielded 9 cases of PL with adequate Lymphoma from CD10-Positive Diffuse Large B-Cell Lymphoma material for further study. Immunophenotypic analyses were performed by both SD Schniederjan, S Li, MJ Lechowicz, DF Saxe, PD Terry, KP Mann. Emory University immunohistochemistry (IHC) and flow cytometry (FC). IHC markers included B-cell School of Medicine, Atlanta; Winship Cancer Institute, Emory University School of antigens bcl-2, bcl-6, CD79a, PAX-5, and MUM1, mTOR pathway proteins 4E binding Medicine, Atlanta; Rollins School of Public Health, Emory University, Atlanta. protein-1 (4EBP-1) and phospho-S6-ribosomal protein (pS6), as well as Notch1. IHC Background: Rapid and accurate differential diagnosis between Burkitt lymphoma for human herpesvirus 8 (HHV8) and in situ hybridization for EBV encoded RNA (BL) and CD10+ diffuse large B-cell lymphoma (CD10+ DLBCL) is imperative as (EBER) were also performed. their treatment differs. Undertreatment of BL results in treatment failure, whereas Results: Patients included 6 males and 3 females with a median age of 42 years (range overtreatment of DLBCL adds toxicity without improving response rate or survival. 31-59). Five patients presented with lymph node involvement, 1 with an extramedullary Distinguishing BL from CD10+ DLBCL can be problematic, especially in cases with mass, and 3 with primary BM involvement. All were HIV positive except one with minimal material for evaluation. Recent studies have characterized several antigens post-transplant immunocompromise and all showed evidence of viral infection by either differentially expressed in these two types of lymphoma. Our goal was to determine EBV(+) (8/9 cases) or HHV8(+) (1/9 case). All cases were characterized by a diffuse whether use of these markers would aid in the differential diagnosis of BL versus CD10+ proliferation of medium-sized to large malignant cells with variable plasmablastic DLBCL by flow cytometric immunophenotyping (FCI). morphology. They exhibited almost uniform loss of B-cell associated markers, including Design: Twenty-five cases of CD10+ B-cell lymphomas with available cryopreserved CD19 (expressed in 0/9 cases), CD20 (0/9), CD10 (2/9), CD79a (2/9), PAX-5 (1/9), samples were identified (15 cases of BL with confirmed MYC gene rearrangements bcl-2 (0/9), bcl-6 (0/9), sIg (2/9), and icIg (3/9). MUM1, required for plasma cell and 10 CD10+ DLBCL). Four normal controls, 10 cases of low grade follicular differentiation, was positive in 9/9 cases. Notch1 was expressed in all 9 cases; the pattern lymphoma, 3 cases of precursor B-cell acute lymphoblastic leukemia, and 3 cases of was mixed nuclear and cytoplasmic in most cases (6/9), predominantly cytoplasmic high grade B-cell lymphoma (not further characterized) were also analyzed. Samples in 2 cases, and predominantly nuclear in 1 case. 4EBP-1 and pS6 were detected in a were thawed and multiparameter FCI was performed using the following antibodies: majority of cases (6/7 and 7/7 respectively). CD18, CD20, CD43, CD44, CD54, and isotype controls. The difference between mean Conclusions: Uniform expression of Notch1 correlated with loss of B-cell markers fluorescence intensity (channel values) and isotype controls was calculated for each and activation of the mTOR pathway in PL. This suggests that activation of Notch1 antibody. Two-tailed T tests were performed comparing antigen expression between is involved in repression of B-cell specific gene expression and global loss of the BL and CD10+ DLBCL. B-cell phenotype. Thus, there might be a role for Notch1 and mTOR pathways in the Results: Expression of CD44 and CD54 was detected at a significantly lower level in pathogenesis and therapy of PL. BL as compared to CD10+ DLBCL (p=0.01 and p=0.005, respectively). There was not a significant difference in expression of CD18 and CD43 (p=0.38 and p=0.40, 1293 Pattern of Cytoplasmic IgM Expression in Hematogones: A Flow respectively). Cytometric Study Conclusions: Distinguishing between BL and CD10+ DLBCL is imperative to rapidly DW Sevilla, A Colovai, FN Emmons, MB Levin, G Bhagat, B Alobeid. Columbia initiate appropriate therapy. Our data shows that expression of CD44 and CD54 differs University, New York, NY. significantly between these lymphomas. Therefore, a flow cytometric immunophenotypic Background: Hematogones are physiologic B-cell precursors in the marrow. panel including these antibodies may prove valuable for differential diagnosis of Despite their well-characterized phenotype and maturation patterns, problems CD10+ B-cell lymphomas. Prospective studies are necessary to confirm that using persist in differentiating hematogones from the lymphoblasts of precursor B-cell these markers, in conjunction with routine antigen analysis, will allow differential acute lymphoblastic leukemia (B-ALL). The pattern of cytoplasmic IgM (cyt IgM) diagnosis of these two entities. expression in the context of the three recognized maturational stages of hematogones has not been characterized. This expression is particularly relevant as a proportion of 1291 EBV Reactivation Syndromes in Adults without Known B-ALL demonstrate cyt IgM expression. In this study, we investigated the pattern and Immunodeficiency maturational sequence of cyt IgM expression in hematogones to provide an additional JA Schrager, S Pittaluga, M Raffeld, ES Jaffe. NCI, Bethesda, MD. resource in differentiating hematogones from lymphoblasts. Background: EBV-related B-cell lymphoproliferative diseases (LPDs) constitute a Design: We prospectively analyzed 12 bone marrow aspirate samples by 3-4 color flow broad spectrum. Recent studies have identified EBV+ B-cell lymphomas in the elderly. cytometry obtained between February and August 2008. In addition to our standard We wished to identify possible precursors of EBV+ B-cell lymphoma, and to characterize comprehensive antibody panel, we included additional antibody combinations: CD34/ the phenotype, genotype, and possible relationship of these cases to classical Hodgkin cyt IgM, TdT/cyt IgM and CD10/cyt IgM to further characterize the patterns of cyt lymphoma (CHL) . IgM expression. Populations were considered negative if <10% of gated cells expressed Design: 116 benign or malignant EBV+ B-LPDs were identified in a 7 yr period. Pts the antigen. with known lymphoma, autoimmune disesease, or immunodeficiency were excluded. 286A ANNUAL MEETING ABSTRACTS

Results: Using CD10/CD19 in combination the percentage of double positive cells representing the entire hematogone population ranged from 2.1 to 23.1% (median 3.7%). Expression of cyt IgM by hematogones varied depending on the level of maturation, however, two clear populations were identified in all bone marrow aspirate samples: cyt IgM negative early hematogones (TdT+/CD34+/CD10+bright/CD19+dim) ranged from 0.1% to 7.5% (median 0.9%) and cyt IgM positive more mature hematogones (TdT-/ CD34-/CD10+dim, CD19+bright) ranged from 0.6% to 20.2% (median 3.1%). Cytoplasmic IgM Expression Early Hematogones Intermediate Hematogones CD34+ TdT+ CD19dim CD10bright CD34- TdT- CD19bright CD10dim Negative Negative Negative Negative Positive Positive Positive Positive (12/12) (7/7) (12/12) (12/12) (12/12) (7/7) (12/12) (12/12) Conclusions: Our findings document a biphenotypic pattern of cyt IgM expression that closely correlates with the stage of hematogone maturation, and indicate that the acquisition of cyt IgM occurs early in transition to the intermediate stage. In contrast to cyt IgM+ B-ALL, there is a spectrum of cyt IgM expression in normal precursor B-cells without aberrant or asynchronous expression. In conjunction with the previously described maturation patterns of B-cell antigens, this highly reproducible pattern of cyt IgM expression should aid in differentiating hematogones from residual or recurrent Design: Immunohistochemistry studies were performed on AML (n=60) and non- leukemic lymphoblasts. neoplastic control bone marrow (n=10) paraffin tissue sections utilizing a monoclonal antibody directed against the N-terminal region of SALL1. Bone marrow samples 1294 Aurora-A Kinase Distinguishes ALK+ Anaplastic Large Cell from each group (AML, n=3; control, n=3) were tested by quantitative real time PCR Lymphoma from ALK Negative and Cutaneous ALCL among Other T-Cell using SALL1-specific primers to determine if SALL1 mRNA expression was affected Lymphomas: Analysis of 72 Cases in AML patients. RK Shamanna, LJ Medeiros, L Whiteley, DS Schultz, DA Chitale, KV Inamdar. Henry Results: We demonstrated that SALL1 was upregulated in 44/60 AML patients and not in Ford Hospital, Detroit, MI; The University of Texas MD Anderson Cancer Center, normal controls. Consistent with its role as a transcription factor, SALL1 histochemical Houston, TX. staining was nuclear. Background: Aurora-A kinase (AA), a cell cycle-regulating Ser/Thr kinase, plays a key role in the tumorigenesis of a variety of solid tumors as well as highly aggressive B-cell non-Hodgkin lymphoma (NHL). Expression of AA has not been assessed to date in T- NHL. Thus, we performed this study to assess AA expression in a variety of T-cell lymphoma types. Design: We assessed 72 T-cell NHL for AA expression by immunohistochemistry (Table). A mouse monoclonal AA-antibody was used (Bethyl Labs, USA). Any cytoplasmic and/ or nuclear staining was considered positive. Each case was semi-quantitatively graded for percentage of positive cells (0-25%; 25-50%; >50%) and staining intensity (1-3+). AA mRNA expression was also assessed by real-time quantitative reverse transcriptase Polymerase Chain Reaction (RT-PCR) in 9 ALCL and 9 PTCL, NOS cases. Results: AA was detected in 48/72 (67%) T-NHL; most frequently in ALKpositive (ALK+)ALCL (100%) and peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS) (100%) compared with other T-NHL. AA expression was primarily cytoplasmic in ALK+ALCL and predominantly nuclear in other T-NHL types (p=<0.001). In the ALCL group, ALK+ ALCL more frequently had cytoplasmic AA compared with ALK negative (ALK-) (p<0.001) or cutaneous ALCL (c-ALCL) (p<0.001). 2+ or 3+ staining intensity was more frequent in ALK+ (67%) compared with ALK- (9%, p=0.03) or c-ALCL (43%, p=0.05). ALK+ALCL (55.6%) more frequently had >50% positive tumor cells than c-ALCL (0%) (p=0.0264). RT-PCR revealed higher AA mRNA expression in ALK+ ALCL compared with ALK-, c-ALCL or PTCL, NOS cases. Table T-NHL AA Expression n (%) Cytoplasmic(%) Nuclear(%) ALCL 22 (81.4) 50 50 ALK+ 9 (100) 100 0 ALK - 8 (73) 25 75 Cutaneous 5 (71.4) 0 100 These findings were confirmed by quantitative real time PCR where SALL1 mRNA was T-ALL 3 (60) 0 100 undetectable in all control samples and significantly upregulated inAML patients. Mycosis Fungoides 4 (67) 0 100 Extranodal NK/T cell, nasal type 3 (50) 0 100 Conclusions: Data in mice and humans has linked the overexpression of SALL1 T-PLL 2 (67) 0 100 to AML neoplasias. Since the nucleosome remodeling complex that associates with ETL 1 (16.7) 0 100 SALL1 has been shown to regulate tumorogenesis in other cancers, it is tempting to Subcutaneous panniculitis-like 1 (33.3) 0 100 speculate that the upregulation of SALL1 is functionally significant for the molecular Angioimmunoblastic 3 (42.8) 33.3 66.7 pathogenesis of AML. PTCL,NOS 9 (100) 33.3 66.7 Conclusions: AA is a useful marker for distinction of AKL+ ALCL from ALK- and c-ALCL. Its overexpression in ALK+ ALCL implicates a key role in the pathobiology 1296 Bcl-6 Coexpression in Mantle Cell Lymphoma: A Rare Finding of ALCL. AA might be a molecular target for treatment of T-NHL. Associated with a Lack of CD5 and/or CD3 Coexpression AJ Shaw, AM Gown, HC Hwang, PL Kandalaft, LC Goldstein, SJ Kussick. PhenoPath Laboratories, Seattle, WA; IMPRIS, Seattle, WA. 1295 Upregulation of the Developmental Regulator, SALL1, in Human Background: The mantle cell lymphoma (MCL) literature contains occasional reports Leukemias on variant immunophenotypes, such as absence of CD5 or coexpression of CD10, but L Shatat, S Kiefer, L Robbins, Q Xie, M Rauchman, L Grosso, H Salman. Creighton does not appear to include publications specifically evaluating bcl-6 coexpression. University, Omaha, NE; St Louis University, St Louis, MO. Because we have seen rare cases of bcl-6-positive MCL, and recognition of such rare Background: SALL1, a human homologue to Drosophila spalt, is a zinc finger cases is diagnostically important, we performed a comprehensive search of our database transcription factor that regulates normal development. In humans, SALL1 is located over the last four years to identify such cases. on chromosome 16q12.1, encodes a protein of 1324 amino acids, and interacts with Design: Our database was searched for all cases of MCL over the last four years. Five a nucleosome remodeling complex. Mutations in human SALL1 cause the autosomal cases of cyclin D1-positive, t(11;14)-positive MCL were identified that also expressed dominant disorder, Townes-Brocks syndrome (TBS). In our lab, we have created a bcl-6 in 10% or more of the tumor cells. These cases were compared to a control mouse model of TBS syndrome that phenocopies the human syndrome and expresses group comprised of our 20 most recent cyclin D1-positive, bcl-6-negative MCL cases. a truncated SALL1 protein. In addition to the TBS limb, ear, anal, and kidney defects, The clinical and immunophenotypic features of the 2 groups were compared. To be we observed that these mice also display severe myelodysplastic syndrome/ acute considered “positive” for expression of a given antigen, at least 10% of the MCL cells myeloid leukemia (AML). had to express that antigen. Results: There were no significant differences between the bcl-6-positive MCL and bcl-6-negative MCL groups in the mean age, male to female ratio, or anatomic site of presentation. By immunohistochemistry, the cases showed comparable intensity of CD20 and cyclin D1 expression, and no significant difference in the Ki-67-defined proliferative rate. All cases in both groups were negative for CD10. Interestingly, 4 of ANNUAL MEETING ABSTRACTS 287A

5 (80%) of the bcl-6-positive MCLs lacked CD5 coexpression, and 3 of 5 (60%) lacked 1299 Three-Dimensional (3D) Image Analysis of Benign vs Malignant CD43 coexpression, in contrast to only 10% CD5-negativity and 19% CD43-negativity Follicular Lymphoid Proliferations (FLPs) among the bcl-6-negative control group. AR Sohani, V Brodsky, AM Ackerman, EP Hochberg, JR Gilbertson, Y Yagi. Massachusetts Conclusions: Bcl-6 expression is a recurrent, variant immunohistochemical finding General Hospital, Boston; Mass General Hospital Cancer Center, Boston. in a small number of MCL cases, and tends to be associated with lack of CD5 and/or Background: Morphologic distinction between florid reactive follicular hyperplasia CD43 coexpression. Given the existence of this rare variant of MCL, and the clinical (RFH) and follicular lymphoma (FL) may be challenging. We studied whether 3D importance of distinguishing MCL from other B cell lymphomas of small to intermediate histologic reconstruction of scanned whole-slide serial sections could aid in this cell size, we would strongly recommend that cyclin D1 staining be performed in any distinction by highlighting aspects of nodal architecture that are difficult to visualize biopsy in which the histologic features suggest the possibility of MCL, even if CD5 on conventional 2-dimensional (2D) sections. and/or CD43 are negative, and bcl-6 is positive, on the neoplastic B cells. Design: A case each of florid RFH involving tonsil and low-grade FL involving a lymph node were selected for 3D analysis. 100 5µm-thick H&E-stained serial sections were 1297 Immunoexpression of Cell Cycle Molecules Participated in P27Kip1 made from a formalin-fixed paraffin-embedded block of each. Serial sections were Degradation in B-Cell Lymphomas scanned with 0.33µm/pixel resolution using Mirax Scan device (3DHISTECH Ltd, C Sirinian, A Symeonidis, N Giannakoulas, H Vlotinou, M Melachrinou. Medical School, Carl Zeiss Microimaging GmbH); 3D reconstruction was performed using 3DHistech’s University of Patras, Patras, Greece. Mirax Viewer software. Images of serial sections were aligned to produce a 3D stack. Background: Evidences suggest that deregulation of p27 plays a critical role in the Limited computational power forced us to decrease the 3D model’s resolution. However pathogenesis of many human tumors. The inactivation of p27 is achieved through either the model could be sectioned in various planes, freely rotated and moved, exposing 3D degradation via the ubiquitin-mediated proteolytic pathway or sequestration by cyclins relationships. Spacing between scanned sections was varied dynamically during the D. Skp2, a component of ubiquitin-ligase SCF complex, interacts with p27 only when analysis to emphasize/explore specific features. it is phosphorylated on Thr-187 (Pp27) by cyclin E/CDK2 and/or cyclin A/CDK2. We Results: Morphologic features that were significantly enhanced upon 3D reconstruction examined the expression of skp2, p27, Pp27, cyclin E, cyclin A and CDK2 in B-cell and analysis over conventional 2D examination included: back-to-back follicles in FL vs lymphomas to determine whether alterations in their relative levels are associated with RFH; higher volume of cortex relative to paracortex in FL vs RFH; patency of vascular changes in cell proliferation and lymphoma groups. sinuses in RFH vs FL; and intact mantle zones with asymmetric polar thickening in Design: Formalin-fixed, paraffin-embedded tissue from 105 cases of B-cell lymphomas RFH vs FL. Polarization and cellular content of individual follicles (eg distinct dark/ [Group A: 66 DLBCLs, Group B: 39 small B-cell lymphomas (9 FLs {3 Grade 1, 6 Grade light zones, # centroblasts vs centrocytes, # mitoses and tingible-body macrophages 2}, 8 MCLs {no blastoid}, 12 MZLs, 10 SLLs] were immunostained with antibodies [TBMs]) were only moderately enhanced by this analysis. The relatively low resolution for skp2, p27, Pp27, cyclin E, cyclin A, CDK2 and Ki67 using Envision detection kit. of the 3D model precluded extensive analysis of cellular interactions (eg lymphocyte- Clinical data were available for 52 cases [29/Group A, 23/Group B]. dendritic cell interactions) within follicles. Results: Group A showed a significant higher immunoexpression of skp2 (30.07% vs Conclusions: Certain low-power morphologic features that help distinguish benign from 5.32%), Pp27 (34.15% vs 6.79%), cyclin E (21.33% vs 5.19%), cyclin A (22.89% vs malignant FLPs are significantly enhanced by 3D image analysis. Such analysis may be 5.31%) and CDK2 (23.11% vs 6.80%), and a higher proliferation index (PI) (69.08% cumbersome for routine diagnostic use in straightforward cases of RFH and low-grade vs 17.27%) as well, compared to Group B. On the contrary, Group B demonstrated a FL, but may help distinguish RFH from grade 3 FL which share many high-power significant more intense immunoreaction for p27 (64.46% vs 31.18%) (p < 0.05). In morphologic (large # centroblasts; high # mitoses and TBMs) and immunohistochemical both groups was detected a positive correlation between 1) skp2 and PI, 2) cyclin A (high Ki67, bcl2 negativity) features within follicles. In the future, computational power and PI, 3) CDK2 and PI, 4) cyclin E and cyclin A, 5) cyclin A and CDK2. In Group A will increase to allow higher resolution 3D analysis. was identified a positive correlation between 1) p27 and Pp27, 2) skp2 and cyclin A, 3) skp2 and CDK2, 4) cyclin E and CDK2. In Group B was observed a positive correlation 1300 MYC Gene Copy Increase Is Common in Diffuse Large B-Cell between 1) skp2 and Pp27, 2) Pp27 and a) PI, b) cyclin A and c) CDK2 (p < 0.05). The Lymphoma studied molecules were not related with patients’ prognosis. CJ Stasik, H Nitta, JR Cook, RR Tubbs, TM Grogan, LM Rimsza. University of Conclusions: Our findings suggest the p27 degradative function of Skp2 in small Arizona, Tucson, AZ; Ventana Medical Systems, Inc., Tucson, AZ; Cleveland Clinic, B-cell lymphomas. Although this relationship is not detected in DLBCLs, the Cleveland, OH. overexpression of P-p27 is indicative that the proteolysis of p27 has been triggered. Background: Recently, mRNA expression levels of candidate genes identified in the Our data demonstrate a positive correlation between Skp2 expression and PI in B-cell literature as having prognostic significance in diffuse large B cell lymphoma (DLBCL) lymphomas. The positive relationship between P-p27 and PI in small B-cell lymphomas were evaluated. HLADRB and MYC emerged as independent indicators of survival and is consistent with the findings of previous studies which detected the expression of formed a strong 2-gene model of outcome prediction (Rimsza et al, Blood 2008). Possible P-p27 in proliferating cells. mechanisms of MYC over-expression in DLBCL include t(8;14)IGH/MYC, increased MYC copy number and chromosome 8 hyperploidy. In this study, cases with known MYC 1298 High Proliferation Rate in Mantle Cell Lymphoma Is Associated expression levels were evaluated in order to evaluate the underlying mechanisms. with p53 Mutations and Blastoid Morphology, but Not with 3’UTR Deficient Design: Thirteen DLBCL cases (labeled A through M) were identified with known Cyclin D1 mRNA or High Total Cyclin D1 mRNA Levels MYC gene expression status and separated into high (7) and normal (6) MYC groups. J Slotta-Huspenina, I Koch, L de Leval, G Keller, K Bink, M Kremer, M Raffeld, F Fend, Mechanisms for MYC over-expression were evaluated using fluorescence in-situ L Quintanilla-Martinez. Institute of Pathology, Technical University, Munich, Germany; hybridization (FISH) for chromosome 8 ploidy, MYC gene alterations using a breakapart Institute of Pathology, Helmholtz Center Munich, Munich, Germany; 3 Institute of MYC probe, or the presence of t(8;14) IGH/MYC using fusion probes. Silver in-situ Pathology, University of Tuebingen, Tuebingen, Germany; University of Liège, Liège, hybridization (SISH, Ventana Medical Systems, Tucson, AZ) for MYC was used to Belgium; National Cancer Institute, Bethesda. evaluate the original 13 cases as well as 40 additional DLBCL cases (tissue microarray) Background: The hallmark of MCL is overexpression of cyclin D1 (CyD1). for increased MYC copy number. Predominance of 3’UTR-deficient CyD1 mRNA and genomic 3’UTR alteration were Results: FISH screening utilizing a break-apart probe demonstrated one case (H) with found to be associated with a “proliferation signature” and poor prognosis. The aim a MYC translocation not involving the 14q32.3 IGH locus. Fusion probes were used to of our study was to correlate the expression of CyD1 mRNA isoforms and genomic evaluate MYC copy number and chromosome 8 (CEP8) and the MYC/CEP8 ratio was 3’UTR alterations, as well as secondary genetic alterations (p53, p16 and c-myc) with calculated. Two cases (H and I) had increased MYC and CEP8 signals with a MYC/ morphology and proliferation rate, as measured by MIB 1 staining in primary MCL. CEP8 ratio near 1. Further FISH analysis revealed 3/13 cases with trisomy 8 (F, H, I), Design: Paraffin-embedded tissue of 62 CyD1+MCL (10 blastoid and 52 typical) two cases with an extra MYC signal and normal chromosome 8 number (F, H) and one were analysed for expression of CyD1 3’UTR isoforms and genomic 3’UTR case with a borderline number of nuclei demonstrating tetrasomy 8 (I). SISH found deletion by real-time RT-PCR and genomic qPCR, respectively. Findings were 2/13 cases with increased MYC copy number (H, I). No cases with normal MYC levels correlated with morphology and proliferation rate (MIB 1 pos <10%, 11-30%, had increased MYC copy number or chromosome 8 hyperploidy. Analysis of additional >30%). P53 overexpression, as surrogate marker for p53 mutations was assessed by cases by SISH found increased MYC gene copy number in 20/40 cases. immunohistochemistry (>20%) and confirmed by DHPLC and sequencing analysis. Conclusions: Trisomy 8 or increased MYC copy number was shown in 43% of cases with P16 deletions and c-myc gene alterations were analysed by FISH. increased MYC expression and 50% of cases with unknown expression. The remaining Results: Predominance of CyD1 3’UTR deficient mRNA was found in 14 (22.6%) cases with increased MYC levels did not show MYC or chromosome 8 alterations samples, including 4 with complete loss of the standard transcript, accompanied by implying MYC upregulation may occur by alternate mechanisms. Chromosome 8 genomic UTR deletion in two cases. P53 overexpression/ mutation was found in 12 hyperploidy and/or increased MYC gene copy number likely represent common cases. 3’UTR predominance was significantly associated with high total CyD1 mRNA mechanisms of MYC gene over-expression in DLBCL. levels (mean CyD1/TBP 87.2 vs 62.1, p=0.01) and blastoid morphology (5/10, p=0.04), but not with high proliferation rate (6/18, p=0.32). Instead, high proliferation rate 1301 Systemic Mastocytosis (SM) Is Frequently Masked by Its (>30%) was significantly correlated with blastoid morphology (9/10, p<0.001) and Associated Clonal Hematological Non-Mast Cell Lineage Diseases p53 overexpression (11/12, p<0.001). In five highly proliferative cases without p53 (AHNMD): Clinicopathologic Study of Five Cases mutations other secondary genetic alterations were investigated. In none of the cases M Stoecker, E Wang. Duke University Medical Center, Durham, NC. p16 deletions or c-myc translocations were identified. Background: Isolated systemic mastocytosis (SM) usually shows significant mast cell Conclusions: Predominance of the 3’UTR CyD1 deficient mRNA and CyD1 3’UTR aggregates, which are relatively easy to identify by careful microscopic examination. deletion in MCL is associated with high total CyD1 mRNA level and blastoid In cases of SM with associated hematological non-mast cell disease (SM-AHNMD), morphology but not with high proliferation rate, as assessed by MIB 1 staining. In however, the overwhelming non-mast cell neoplasm frequently obscures the mast contrast, proliferation rate is highly associated with blastoid morphology and p53 cell infiltrates and the SM component is missed on primary diagnosis. We report our overexpression. experience with the diagnosis of SM in five cases of SM-AHNMD and discuss the clinical implications of mastocytosis in SM-AHNMD. 288A ANNUAL MEETING ABSTRACTS

Design: Cases of SM-AHNMD were identified from our databases, using the search There were some segmental gains and some segmental loss that occurred in PL but not word “mastocytosis”. In addition to morphologic examination, all cases were also in the other types of lymphoma suggesting that these foci may contain genes responsible evaluated by flow cytometry, cytogenetics, and immunohistochemistry with mast cell for the differentiation of this lymphoma. Additionally, some segmental gains and some tryptase, CD117, CD25, CD2, etc. Mast cell loads were assessed on consecutive bone segmental loss occurred only in PL and AIDS associated DLBCL suggesting that these marrow biopsies in each patient. foci may be associated with HIV infection. Furthermore, some segmental gains and Results: Of the 5 cases of SM-AHNMD, the AHNMD component was acute myeloid some segmental loss occurred only in PL and PCM suggesting that these lesions may leukemia (AML) in 2 cases and chronic myelomonocytic leukemia (CMML), be related to plasmacytic differentiation. myelodysplastic syndrome (MDS), and marginal zone B-cell lymphoma (MZL) in 3 Conclusions: To the best of our knowledge, the current study represents the first separate cases. The component of SM was missed in primary diagnosis in 4 cases. SM genomic exploration of PL. The genomic aberration pattern of PL appears to be more was unmasked after induction chemotherapy with profound aplasia of leukemic blasts similar to that of DLBCL (AIDS-related or non AIDS-related) than to PCM. Our and hematopoiesis in the AML cases. Mastocytosis persisted during the entire course findings suggest that PL may remain best classified as a subtype of DLBCL at least of chemotherapy, and AML relapsed after a short remission. SM in MZL was noted at the genome level. after successful treatment of lymphoma. The original biopsies in these cases were retrospectively evaluated, and the SM component was confirmed. SM in the CMML case 1304 Objective Quantification of Osteosclerosis by Area Pixel Count was identified in a repeat biopsy before any intervention. In the MDS case, a compact of Bone Marrow Biopsy as a Diagnostic Tool in Myeloproliferative mast cell infiltrate obscured myelodysplasia; however, it was identified along with a Neoplasms clonal cytogenetic abnormality after brief treatment for SM. CJ Teman, AR Wilson, SL Perkins, JT Prchal, M Salama. University of Utah, Salt Lake Conclusions: SM component in SM-AHNMD is frequently masked by the AHNMD City, UT; ARUP Laboratories, Salt Lake City, UT. and revealed by therapy-induced aplasia or repopulation of hematopoietic elements. This Background: Myeloproliferative neoplasms (MPN) are a heterogeneous disease group indicates mast cells’ resistance to current chemotherapy, and persistent mastocytosis with overlapping clinical and histological features, leading to frequent diagnostic in cases of myeloid malignancies may predict an imminent relapse of leukemia. The difficulties. Fibrosis can occur in late stages of several MPN, including primary therapeutic approach for SM-AHNMD should be targeted to both components to myelofibrosis (PM), essential thrombocythemia (ET), chronic myelogenous leukemia achieve sustained remission; however, the optimal treatment remains to be further (CML) and polycythemia vera (PV). Osteosclerosis frequently accompanies fibrosis, but investigated. has not achieved widespread recognition in diagnostic algorithms of MPN, reflecting a lack of objective quantification methods. We propose a method of osteosclerosis 1302 Clinical Utility of Fluorescence In Situ Hybridization for Detection quantification in marrow cores, and hypothesize that trabecular volume (TV) can be of Del(5q) in Myelodysplasia and Acute Myeloid Leukemia used as a diagnostic criterion to differentiate MPN. Y Sun, JR Cook. Cleveland Clinic, OH. Design: The study group included 68 patients with established MPN, including PM Background: Fluorescence in situ hybridization (FISH) studies for del(5q) may assist in (n=24), ET (n=21), PV (n=15), and untreated CML (n=8). The control group consisted the diagnosis and classification of myelodysplastic syndromes (MDS), and may also help of 47 negative lymphoma staging marrows in age and gender-matched patients. Biopsies guide the choice of therapy, given the efficacy of lenalidomide in cases containing this were digitally scanned with the ScanScope XT system (Aperio, Vista, CA). The entire abnormality. It is currently unclear which chromosomal loci are the most appropriate to hematopoietic area and each individual bony trabecula were circled manually; and examine for detection of del(5q) in routine practice, and it is unclear in which situations areas of cortical bone, fragmentation, and crush artifact were excluded. Areas were FISH studies may increase the diagnostic yield over metaphase cytogenetics (MC) quantitated using the ImageScope software’s pixel count algorithm, and trabecular area alone. Two commonly deleted regions have been described on chromosome 5q: one was divided by total area to calculate the TV. occurs in acute myeloid leukemia (AML) and high grade MDS and includes EGR1 at Results: The control group had an average TV of 15.7 ± 4.7%. The categories of MPN 5q31, while the second, occurring in at least some cases reported as 5q- syndrome, is with higher TV included PM (33.8 ± 11.4%, p<0.0001), myelofibrosis secondary to PV centered around CSF1R at 5q33. We first examined whether FISH for EGR1, CSF1R (29.7 ± 8.5%, p=0.005), and myelofibrosis secondary to ET (24.8 ±11.7%, p=0.037). or both provides the greatest sensitivity for detection of del(5q). Next, we examined PV without fibrosis (20.0 ± 7.1%), ET without fibrosis (21.6 ± 8.7%), and CML (18.9 ± the additional diagnostic yield of FISH studies over MC alone. 5.9%) had greater TV than the control group, but not to a statistically significant degree. Design: First, FISH for EGR1 and CSF1R (Abbott Molecular, Abbott Park, IL) were Although the PM group had the greatest overall TV, there was no statistically significant performed on 51 archived bone marrows (AML, n=21; MDS, n=30) containing del(5q) difference when compared to groups with myelofibrosis secondary to either ET (p=0.055) by MC. Next, results of FISH for EGR1 (performed at a reference laboratory or at the or PV (p=0.32). However, PM displayed a striking increase in TV compared to prefibrotic Cleveland Clinic) were compared to results of MC in 183 bone marrow samples obtained PV (p=0.0007), prefibrotic ET (p=0.0018), and CML (p<0.0001). for known or suspected MDS or AML. Conclusions: These findings suggest that trabecular volume can serve as an objective Results: Of 51 cases containing del(5q) by MC, FISH studies were positive for EGR1 tool for quantification of osteosclerosis in MPN, and that the degree of osteosclerosis deletion in 49 (96%), including each of 8 cases of 5q- syndrome. CSF1R deletion was can help differentiate PM from other MPN. identified in 44/48 cases (92%), each of which also showed deletion ofEGR1 . 2 cases (4%) were positive for EGR1 deletion but normal for CSF1R, while 2 cases showed 1305 A Characterization of Angiogenesis and VEGF Expression in del(5q) by MC only. In 183 cases of known or suspected MDS/AML, MC showed an Gastric MALT Lymphoma, Chronic Gastritis, and Normal Stomach abnormal karyotype in 57 (31%) including del(5q) in 15 cases (8%). FISH studies were G Turner, MJ Morgan, RK Orr, M Eldibany. Evanston Northwestern Healthcare, positive for EGR1 deletion in 18 cases overall, including each case with del(5q) by MC Evanston, IL. and 3 cases that showed no growth or fewer than 20 normal metaphases by MC. Background: Vascular Endothelial Growth Factor (VEGF) has been implicated in the Conclusions: FISH for EGR1 deletion detects del(5q) in the vast majority of cases development of solid tumor and hematological malignancies. In addition to promoting of MDS or AML containing this abnormality, including at least most cases of 5q- angiogenesis, VEGF increases microcapillary permeablility to tumor cell metastasis. syndrome. Additional studies for CSF1R deletion rarely increase the diagnostic yield. Recent research in a mouse model has suggested that VEGF mediated angiogenesis is In routine practice, MC alone provides reliable detection of del(5q) in most cases, and important in the development of Helicobacter-related, low-grade MALT lymphoma. This a small percentage of cases are detectable only by MC. However, FISH studies assist study examines vascular density and VEGF expression in gastric MALT lymphoma, in the identification of del(5q) when suboptimal numbers of metaphases are obtained chronic gastritis lymphoid aggregates and normal stomach. for MC. Design: The L.I.S. was searched for gastric biopsy specimens diagnosed as “MALT lymphoma” and “chronic gastritis.” 16 paraffin embedded tissue blocks from 13 cases 1303 Genomic Profiling of Plasmablastic Lymphoma Using BAC Array of MALT lymphoma and 17 blocks from 15 cases of chronic gastritis were retrieved. CGH – Revealing Significant Overlapping Genomic Lesions with DLBCL 9 cases of normal stomach were retrieved as controls. Sections from each block J Taylor, W Huang, F Facchetti, E Jaffe, C Chang. The Methodist Hospital/TMHRI, were stained with antibodies against CD34 to highlight vascular endothelial cells. A Houston, TX; Chang-Gung Memorial Hospital, Taiwan; University of Brescia, Brescia, Chalkley counting grid was used to assess vascularity within areas of MALT lymphoma Italy; NCI/NIH, Bethesda, MD. and chronic gastritis lymphoid aggregates. The average diameter was calculated for Background: Plasmablastic lymphoma (PL) is a subtype of diffuse large B-cell vessels within areas of MALT lymphoma and chronic gastritis lymphoid aggregates. lymphoma (DLBCL). Studies have suggested that tumors with PL morphology represent The MALT lymphoma, chronic gastritis and normal stomach specimens were stained a group of neoplasms with clinopathologic characteristics corresponding to different with antibody against VEGFA (Chemicon #MAB3734) and staining was assessed via entities including extramedullary plasmablastic tumors associated with plasma cell light microscopy. myeloma (PCM). The goal of the current study was to evaluate the genetic similarities Results: The Chalkley counting grid overlayed CD34 stained vessels an average of and differences among PL, DLBCL (AIDS-related and non AIDS-related) and PCM 11.0 times per field in the cases of MALT lymphoma and an average of 5.6 times per using array-based comparative genomic hybridization. field in the cases of chronic gastritis (P = <0.001). The average diameter of vessels in Design: Paraffin blocks of PL (n=16), DLBCL (AIDS-related n=13; non AIDS-related the cases of MALT lymphoma was 29.6 µm and was 16.8 µm in the cases of chronic n=13) and PCM (n=8) were retrieved. One H&E section of each case was analyzed. gastritis (P= <0.005). There was no significant difference between VEGF staining of DNA was extracted. Tumor DNA and control DNA was labeled with Cy5 or Cy3, MALT lymphoma aggregates and VEGF staining of lymphoid aggregates in chronic hybridized to array slides and imaged using an Axon 4000B scanner and GenePix Pro gastritis. Gastric epithelium stained focally positive with antibodies against VEGF in 6.0 scanning software. Differences between the log base 2 median minus background most cases of MALT lymphoma, chronic gastritis and normal stomach. values were used as raw data for analysis. After normalization, values for duplicated Conclusions: Vascular density is increased in gastric MALT lymphoma as compared spots representing one clone were averaged. The top 75 clones showing highest degrees to chronic gastritis lymphoid aggregates. The average diameter of vessels in MALT of gain or loss were selected for each case. lymphoma is approximately twice that seen in chronic gastritis. VEGF staining does Results: Examination of genomic data in PL revealed that the most frequent segmental not appear to be increased in gastric MALT lymphoma as compared to chronic gastritis gain (> 40%) include: 1p36.11-1p36.33, 1p34.1-1p36.13, 1q21.1-1q23.1, 7q11.2- lymphoid aggregates. Uninvolved gastric epithelial cells in MALT lymphoma, chronic 7q11.23, 11q12-11q13.2 and 22q12.2-22q13.3. This correlated with segmental gains gastritis and normal stomach stain focally with antibody against VEGF. occurring in high frequency in DLBCL (AIDS-related and non AIDS-related) cases. ANNUAL MEETING ABSTRACTS 289A

1306 MGMT in Primary Nodal Diffuse Large B-Cells Lymphomas: situ hybridization (FISH), we recently showed that some cALCLs have translocations Immunohistochemical Expression and Methylation Status involving IRF4 (interferon regulatory factor-4, or multiple myeloma oncogene-1 S Uccella, C Placidi, S Marchet, R Cerutti, I Carnevali, D Furlan, G Pinotti, C [MUM1]). We undertook the current study to see if IRF4 translocations are specific Capella. University of Insubria - Ospedale di Circolo, Varese, Italy; Ospedale di for cALCL in skin biopsies involved by TLPDs. Circolo, Varese, Italy. Design: Skin biopsies involved by TLPDs from 68 patients were classified by WHO/ Background: Loss of MGMT function has been proposed as favourable prognostic EORTC criteria. Clinicopathologic data for classification included progression/ marker in diffuse large B-cell lymphomas (DLBCLs). MGMT silencing is frequently regression of lesions, history of mycosis fungoides (MF) or other cutaneous TLPD, caused by the hypermethylation of the promoter region. However, the relatioships anatomic site and timing of extracutaneous disease, morphology, immunophenotype, and between MGMT methylation status (MS) and protein expression are not clear and T-cell clonality if needed. Cases that could not be classified definitively were excluded. there are very few studies which compare immunohistochemical expression of MGMT FISH for IRF4 was performed using a home-brew breakapart probe. Most positive cases and MS of its promoter. also were screened for T-cell receptor (TCRA, TCRB, and TCRG) rearrangements. FISH Design: We studied 71 patients with primary nodal DLBCL, treated with was scored by a cytogeneticist using previously established normal ranges based on cyclophosphamide-containing regimens and with available follow-up. MGMT-IR was 95% confidence intervals. detected using a specific monoclonal antibody. DLBCLs showing less than 5% of IR Results: Among cALCLs, 9 of 22 (41%) demonstrated abnormal separation of the neoplastic cells were considered as MGMT-negative. The MS of MGMT promoter IRF4 breakapart probe, indicating an IRF4 translocation. None of the 46 remaining was analyzed in a subset of cases using a quantitative method based on Real Time TLPDs showed IRF4 translocations, including: 12 LyPs; 6 systemic ALK-negative methylation-specific PCR. For each sample we determined a percentage of methylated ALCLs; 3 systemic ALK-positive ALCLs; 12 cases of MF (2 transformed); 2 cases of reference (PMR) using ActB as reference gene and we considered cases with PMR < Sezary syndrome; 1 CD4-positive small/medium-sized pleomorphic T-cell lymphoma; 4 as methylated. The survival analysis was performed using the survanXL program, 2 extranodal NK/T-cell lymphomas, nasal type; 1 subcutaneous panniculitis-like T-cell version 1.14. lymphoma; and 7 peripheral T-cell lymphomas, unspecified. No rearrangements of Results: 20 /71 DLBCL (28%) were negative for MGMT at immunohistochemistry, TCRA, TCRB, or TCRG were identified in cases withIRF4 translocations. and 19 of them were studied for MGMT methylation status. 7 of these cases (41%) Conclusions: Our findings suggestIRF4 translocations are specific for cALCL in skin showed high levels MGMT promoter methylation (M), 5 cases (26%) showed low levels biopsies involved by TLPDs, and support the utility of clinical FISH testing in such cases. of MGMT promoter methylation (U*) and 7 (33%) were unmethylated (U). We also Because not all cALCLs harbor IRF4 translocations, and because such translocations analyzed the methylation status of 20 cases which showed MGMT immunoreactivity rarely occur in extracutaneous peripheral T-cell lymphomas, FISH results should be and 17 of them were U, 2 were U* and 1 was M. The survival analysis showed interpreted in the context of morphology, phenotype, and clinical features. The gene significantly longer event-free survival (EFS) in DLBCLs with high levels MGMT partner(s) and biologic significance ofIRF4 translocations in cALCL remain unknown promoter methylation compared to U and U* DLBCLs. In addition, the presence and merit further study. of immunohistochemical immunoreactivity for MGMT was also associated with a significantly longer EFS. When overall survival was considered, only methylation 1309 CD23 Expression in Plasma Cell Myeloma Is Specific for status was a significant predictive factor. Abnormalities of Chromosome 11, and Is Associated with Primary Plasma Conclusions: These data suggest that both MGMT promoter methylation status and Cell Leukemia in This Cytogenetic Sub-Group protein expression are prognostic factors in primary nodal DLBCLs treated with MP Walters, H Olteanu, P Van Tuinen, SH Kroft. Medical College of Wisconsin, cyclophosphamide-containing regimens. Cases with low levels of methylation have the Milwaukee, WI. same behaviour of unmethylated DLBCLs in the survival analysis. Immunohistochemistry Background: CD23, a low-affinity Fc receptor for IgE, plays a pivotal role in IgE seems to be an useful tool to identify unmethylated cases, while a methylation analysis homeostasis. It is present on a subset of B-cells, as well as on some activated T cells, should be performed when there is no or low expression of MGMT. monocytes, Langerhans cells, eosinophils, and macrophages. CD23 expression has been well-characterized in non-Hodgkin lymphoma, and has diagnostic utility in the 1307 Classification of Amyloidosis in Fat Aspiration Specimens distinction of chronic lymphocytic leukemia and mantle cell lymphoma. However, Using Mass Spectrometry Based Proteomics to our knowledge, CD23 expression has not been previously studied in plasma cell JA Vrana, SR Zeldenrust, JD Theis, JD Gamez, PJ Kurtin, A Dogan. Mayo Clinic, myeloma (PCM). Rochester, MN. Design: Fifty diagnostic bone marrows containing PCM (including 4 cases of Background: Abdominal subcutaneous fat aspiration (SFA) is one of the most practical, primary plasma cell leukemia (PCL)) were evaluated for CD23 expression by sensitive and specific methods for the diagnosis of systemic amyloidosis. One limitation immunohistochemistry (IHC). CD23 was also evaluated by flow cytometry (FC) in of this method, compared to more invasive tissue biopsy based approaches, remains select cases. Additional immunophenotypic features were derived from routine FC the technical difficulties in further classification of the amyloidosis as commonly performed at diagnosis, and included assessment of CD19, CD20, CD45, and CD56 used methods, such as immunohistochemistry, are not readily applicable to SFA in most cases. Expression of CD23 was correlated with presenting laboratory data and specimens. To overcome these difficulties we developed a method using nano-flow cytogenetic findings. liquid chromatography electrospray tandem mass spectrometry (LC-MS/MS) that Results: Five PCM cases (10%) expressed CD23 by IHC; FC analysis performed in two could identify amyloid subtypes in freshly obtained Congo Red positive SFA specimens of these confirmed the CD23 expression. In all 5 cases, CD23 expression was strong with great accuracy. and uniform on the PCM cells. Compared to negative cases, CD23(+) PCM was more Design: Abdominal SFA specimens were obtained from 73 patients with clinical likely to express CD19 (2/5 vs 0/41; p=.01) and less likely to express CD56 (1/5 vs suspicion for systemic amyloidosis. One half of the specimen was stained with Congo 33/43; p=.021). Three of 4 cases of primary PCL expressed CD23 (p=.002). Four of the red and used for diagnosis of amyloidosis and the other half was processed and enzyme 5 CD23 (+) harbored a t(11;14), and the fifth had a +11. Six CD23 negative cases also digested for LC-MS/MS analysis. The resulting LC-MS/MS data was correlated to contained a t(11;14). Among t(11;14) (+) cases, there were no significant differences theoretical fragmentation patterns of tryptic peptide sequences from the Swissprot in immunophenotype and presenting laboratory data (blood counts, M-protein level, database using Scaffold (Mascot, Sequest, and X!Tandem search algorithms). Peptide creatinine, beta-2 microglobulin, calcium) between CD23(+) and CD23(-) cases. Overall, identifications were accepted if they could be established at greater than 90.0% t(11;14) (+) PCM was more likely to express CD20 (4/10 vs 2/35, p=.016) and less probability and protein identifications were accepted if they could be established at likely to express CD56 (4/10 vs 29/36, p=.02). Compared to non-leukemic PCM, PCL greater than 90.0% probability and contain at least 2 identified spectra. The identified was associated with expression of CD45 (4/4 vs 16/39, p=.039) and CD19(2/4 vs 0/42, proteins were subsequently examined for the presence or absence of amyloid related p=.006), and less frequent CD56 (1/4 vs 33/44, p=.069). peptides. Conclusions: CD23 is expressed by neoplastic plasma cells of PCM in 10% of cases, Results: Of the 73 cases studied, 41 were positive for Congo red consistent with systemic and shows a strong association with abnormalities of chromosome 11, particularly the amyloidosis. In Congo red positive cases, LC-MS/MS peptide profiles consistent with t(11;14), and with primary PCL. Our study corroborates previously reported associations AL-lambda (28/31), AL-kappa (6/7), and ATTR (2/3) were observed. Only one case of t(11;14) with expression of CD20 and lack of CD56 in PCM. The biologic role of in the Congo red negative control group (n=32) gave a peptide profile containing an CD23 in PCM remains to be determined. CD23 could represent a therapeutic target in amyloidogenic protein (AL-kappa) which was attributed to a high level of kappa in the the subset of positive cases. serum . Of the 35 out of 41 cases of systemic amyloidosis successfully classified by LC- MS/MS, additional clinical and pathology data validating the amyloid type was available. 1310 Notch 1 in Primary Effusion Lymphoma: A Clinicopathologic In each of these cases the MS/MS results accurately predicted the amyloid type. Study Conclusions: LC-MS/MS proteomic analysis of abdominal SFA specimens involved HY Wang, F Fuda, NJ Karandikar. UT Southwestern Medical Center at Dallas, Dallas, by amyloidosis provides a highly specific (97% specificity) and sensitive (>85% TX. sensitivity) method for diagnosis and classification of amyloidosis. The method is Background: Primary effusion lymphoma (PEL) is a human herpes virus 8 (HHV8) rapid and readily applicable in a clinical setting and will greatly improve the clinical [also known as Kaposi-sarcoma associate herpes virus (KSHV)] -associated large cell management of amyloidosis patients. lymphoma of body cavities. In vitro studies using PEL cell lines have defined the linkage between latent infection of HHV8 and the induction of oncogenesis through Notch1 (J 1308 IRF4 Translocations Are Specific for Cutaneous Anaplastic Large Virol. 2006;80:6411-6419 and Virology. 2006;351:393-403). However, the potential Cell Lymphoma in Skin Biopsies Involved by T-Cell Lymphoproliferative pathogenic role of Notch1 has not been evaluated in ex vivo patient material. Disorders Design: A total of 11 cases of PEL were retrieved from the Flow Cytometry database of D Wada, ME Law, A de Souza, R Weenig, N Comfere, WR Macon, LA Erickson, A Dogan, the UT Southwestern Medical Center at Dallas from 1996-2007. Clinical, laboratory and AL Feldman. Mayo Clinic, Rochester, MN. other relevant demographic data were reviewed; detailed flow cytometric analysis was Background: Reliable pathologic criteria are lacking to differentiate cutaneous performed. Immunohistochemical staining of Notch1 (mN1A, Chemicon International), anaplastic large cell lymphoma (cALCL) from other T-cell lymphoproliferative disorders using the monoclonal antibody with a high affinity for the “activated intracellular form (TLPDs) involving skin, particularly lymphomatoid papulosis (LyP) and systemic of Notch1”, was carried out on 6 cases from which cell blocks were available. ALK-negative ALCL with secondary cutaneous involvement. Using fluorescent in 290A ANNUAL MEETING ABSTRACTS

Results: HIV was positive at the time of diagnosis in 72.7% (8/11) of cases. Three Conclusions: EBV-T/NK-LPD in children and adolescents is a distinctive entity HIV-negative PEL patients included a post heart transplantation patient, an elderly associated with aggressive behavior and high mortality. Categorization based on patient with asbestosis exposure, and an elderly patient with Hepatitis C. The longest morphology, clonality and clinical presentation, according to the novel CAEBV Study follow-up was 113 months. Flow cytometry analysis detected the expression of following Group classification, appears to be useful in predicting clinical outcome. surface antigens (in descending order): CD38(100%), CD45(100%), CD71(100%), CD45RO(88%), HLA-DR(86%), CD30(82%), CD7(36%), CD4(18%), CD20(18%) 1313 Bone Marrow Microenvironment-Sustained Lymphoma Persistence and CD22(14%). HHV8 was detected in all 8 tested cases (100%) by either polymerase in Follicular Lymphoma with Multidirectional Cell Migration between chain reaction (PCR), or in situ hybridization, or both. Notch1 was positive in 83.3% Lymph Node and Bone Marrow (5/6) of cases with both the nuclear and cytoplasmic staining pattern with the former M Wartenberg, C Meyer zum Bueschenfelde, G Ott, A Rosenwald, F Fend, M Kremer. stronger than the latter. Technical University, Munich, Germany; Robert-Bosch Krankenhaus, Stuttgart, Conclusions: In consistent with in vitro studies using PEL cell lines, we found Notch1 Germany; University of Wuerzburg, Wuerzburg, Germany; University of Tuebingen, expression in the vast majority of PEL specimens, corroborating the notion that it Tuebingen, Germany. may play an important role in PEL pathogenesis. Other mechanism(s) in the setting Background: In follicular lymphoma (FL), the initial t(14;18) translocation is believed of HHV8 should also be considered. As rarely reported, PEL can occur in HIV(-) but to occur in the bone marrow (BM), whereas evolution to definite lymphoma is assumed immuosuppressed as well as in non-immunosuppressed patients. In agreement with to proceed within the germinal centers of involved lymph nodes (LN). However, details previous studies, PEL has high expression of cell surface activation markers (CD38, of tumor cell dissemination to or from the BM remain largely unknown. Goals:We CD71, HLA-DR and CD30) with incomplete expression of B-cell antigens and aberrant performed simultaneous mutational analysis of the IgH rearrangement of LN and BM expression of T-cell associated antigens. clones. By generating genealogical trees, we delineated the dissemination and migration of FL cells between LN and BM, and revealed the clonal evolution of BM involvement. 1311 Differentiation-Associated Antigen Expression Profile of Primary Furtheron, we analyzed ongoing somatic hypermutation (SH) for the presumed influence Bone Diffuse Large B Cell Lymphoma (DLBCL) – Analysis of 31 Cases of BM microenvironment on clonal tumor cell evolution. Q Wang, S Aisner, J Benevenia, P Patterson, K Beebe, L Pliner, M Hameed. UMDNJ- Design: Fresh frozen and formalin-fixed sequential biopsies of three FL with New Jersey Medical School (NJMS), Newark, NJ; UMDNJ-NJMS, Newark. simultaneous infiltration of LN and BM were studied, by amplification of the IgH gene Background: Primary bone lymphoma is a rare disease and comprises less than 1% of with family specific primers (VHL1-6) against FR1, and FR2. Microdissection was used all non-Hodgkin lymphomas, with the majority being diffuse large B cell type. Because when necessary in BM trephines. Amplification products were cloned, transfected and of the rarity of the disease, limited study on this entity has been done. The aim of this sequenced. Hierarchical genealogical trees were generated by comparative analysis with study is to analyze the expression profile of the prognosis-related differentiation- the germline sequence (NCBI Blast) and by interclonal comparison. Ongoing mutations associated antigens. were analyzed using the modified multinomial Chang and Casali formula. Design: Thirty one cases of primary bone DLBCL were retrieved from UMDNJ-New Results: The IgH families of the IgH rearrangements were identical in all LN and BM Jersey Medical School from 1997-2007. Immunohistochemical staining for CD20, CD3, clones of each case, showing the common clonal origin of tumor clones. The cases CD10, bcl-6, MUM-1, BCL-2, p53, Ki-67 were performed. Differentiation-associated showed tumor cell clusters for LN and BM clones created by SH. The clones of each antigen expression profile was analyzed and the cases were subclassified into two types: tissue shared different numbers of mutations (ranging from 0-16), with early migration Germinal Center B cell (GCB) (CD10+ or CD10-/bcl-6+/MUM-1-), or non-Germinal from the LN to the BM, and vice versa. All IgH sequences from LN and about 90% of Center B cell (non-GCB) (CD10-/bcl-6- or CD10-/bcl-6+/MUM-1+). Additional related the BM showed ongoing somatic hypermutation with evidence for antigen selection. markers such as FOXP1, HGAL, BLIMP1 are also being analyzed. Conclusions: BM involvement in FL is characterized by infiltration of early descendants Results: The study included 19 males and 12 females with median age 44 years. The of LN clones. An exchange of BM derivates back to the LN was observed. BM most common site was femur, followed by pelvis, humerus, tibia, fibula, radius, clavicle, may display a niche for tumor cells during therapy. All LN and most BM infiltrates and sacrum. Majority of the cases revealed a centroblastic morphology. All cases were showed ongoing somatic hypermutation, indicating that the BM provides a mutagenic diffusely positive for CD20. Twenty three of thirty one (74 %) cases were GCB type, of microenvironment, similar to the germinal centers in LN. which 18 (78%) were CD10 positive, and 5 (22%) were CD10-/bcl-6+/MUM-1-. Eight of thirty one (26%) cases were non-GCB type, of which 5 (62%) were CD10-/bcl-6+/ 1314 Nodular Pattern of Bone Marrow Infiltration by Diffuse Large B-Cell MUM-1+, and 3 (38%) were CD10-/bcl-6-. Among the 23 GCB cases, 6 (26%) were Lymphoma: Clinicopathologic Characteristics positive for p53 and 8 (34%) were positive for bcl-2, whereas in the 8 non-GCB cases, 5 EM Weeden, DW Sevilla, S Alexander, VV Murty, B Alobeid, G Bhagat. Columbia (62.5%) were positive for p53 and 6 (75%) were positive for bcl-2. The mean positivity University, New York, NY. for the proliferation marker Ki-67 in GCB cases was 34.0% and that in non-GCB was Background: Different patterns of bone marrow (BM) infiltration by diffuse large 40.3%. In non-GCB lymphomas, the percentage of Ki-67 positivity was much higher B-cell lymphomas (DLBCL) have been described. A pure nodular pattern is considered in CD10-/bcl-6+/MUM-1+ (62%) than that in CD10-/bcl-6- (4%) cases. uncommon and is not well-characterized. We thus undertook this study to assess the Conclusions: In primary bone DLBCL, majority of the cases are GCB type. The morphology, phenotype, cytogenetic abnormalities, and clinical features of DLBCL non-GCB type has a higher expression of p53 and Bcl-2. The percentage of Ki-67 associated with this pattern. expression is independent of the GCB or non-GCB subtype of DLBCL. However, in Design: We searched our departmental database to identify BM biopsies (bxs) involved non-GCB subtype, Ki-67 expression is much higher in cases with bcl-6 expression than by DLBCL from 1997-2008. H&E stained sections were reviewed to characterize that without bcl-6 expression. the pattern of BM involvement and identify cases with a discrete nodular pattern. Immunohistochemical stains and in situ hybridization for EBER were performed on 1312 EBV-Associated T/NK-Cell Lymphoproliferative Disorders in all cases. Patient demographics and clinical data were obtained from our laboratory Children and Adolescents: A Clinicopathologic and Molecular Study of information system. 10 Cases Results: A pure nodular pattern of infiltration was noted in 12/52(23%) BM bxs involved S Wang, SEC Quek, B Mow, WJ Chng, A Yeoh, TC Quah, PL Tan, SB Ng. National by DLBCL. We identified 7 cases of EBV+ DLBCL, of which 6 (86%) showed a University Hospital, Singapore, Singapore; King’s College, London, United nodular pattern. Only 6 of the 45 EBV- DLBCL (13%) demonstrated nodular infiltration Kingdom. (p=0.00028). The nodules in all 6 EBV+ DLBCL (4M, 2F, aged 3-72 yrs, median 60 yrs) Background: Epstein-Barr virus-associated T/NK-cell lymphoproliferative disorders had rounded contours, numerous admixed histiocytes, without obvious angiocentricity. (EBV-T/NK-LPD) in children and adolescents is a collective term referring to a In 4/6(67%) cases, the neoplastic cells were pleomorphic and scattered Reed-Sternberg heterogeneous group of entities characterized by a proliferation of EBV-associated like cells were present. 2/6(33%) cases showed centroblastic morphology. All had non- cytotoxic T/NK-cells. The perplexity in nomenclature and diagnosis of EBV-T/NK-LPD germinal center (GC) phenotype. In contrast, the nodules of 6 EBV- DLBCL (3M, 3F, stems from the lack of well-defined diagnostic criteria and overlapping clinicopathologic aged 54-84 yrs, median 64 yrs) had irregular contours and fewer admixed histiocytes. features. A classification system for EBV-T/NK-LPD was recently proposed by the The neoplastic cells had centroblastic (n=6) morphology; 4/6(66%) and 2/6(33%) had CAEBV Study Group. This study describes the genetic and clinicopathologic features non-GC and GC phenotype, respectively. 5/6(83%) EBV+ DLBCL expressed CD30 of 10 cases of pediatric EBV-T/NK-LPD and assesses the predictive value of the novel compared to 1/6(17%) of EBV- DLBCL (p=0.4). No recurrent cytogenetic abnormalities classification system. were detected in either the EBV+ or EBV- DLBCL. All patients with EBV+ nodular Design: Immunohistochemistry for CD3, CD20, CD4, CD8, CD56, granzyme B and DLBCL were immunocompromised (2 HIV+, 3 post transplant lymphoproliferative TIA1, EBER in-situ hybridization and TCR-gene clonality assay were performed. Cases disorders, 1 old age), compared to only 1/5(20%) individuals with EBV- nodular were classified as follows: category A1, polymorphic LPD without clonal proliferation DLBCL (old age). of EBV-infected cells; category A2, polymorphic LPD with clonal proliferation of Conclusions: A pure nodular pattern of BM infiltration by DLBCL is not infrequent EBV-infected cells; category A3, monomorphic LPD with clonal proliferation of EBV- (23%) and the vast majority of such cases have a non-GC phenotype (83%). We observed infected cells; category B, monomorphic LPD with clonal proliferation of EBV-infected a marked enrichment of EBV+ pleomorphic DLBCL occurring in immunocompromised cells and fulminant course. patients with a significant propensity to involve the bone marrow in a purely nodular Results: Patients included 3 females and 7 males with a median age of 13 years (range, pattern. This association suggests that evaluation for EBV infection and investigation 16 months to 21 years). The clinical presentation included fever, hepatosplenomegaly, into the patient’s immune status is warranted when a nodular pattern of BM infiltration pancytopenia, liver dysfunction, skin rash and lymphadenopathy. Six cases had is encountered. monomorphic LPD, and four showed polymorphic LPD. All cases had an EBV- associated cytotoxic T/NK-cell infiltrate with the following CD8/CD4 profile: CD8+/ CD4- (7/10), CD8-/CD4+ (1/10), CD8-/CD4- (2/10). Clonal TCR gene rearrangements were detected in 6 of 9 patients. All patients in categories B (5 cases) and A3 (1 case) died within 2 months of diagnosis. One patient in category A2 survived for 3 years. Patients in categories A1 (1 case) and A2 (2 cases) remain alive (range, 4 to 40 months after diagnosis). ANNUAL MEETING ABSTRACTS 291A

1315 Acute Myeloid Leukemia with Myelodysplasia-Related Changes Sezary cell line SeAx were plated at 5 x 105 cells/mL and treated with either R406 (8 as Defined by the 2008 WHO Classification System mM) or vehicle alone. Total and phosphorylated Syk were assessed at 24 h by western OK Weinberg, M Seetharam, L Ren, L Ma, K Seo, J Merker, J Gotlib, J Zehnder, DA blot and flow cytometry. Apoptosis was assessed at 96 h using an annexin V assay, and Arber. Stanford University, Stanford, CA. expressed as percent apoptotic cells ± standard deviation. P values were determined Background: The 2008 WHO classification system has created a new category “Acute using a t-test. myeloid leukemia with myelodysplasia-related changes” (AML-MRC) that includes Results: SUDHL-1, SR786, and SeAx all expressed total Syk. SUDHL-1 and SR786 1) AML arising from myelodysplastic syndrome (MDS), 2) AML with MDS-related showed phosphorylation of Syk at tyrosine residues Y348 and Y525/526. Treatment cytogenetic abnormalities and/or 3) AML with multilineage dysplasia. The goal of this with R406 completely dephosphorylated Syk in both cell lines, and caused a significant study is to characterize the newly defined AML-MRC group. induction of apoptosis (SUDHL-1: 42.8 ± 9.8 vs. 17.9 ± 3.6 untreated, P=.01; Design: One-hundred AML patients, including 57 males and 43 females diagnosed at SR786: 41.4 ± 16.2 vs. 12.3 ± 6.0 untreated, P=.04). In SeAx cells, Syk was partially Stanford University, were tested for NPM, FLT3 (ITD and D835) and CEBPA mutations phosphorylated at Y525/526 but showed no phosphorylation at Y348. R406 induced and stratified into cytogenetic risk groups. Overall survival (OS), progression free significant apoptosis in these cells also, though the mean apoptotic fraction was lower survival (PFS) and complete remission (CR) rates were retrospectively determined and (22.0 ± 5.8 vs. 10.6 ± 3.2 untreated, P=.04). compared using Kaplan-Meier and multivariate Cox proportional hazards regression. Conclusions: R406 inactivates Syk and kills PTCL cells that express phospho-Syk. Results: Using the 2008 WHO criteria, there were 48 AML-MRC (16 with prior MDS; These findings suggest that Syk may play a critical biologic role in some PTCLs, and 14 with MDS-related cytogenetics; 32 with multilineage dysplasia), 40 AML not represents a feasible therapeutic target for clinical tyrosine kinase inhibition. The relative otherwise specified (AML-NOS), 9 AML with either t(8;21), inv(16) or t(15;17) and 3 contribution of phosphorylation of Syk at Y348 and Y525/526 to the functional role of therapy-related AMLs. Clinically, patients with AML-MRC, compared to AML-NOS, Syk in PTCL merits further study. were significantly older (59 vs 51 years, p=0.014) and presented with lower hematocrit (28 vs 33%, p=0.014). AML-MRC, compared to AML-NOS, was also associated with 1318 Clinicopathologic Investigation of Early Post-Transplant unfavorable cytogenetics (14/48 vs 3/36, p=0.014) and a lower frequency of CEBPA Lymphoproliferative Disease (EPTLD) mutations (0/46 vs 7/40, p=0.001), but no difference in NPM1 or FLT3 mutations. KL Wolniak, MA Proytcheva, BP Nelson. Northwestern University, Feinberg School of Patients with AML-MRC had significantly worse OS, PFS and CR compared to Medicine, Chicago, IL; Children’s Memorial Hospital, Chicago, IL. AML-NOS (all p<0.0001). To evaluate the significance of multilineage dysplasia, Background: PTLD is a potentially lethal complication of organ transplantation. 14 patients with unfavorable cytogenetics were excluded from the AML-MRC group PTLDs fall into 3 main groups: early, polymorphic, and conventional lymphomas. and the remaining patients with AML-MRC still had worse outcomes compared to all PTLDs grouped as lymphomas often require aggressive therapy, but the clinical AML-NOS (OS p=0.013; PFS p=0.012; CR p=0.008). Within the AML-MRC group, behavior of PTLD is unpredictable. Data regarding EPTLDs are limited. Recent studies low platelets (<20K/m3), FLT3-D835 and MDS-related cytogenetics correlated with showed increased mammalian target of rapamycin (mTOR) activation in PTLDs, but worse OS (p=0.045, p=0.026, p=0.002). Multivariate Cox proportional hazard analysis clinical correlation was not provided. We investigated the clinical, morphologic, and performed on all cases identified unfavorable cytogenetics, age >60y, FLT3-ITD and immunophenotypic features as well as mTOR activation in 16 EPTLD (14 patients) AML-MRC status as predictors of worse OS (hazard ratios: 2.82, 2.11, 1.98, 1.92). cases. Conclusions: The newly defined WHO category of AML-MRC, when compared to Design: Sixteen cases of EPTLD were identified. H&E, kappa/lambda, and EBER-ISH AML-NOS, is significantly associated with advanced age, lower hematocrit, lack of stained sections, and clinical data from the medical records were reviewed in all cases. CEPBA mutation, presence of high risk cytogenetics and worse clinical outcome. The Activation of the mTOR pathway was assessed with immunostain (IS) of formalin fixed, poor overall survival of this group in all AML, independent of age or cytogenetic risk paraffin-embedded tissue with an antibody specific for phosphorylated S6 ribosomal group, supports the clinical relevance of AML-MRC. protein (pS6), a downstream effector of activated mTOR; cases were scored using a formula to combine the % positive cells and staining intensity. 1316 Biological Characterization of Stage I Follicular Lymphoma Results: Cases included 3 adults and 11 children (2 to 660 months (mo) old). Allografts According to Extranodal or Nodal Primary Origin and t(14;18) Status were 6 livers, 4 hearts, 3 kidneys, 1 pancreas. Immunosuppressive therapy (IM) was Using High-Resolution Array-Based Comparative Genomic Hybridization cyclosporine 4 cases, tacrolimus 4, mycophenolate mofetil & tacrolimus 6. Median time (aCGH) from transplant to PTLD was 50 mo. (8-135 mo.) in the 12 cases with known data. PTLD OK Weinberg, L Ma, K Seo, R Hoppe, DA Arber. Stanford University, Stanford, CA. sites were tonsil/adenoids 12, lymph node (LN) 3, LN & small bowel 1. Pre-transplant Background: We have recently shown that in patients with low stage follicular EBV status was available in 5 children: 1+, 4-. All 16 cases were EBER+. PTLD groups lymphoma (FL), the status of t(14;18) appears to be more predictive of clinical outcome were: 11 plasmacytic hyperplasia (PH), 1 infectious mononucleousis, 4 PH & follicular than origin from an extranodal versus nodal site. Little is know of the molecular genetics hyperplasia. 13/13 had polytypic plasma cells by IS (12) & flow cytometry (FC;1), 3 of t(14;18)-negative FL, or of the genetic differences between nodal and extranodal FL. had polytypic B-cells by FC. 14/14 cases had increased pS6 staining as compared to The goal of the current study was to characterize stage I FL using aCGH. normal tonsil (p=.002, F-test). Positive staining was in plasma cells and large and small Design: Twenty stage I FL cases, 10 each with and without evidence of t(14;18), lymphocytes, in the germinal centers and interfollicular regions. Therapy was known in diagnosed at Stanford University were studied, including 9 primary extranodal (thyroid, 10 cases: IM was reduced in 6, 1 received steroids, 3 had surgery only. All 14 patients ocular, submandibular gland, parotid, stomach, duodenum) and 11 nodal cases. Five are alive without PTLD; the pancreas was rejected. extranodal FL cases were t(14;18)+ and 4 were negative for the translocation. DNA was Conclusions: Early PTLD developed a median of 50 mos. after transplant in 14 patients, isolated from paraffin blocks and all 20 cases were hybridized to the Agilent Human involved mostly tonsils/adenoids in children, were EBER+, and had good clinical Genome CGH 4 x 44 K Microarray (Agilent Technologies, Santa Clara, CA). The outcome with reduction of IM or surgery. Increased phosphorylation of S6 occured obtained data was analyzed using both Agilent DNA Analytics 4.0 Software and CGH- in EPTLD over normal tonsil suggesting investigation of the inhibitor of mTOR, miner (Stanford, CA) software, and only gains and losses identified by both methods rapamycin, may be of value in EPTLD. were considered significant. Cases were compared both by origin from extranodal versus nodal site and by t(14;18) status. 1319 Persistent Tumor-Specific Expression of CD52 but Not CD20 Results: The t(14;18)-negative FL contained gains on chromosome 1q42, 3q13, 3p22, Following Alemtuzumab and Rituximab Treatment in Chronic Lymphocytic 14q12 and deletion of 6q12 (regulating synaptic membrane, RIMS1) as compared to Leukemia: Implications for Resistance to Antibody-Based Therapies t(14;18)+ cases. The region with the most gains was 14q12, and this gain has not been D Wu, JL Kutok, SJ Rodig. Brigham & Women’s Hospital, Boston, MA. reported in prior CGH studies of FL. Genes in chromosomal regions with established Background: Alemtuzumab (Campath) is a humanized monoclonal antibody that importance gained in t(14;18)-negative cases include germinal center expressed targets CD52, a GPI-linked glycoprotein expressed by B cells, T cells, monocytes transcript 2 (GCET2), interferon regulatory factor 9 (ISGF3G), ring finger protein and macrophages. Alemtuzumab is FDA-approved for the treatment of relapsed or (RNF31), and B and T lymphocyte attenuator (BTLA). The extranodal FL cases differed refractory chronic lymphocytic leukemia (CLL), and its role in the treatment of other from nodal by gain on chromosome 3p22, 4q34, 7p15 and 17p13. Genes in these regions hematopoietic malignancies is being actively explored. We have previously reported with established importance gained in extranodal FL include transforming growth factor a survey of CD52 expression among the various classes of hematopoietic neoplasms. beta receptor (TGFBR2), interferon regulatory factor 2 (IRF2), methyl-CpG binding However, the expression of CD52 in tumors that recur following alemtuzumab treatment domain protein (MBD4) and TP53. has not been studied in detail. Knowledge of post-treatment expression of CD52 antigen Conclusions: In stage I FL, cases lacking t(14;18) contained significant gains in may provide insight into mechanisms of tumor evasion and suggest optimal diagnostic chromosome 1q, 3q, 3p, 14q and a loss in 6q as compared to t(14;18)+ cases. Extranodal approaches for monitoring response to therapy. FL cases were characterized by significant gains in 3p, 4q, 7p and 17p as compared to Design: Paraffin-archived bone marrow biopsies from twelve patients with recurrent/ nodal FL. These findings provide clues for possible biological differences associated relapsed CLL following treatment with both alemtuzumab and rituximab were retrieved with t(14;18) status and origin from extranodal versus nodal site in low-stage FL. from the Brigham and Women’s Hospital repository. For five of the twelve patients, pre-treatment bone marrow biopsies were also available for study. Cases were stained 1317 An Orally Available Syk Tyrosine Kinase Inhibitor Induces using standard and published immunohistochemical methods (IHC) for CD52 and Apoptosis in Peripheral T-Cell Lymphomas Overexpressing Activated CD20. Cases were reviewed for antigen expression by two hematopathologist in a Syk blinded fashion. Correlation with clinical records to determine treatment responses R Wilcox, D Sun, ED Remstein, A Dogan, S Ansell, AL Feldman. Mayo Clinic, was then performed. Rochester, MN. Results: CD52 and CD20 were expressed by all tumor cells prior to treatment- in Background: Syk is a tyrosine kinase that promotes growth of B cells. An orally agreement with prior reports. Among cases of recurrent/ relapsed CLL following available Syk inhibitor, R406, is in clinical trial for B-cell lymphomas. Though absent treatment with both alemtuzumab and rituximab, 11 cases (92%) showed strong in normal T cells, Syk is overexpressed in most peripheral T-cell lymphomas (PTCLs; expression of CD52 indistinguishable from untreated tumor. One case showed Feldman et al, Leukemia 2008;22:1139-43). We wanted to see if (1) Syk was activated heterogeneous expression of CD52 among the tumor cells. In contrast, all cases (100%) in PTCLs; (2) R406 inactivated Syk; and (3) R406 killed the tumor cells. Design: The anaplastic large-cell lymphoma cell lines SUDHL-1 and SR786 and the 292A ANNUAL MEETING ABSTRACTS showed evidence for downregulation of CD20, with 11 of 12 cases (92%) showing 1322 Lymphoma in Northern Iraq, a World Health Organization undetectable CD20 by immunohistochemical techniques. Undetectable CD20 expression Classification of 111 Cases in these cases was also confirmed by flow cytometry. RT Yaqo, FK Sulayvani, N AlAllawi, M Hughson. Dohuk University Medical College, Conclusions: Among patients with recurrent CLL after treatment with alemtuzumab and Dohuk, Iraq; Hewa Hematology and Oncology Hospital, Sulaimaniyah, Iraq. rituximab, there is a strong selection bias for tumor cells that downregulate the target Background: The prevalence of diagnostic categories of lymphoma differs across antigen CD20; however, there is not a corresponding decrease in CD52 expression in geographical regions. In developing countries, diffuse large B-cell lymphomas (DLBCL) these same tumor cells. These findings indicate that, within a single tumor type, there are uniformly common. In contrast to Western countries, follicular lymphomas (FL) are distinct adaptive responses to individual antibody-based therapies and suggest are rare, and mixed cellularity (MC) is the most common form of Hodgkins lymphoma the possibility of sustained sensitivity to alemtuzumab but not rituximab in relapsed (HL). Our objective is to compare lymphoma diagnoses in Northern Iraq with reported disease. series in nearby Mid-Eastern countries. Design: All lymphomas referred to Azadi teaching hospital, Dohuk, Northern- 1320 Biased Immunoglobulin VH Recombination and Increased Iraq between 2003 and 2008, were categorized using the WHO classification. Expression of Chemokine Receptor XCR1 Are Distinct Features of Bone Immunohistochemistry was performed with the following panel of antibodies: CD3, Marrow-Derived Diffuse Large B-Cell Lymphoma CD4, CD5, CD7, CD8, CD10, CD15, CD20, CD23, CD30, CD57, CD79a, ALK-1, Y Yamashita, D Kajiura, S Nakamura, S Toyokuni, N Mori. Nagoya University Graduate Cyclin D1, BCL2, BCL6, and TdT. School of Medicine, Nagoya, Aichi, Japan; Nagoya University Hospital, Nagoya, Results: There were 90 (81%) non-Hodgkin lymphomas (NHL) and 21 (19%) HL. The Aichi, Japan. age range of NHL patients was 3 to 75 years with a median age of 39 years. Of the NHL, Background: Bone marrow-derived diffuse large B-cell lymphoma (DLBCL) 93% had a B-cell phenotype and 7% were T- cell neoplasms. DLBCL comprised 64% presents a poor prognosis, often associated with hemophagocytic syndrome, and of NHL. Burkitt’s lymphoma (BL) was the second most common NHL at 17% with shares the pathologic entity with Asian-variant intravascular large B-cell lymphoma all BL being found in children as abdominal primaries. Small lymphocytic lymphoma, (IVL) in which bone marrow involvement is observed in 75% of the cases. However, mucosa associated lymphoid tissue lymphoma, mantle cell lymphoma, and FL were immunohistochemical marker molecules distinguishing these entities from nodal represented at 7%, 3.3%, 1.1%, and 1.1% respectively. T-cell neoplasms consisted DLBCL are not established. of 2 mycosis fungoides, 2 anaplastic large T-cell lymphomas, and 2 lymphoblastic Design: PCR and sequence analyses of the immunoglobulin VH gene were performed lymphomas. For 21 HL, 10 were MCHL, 9 nodular sclerosis (NS), and 2 nodular in 14 patients of bone marrow-derived DLBCL to evaluate the VH family usage and lymphocyte predominant HL. differentiation status of the neoplastic B-cells. Oligonucleotide microarray restricted to Conclusions: This study revealed a high frequency of DLBCL and childhood abdominal the expression of chemokines and chemokine receptors were performed in comparison BL and a single FL. MCHL exceeded the frequency of NSHL. These distributions are of 4 samples of bone marrow-derived DLBCL with 8 samples of control nodal DLBCL. similar to those reported from Jordan, Kuwait, Saudi Arabia, and Turkey and indicate Statistical analyses were performed by the Mann-Whitney U Test. Immunohistochemical a shared geographic influence on lymphoma prevalence with Northern Iraq. analyses were performed in 25 cases of bone marrow-derived DLBCL and 23 cases of control nodal DLBCL. 1323 Hemoglobins S, C and E Masquerade as Spurious Monoclonal Results: Eleven of the 14 cases had VH recombinations previously reported to encode Bands on Serum Protein Electrophoresis autoreactive antibodies, with 5 cases of VH3-7 and 3 cases of VH4-34. Others included S Zhang, PJ Howanitz, JH Howanitz, I Gashinsky. SUNY Downstate Medical Center, 3-23, 3-48 and 4-39. Somatic hypermutation was detected in all the 14 cases, suggesting Brooklyn, NY; Kings County Hospital Center, Brooklyn, NY. the post-germinal center status of the neoplastic B-cells. Chemokine/chemokine receptor Background: Identification and quantification of monoclonal (M) immunoglobulins are microarray revealed that chemokine receptor XCR1 was significantly overexpressed critical for diagnosis and monitoring of plasmacytomas. Serum protein electrophoresis (p<0.05) in the cases of bone marrow-derived DLBCL, which was confirmed by (SPE) is used routinely for identification and quantification, and few interferences have immunohistochemistry in 80% of the cases. been reported. A partially hemolyzed non-hemoglobin A (non-HbA) specimen apparently Conclusions: Biased immunoglobulin gene recombination to autoreactive VH families containing a M-protein on SPE prompted us to examine the migration of commonly and overexpression of chemokine receptor XCR1 are distinct features of the neoplastic occuring abnormal hemoglobins. B-cells in bone marrow-derived DLBCL. This suggests a distinct differentiation of Design: Specimens from patients with homozygous Hb SS, CC, and EE were the neoplastic B cells between bone marrow-derived DLBCL and nodal DLBCL. Our identified using high performance liquid chromatography (Bio-Rad Variant II, Bio-Rad findings will be helpful for the differential diagnosis of these entities. Laboratories, Hercules, CA). Red blood cells (RBCs) were separated from plasma by centrifugation, washed three times in phosphate buffered saline, lysed and mixed with 1321 Added Value of FISH as an Adjunct to G-Band Karyotyping in equal volumes of normal control serum. We then used the Helena SPIFE 2000 and Evaluation of Myelodysplastic Syndromes corresponding SPE membranes and reagents (Helena Laboratories, Beaumont TX) for W Yang, V Murty, FN Emmons, B Alobeid, G Bhagat. Columbia University, New electrophoresis of the specimens as well as known control specimens from patients York, NY. who had IgG kappa, IgA lambda, and IgM kappa monoclonal proteins. The same Background: Cytogenetic analysis provides important diagnostic and prognostic specimens were subjected to immunofixation electrophoresis (IFE) using the Helena information in patients with myelodysplastic syndromes (MDS). G-band karyotyping instrument and IFE membranes and reagents. Specimens from patients with heterozygous detects cytogenetic abnormalities in 50% primary and >90% secondary MDS. hemoglobinopathies were treated in the same manner as specimens from patients with Fluorescence in situ hybridization (FISH) is more sensitive in detecting aberrations in homozygous hemoglobinopathies. minor clones and non-dividing cells. However, prior studies of FISH in evaluating MDS Results: Hb SS, Hb CC, and Hb EE each migrated as a unique single protein band in with normal karyotype have been contradictory. We thus assessed the added value of the gamma region of the SPE membrane. IFE results indicated that each single unique FISH in evaluating patients with suspected MDS. protein band in the gamma region did not react with IgA, IgM, IgG, kappa or lambda Design: We searched for all cases submitted as bone marrow (BM) “rule out MDS” at antiserum. Specimens from heterozygous hemoglobinopathy patients gave similar our institution from 2003-2008. MDS diagnoses were made according to current WHO findings for the variant hemoglobin. criteria. MDS cytogenetic studies included G-band karyotyping and MDS FISH panel Conclusions: We conclude that hemoglobin S, C, and E migrate in the gamma region with probes of EGR1/D5S23, D7S486/CEP7, D20S108 and CEP8. Cases were divided on serum protein electrophoresis, react with the protein stain on SPE, but do not react into 3 groups: normal karyotype, karyotype failure and abnormal karyotype. FISH for with IFE antiserum against IgG, IgA, IgM, kappa or lambda. We suggest that non- MDS was performed in each group. HbA hemoglobins such as S, C, and E in hemolyzed specimens may masquerade as a Results: Total 254 cases suspected of MDS were worked up, 117 cases were confirmed false M-protein on SPE. We recommend that pathologists interpreting serum protein MDS and 137 cases were not diagnostic of MDS. Of the total 254 cases, 167 had normal electrophoresis consider the occurrence of a hemoglobinopathy when hemolysis is karyotype (58 MDS/109 non-MDS), 18 had karyotype failure (7 MDS/11 non-MDS) present, an apparent monoclonal band is seen on SPE, and the band is not confirmed and 69 had abnormal karyotype (52 MDS/17 non-MDS). Karyotypes and FISH results as an immunoglobulin on IFE. are shown in Table below. 7/58 (12%) MDS cases with normal karyotype and 2/7 (29%) MDS cases with karyotype failure had abnormal FISH results. 5/52 (9%) MDS cases with 1324 MicroRNA-155 Targets the Transcription Repressor ZNF652 in abnormal karyotypes showed additional cytogenetic abnormalities by FISH: 4 cases had Chronic Lymphocytic Leukemia (CLL) recurrent MDS abnormal karyotypes and 1case had non-specific abnormal karyotype. T Zhang, K Nie, R Furman, A Chadburn, DM Knowles, W Tam. Weill Cornell Medical Karyotypes vs. MDS FISH Panel Results College, New York, NY; Northwestern University Feinberg School of Medicine, Karyotype Total FISH Normal FISH Abnormal Chicago, IL. Normal Karyotype 167 160(96%) 7(4%) Background: The pathogenesis and prognosis of CLL has been linked to deregulation Abnormal Karyotype 69 10(14%) 59(86%) of microRNAs (miRNA). MicroRNA-155 (miR-155) has been identified as one of the Karyotype Failure 18 14(78%) 4(22%) Total 254 184 70 miRNAs in a miRNA gene signature associated with worse prognostic factors and more aggressive clinical course in CLL. Our current study aims at elucidating the mechanisms Conclusions: Based on this study, MDS FISH panel will most likely be normal if of how miR-155 contributes to its pathogenesis and disease progression. karyotype is normal. Abnormal FISH will be found more likely in cases with abnormal Design: B cells purified from 22 IgVh unmutated and 25 IgVh mutated CLL samples karyotypes and also (though to a less extent) in cases with karyotype failure. FISH were analyzed for miR-155 expression by a modified Invader assay and compared to 6 may find additional recurrent MDS cytogenetic abnormalities in cases with abnormal memory B cell samples isolated by flow sorting from independent reactive tonsils. The karyotypes. Since FISH is labor intensive, it is advisable to limit FISH to cases with 6 samples with the highest or lowest miR-155 expressions from each of the two IgVh abnormal karyotype or karyotype failure. However, our study showed that FISH was groups were selected for cDNA microarray analysis. Differentially expressed genes abnormal in 12% MDS cases with normal karyotype. Therefore, MDS FISH panel is between these two groups (12 samples each) were analyzed to search for miR-155 probably recommended in cases highly suspicious for MDS even when their G-band target genes and miR-155-associated gene signatures. karyotypes are normal. Results: 10 of 22 IgVh unmutated CLLs and 10 of 25 IgVh mutated CLLs express miR- 155 at levels 2-fold or higher compared to normal memory B cells. Unmutated CLLs ANNUAL MEETING ABSTRACTS 293A tend to have higher miR-155 expressions compared to mutated CLLs (p=0.09). Global karyotypic changes are similar to those seen in MDS. Further characterization of AEL expression profiling identified a set of genes differentially expressed between CLLs will allow a distinction from or inclusion in the appropriate categories of MDS. with high and low miR-155 levels. Among the 8 genes detected above the significance threshold after multiple comparison corrections, 2 were down-regulated and 6 were up-regulated, including BIC, the miR-155 precursor. One of the two down-regulated Infections genes, ZNF652, encodes a potential tumor suppressor with transcription repressor activity and harbors two miR-155 binding sites. Both its mRNA and protein levels 1327 Plasmodium Vivax Malaria: A Native American Disease negatively correlate with miR-155 levels. In addition, reporter assays demonstrated specific interaction of miR-155 with its 3’ untranslated region. High miR-155 in CLL MJ Allison, S Guillen, E Gerszten. MCV Campus-VCU, Richmond, VA; Instituto is also associated with a higher level (∼8 fold) of CB1 cannibinoid receptor (CNR1) Mallqui, El Algarrobal, Moquegua, Peru. mRNA and a lower (∼6 to 30 fold) Kruppel-like factor (KLF3) mRNA expression. Background: Malaria is probably one of the most ancient human diseases. It is currently Conclusions: ∼40% of CLL over-express miR-155. Our study identified a putative on the increase with half a billion cases in 2007/in 106 different countries. Whereas Pl. miR-155 target gene that is likely to mediate the pathogenic function of miR-155 in falciparum was a recent import carried by imported African slaves, Pl. vivax may well CLL. Studies of other differentially expressed genes may uncover novel mechanisms be an American resident for thousands of years. of miR-155 over-expression in CLL. Design: This current study was the examination of 155 spleens or livers from peruvian mummies belonging to seven cultural groups dating from 3000 to 600 years before present (B.P.) They were studied using U.S.Biological antibodies Pl.vivax 75/76 1325 Phospho-p70s6k and cdc2/cdk1 Are Associated with Diffuse and Pl.falciparum 70/71 with ELISA in situ hybridization using a Biogenex Alkaline Large B-Cell Lymphoma and Are Potential Targets for Combination Phosphatase conjugated Streptaviden Reagent System. Histological sections were cut Chemotherapy and stained with H&E for malaria pigment. MY Zhao, A Auerbach, A DCosta, AP Rapoport, AM Burger, F Jiang, SA Stass, AK Results: Table 1 shows the results: Sands, N Aguilera, EA Sausville, XF Zhao. University of Maryland School of Medicine, Baltimore, MD; Armed Forces Institute of Pathology, Washington, DC; University of Maryland Marlene and Stewart Greenebaum Cancer Center, Baltimore, MD; University at Buffalo, SUNY, Buffalo, NY. Background: Diffuse large B-cell lymphoma (DLBCL) is the most common non- Hodgkin lymphoma in adults. However, only 40-60% of the DLBCL patients respond to the current standard therapy and recurrence after the initial remission is quite common. This study was to identify and evaluate novel molecular targets for the development of novel combination chemotherapy to treat the refractory and recurrent DLBCL. Design: Lymphoma samples from 38 cases of primary and recurrent DLBCL were analyzed using real time quantitative PCR of the RPS6KB1 and CDC2 genes, and immunohistochemistry for their gene products p70S6K/p85S6K and cdc2/cdk1. The Farage, Karpas422, Pfeiffer and Toledo DLBCL cell lines were subsequently treated with combined rapamycin and UCN-01. The cell proliferation, apoptosis and cell cycle progression were analyzed after the drug treatment. Several key protein kinases involved in the PI3K/Akt/mTOR pathway, apoptosis and cell cycle progression were analyzed after the drug treatment. Results: Amplification of RPS6KB1 and CDC2 genes was found in both primary and recurrent DLBCL. Moreover, the vast majority of these lymphomas (∼94%) were strongly positive for phospho-p70S6K and cdc2/cdk1 proteins. Combination of rapamycin and UCN-01 (R+U) synergistically inhibited the DLBCL cell proliferation Conclusions: Studies by Sulzier et al in 1975 on a group of isolated Campa Indian by inducing G1 arrest as well as apoptosis of the DLBCL cells through suppressing the showed a high rate of malaria (63%-85%) with an etiology of only Pl.malariae and Pl. phosphorylation of p70S6K/p85S6K and CDC2 expression. vivax . no Pl. falciparum was found. These two species are said to have evolved from Conclusions: The RPS6KB1 and CDC2 overexpression is common in DLBCL. a primate and cross react with some monkey malaria species. Oliveira, 1995, found 17 Simultaneously targeting the RPS6KB1 and CDC2 products phospho-p70S6K/p85S6K species of South American monkeys positive for malaria with an infection rate 2.1% and cdc2/cdk1 is very effective in inhibiting DLBCL proliferation and overcoming drug - 59%. the red howler monkey was among the highest. Some monkey malaria can be resistance. This work suggests that multi-level inhibition of PI3K/Akt/mTOR pathway contracted by humans, Howler monkey mummies are found in human cemeteries and and double block of cell cycle progression are effective strategies for the DLBCL were probably pets when the mother was hunted and killed locally in coastal valleys for combination chemotherapy. food. One had his food dish with him. As coastal valley forests were cut for agriculture and coastal monkeys were all killed for food the only monkeys ar now mainly in the Amazon. Currently this practice is in effect in Columbia. Human malaria was a common 1326 Acute Erythroid Leukemias – Histologic, Cytogenetic and disease in Pre Columbian Peru and may have had its origen in local monkey malaria. Molecular Characterization Z Zuo, A Kasyan, P Chandra, J Medeiros, H Koeppen. University of Texas M.D. Anderson Cancer Center, Houston, TX. 1328 Clinical, Pathological and Immunohistological Study of Background: Acute erythroid leukemias (AEL) are uncommon types of acute Tularemic Skin Lesion and Lymphadenopathy myeloid leukemias with markedly heterogeneous morphologic, immunophenotypic S Asano, M Kojima, F Shinya, H Fujita. Iwaki Kyoritsu General Hospital, Iwaki, and molecular features. Fukushima, Japan; Gunma Cancer Center Hospital, Ohta, Gunma, Japan; Ohara General Design: We reviewed the clinical, histologic, cytogenetic and molecular data of 47 Hospital, Fukushima, Japan. patients with AEL seen at our institution. All cases strictly met the criteria outlined Background: Tularemia is an infectious disease caused Francisella tularensis-infected in the WHO classification . Molecular studies included mutational analysis for Ras, hare. It has been well known that hare-cooking persons suffered from regional KIT and FLT3 genes. lymphadenopathy. But, there were little reports concerning skin lesions in contrast to Results: The male to female ratio was 3.7, The mean age of the patients was 56 years lymph node lesions. In this section, we show relationship between tularemic skin lesion with a range from 18 to 84. 80% were classified as primary AEL, while the remaining and regional lymphadenopathy. cases arose in patients with an antecedent MDS. Morphologically, the bone marrow was Design: Nineteen cases of finger skin lesions, two tonsills and fifty-four cases of lymph hypercellular (mean cellularity of 73%) with mean bone marrow and peripheral blood nodes of tularemia are clinico-pathologically studied by histology and immunohistology. blast counts of 45% of non-erythroid precursors and 8%, respectively. Dysplastic changes We analyzed relationship between time-course and skin lesions or lymphadenopathy. in the erythroid, megakaryocytic and granulocytic lineages were seen in 84%, 55% and Results: (1).Serum antibody titer; It shows positivity at the first week and reaches 31%, respectively; ringed sideroblasts were seen in 6% of cases. Two cases fulfilled the the peak around the third week. The positive reaction persists till the twentieth week WHO criteria of pure erythroleukemia. Karyotypic abnormalities were seen in 67% of after infection. (2).Skin; Inflammatory cells and small necrotic focus appear in the cases; 18% demonstrated a single chromosomal abnormality and 49% showed complex subcutaneous site till the second week after infection. Through the second to the sixth changes involving two or more chromosomes. Aberrancies of chromosome 5 (33%), week, it appears ulcers and there are a lot of antigen presenting cells such as lymphocytes 7 (35%) and 11 (16%) were seen in the context of complex cytogenetic abnormalities. and dendritic cells in dilated lymph vessels in dermis. There are many small epithelioid Trisomy 8 represented 50% of cases with a single chromosomal abnormality. Mutations granulomas with central necrosis in dermis as detected in subcutaneous. After the sixth of the FLT3 gene were seen in 2 of 36 patients (one internal tandem duplication, one week, inflammatory cells increase and it appears irregular shaped fused granulomas D835 mutation). None of the cases showed mutations in the RAS or KIT genes. The with central homogenous lesions in subcutaneous. (3). Lymph node; It appears primitive average survival of patients with a diploid karyotype, a single cytogenetic abnormality abscess surrounded by rough arranged mononuclear cells and histiocytic cells till the or complex cytogenetic changes were 18.5, 20 and 7.6 months, respectively. Statistical second week after infection. Many small epithelioid granulomas with central necrosis and analysis revealed that patients with complex cytogenetics had significantly shorter aggregation of CD20+ lymphocytes contacted with granulomas are detected through the survival when compared to patients a with diploid karyotype (p=0.045) or patients with lymph node till the sixth week. CD4+ lymphocytes are richer than CD8+ cells through a single chromosomal abnormality (p=0.017). The two patients with pure erythroid granulomas. After the sixth week, there appear granulomas with central caseous necrosis leukemia showed survival times of 4 months and less than 12 months, respectively, like lesions and aggregation of CD20+ lymphocytes as early phase. complex cytogenetic abnormalities including monosomy 5 and 7 and trisomy 8 and Conclusions: The bacteria enter from the finger skin immediately after contact to wildtype FLT3 and RAS genes. the infected hare and it makes abscess forming granulomatous lesion in skin and Conclusions: Our results demonstrate a high incidence of complex karyotypic regional lymph nodes. It may conclude that regional lymph node is draining from the abnormalities in AEL and a low frequency of FLT3, KIT or RAS gene mutations; the involved cutaneous sites via lymph vessel and the antibody production period coincide