Letters to the Editor 885 Biclonal origin prevails in concomitant chronic lymphocytic and multiple myeloma

Leukemia (2010) 24, 885–890; doi:10.1038/leu.2009.294; compartment were not measurable by flow cytometry. Copy published online 21 January 2010 number analyses using Affymetrix 50K and 250K NspI SNP arrays revealed no chromosomal aberration in unselected BM B-cell chronic lymphocytic leukemia (B-CLL) is a common and CD19 þ selected PB cells (Table 1). Despite rigorous hematological malignancy that typically follows an indolent analysis, the clonal relationship of concomitant MM/B-CLL in course over many years. However, with longer disease course this patient remained unclear. Because of stage IIIB MM and and recurring anti-B-CLL therapy, in addition to yet undefined B-CLL Rai 0 disease, he received two cycles of vincristine, causes, transformation to aggressive B-cell may doxorubicin, dexamethasone (VAD), PB stem cells (PBSCs) were occur. Coincidence of B-CLL with other hematological disorders mobilized with epirubicine, etoposide, cyclophosphamide has also been observed, nevertheless, the simultaneous (EVC), followed by an autologous PBSC (auto-PBSC) transplan- appearance of B-CLL and multiple myeloma (MM) is a rare tation. According to European Group for Blood and Marrow phenomenon.1,2 The clonal relationship between B-CLL and Transplantation (EBMT) criteria, the patient obtained a partial MM remains controversial. Two hypotheses have been postu- remission of his MM, and has remained stable of both his MM lated, either that both malignancies develop from two distinct and B-CLL with thalidomide maintenance therapy (Supplemen- clones, or that both evolve from the same B progenitor. tary Table 2). To unravel the clonality of concomitant B-CLL and MM, different The second 76-year-old male patient presented with leuko- experimental strategies have been pursued. Early publications cytosis and IgG lambda (l) paraprotein in January 2006. Via BM focused on different vs identical light chain (LC) expression in biopsy, l-positive mPCs (30%) and k-positive B-CLL-infiltrates both diseases.1 Hoffmann and Rudders3 described a patient in (10%) were detected. IgVH analysis from cDNA showed a whom B-CLL and malignant plasma cells (mPCs) expressed mutated clonal transcript in PB cells. The 50K SNP assay different LCs, suggesting a different clonal origin. Nevertheless, performed on unselected PB cells revealed a 5.8 Mb deletion of despite different LCs and immunglobulin (Ig) isotypes with the 13q14 region (Table 1). The quality and quantity of the BM B-CLL-cells expressing IgM kappa (k) and mPCs IgA lambda (l), sample did not allow CD138 þ selection of mPCs, nevertheless, Saltman et al.4 postulated a monoclonal B-CLL/MM origin, as FISH using multicolor DNA probes on BM cytospins revealed they detected identical heavy chain gene rearrangements in deletion of at least two regions on chromosome 13 (one signal B-CLL cells and mPCs. The second approach to solve the clonal for 13q14 and one signal for 13q34) in 19 of 20 analyzed mPCs relationship is based on different cytogenetic aberrations (Figure 1a). The DNA probe used in the 13q14 locus D13S319 discovered in B-CLL and MM.5 As B-CLL and MM are difficult overlapped with the 5.8 Mb deletion detected by the SNP array to examine by conventional cytogenetics (due to their low (UCSC Genome Browser, http://genome.ucsc.edu). However, proliferation and metaphase quality), interphase fluorescence the more telomeric DNA probe at 13q34 was outside the in situ hybridization (FISH) technology, together with simulta- detected 5.8 Mb deletion, indicating monosomy of chromosome neous detection of cytoplasmatic LCs, have been used recently 13 in mPCs. Immunophenotyping showed different expression to prove a biclonal origin of both diseases.2 In this study, we of LCs for B-CLL (k) and mPCs (l), suggesting biclonality describe five Caucasian male patients with concomitant B-CLL of B-CLL and MM diseases. Because of concurrent stage Rai 0 and MM diagnosed at our center within a 4-year period B-CLL and stage IIIA MM by S&D, the patient received (2005–08). To resolve the clonal relationship, we combined melphalan, prednisone (MP) and achieved a partial remission Affymetrix (Santa Clara, CA, USA) single-nucleotide polymor- of his MM. At 17 months after the diagnosis of B-CLL and MM, phism (SNP) mapping array and FISH analyses on bone marrow the patient acquired acute myeloid leukemia (AML) (French- (BM) smears. The SNP technology is highly efficient in the American-British (FAB) M0), classified as therapy-induced detection of recurrent and new chromosomal aberrations in B-CLL (t-AML). The analysis of AML blast cells did not show del and MM,6 and FISH on BM smears allowed us to connect specific 13q14/monosomy 13, arguing for oligoclonality as well. chromosomal aberrations to different cell compartments. Because of t-AML, mitoxantrone, etoposide, cytarabine (MICE) Among our five patients, the first patient was a 64-year-old induction and consolidation was performed, male, being evaluated in August 2005 because of weight loss, achieving a complete remission. After 10 months, AML relapse leukocytosis, anemia, foamy urine and increased creatinine. occurred, and the patient eventually died of AML progression Detailed diagnostics revealed a Bence-Jones k-proteinuria, and septic complications. Autopsy revealed overt AML, but of elevated serum k-LCs and via BM biopsy mPCs (40%) as well note no MM or B-CLL residues (Supplementary Table 2). as lymphocytic infiltrates (45%). Immunohistology confirmed The third patient was a 73-year-old male diagnosed with stage the simultaneous BM infiltration of mPCs and B-CLL cells. The Rai II B-CLL in 2003. Flow cytometry revealed a typical B-CLL diagnosis of concomitant MM stage IIIB (Salmon & Durie) and phenotype, with monoclonal k-positive PB and BM B-CLL cells. B-CLL stage Rai 0 was made (Supplementary Table 1). Identical In August 2005, the patient was admitted to the orthopedic unmutated clonal IgVH transcripts (VH 4-39) were present in department due to progressive lumbago and deteriorating both peripheral blood (PB) and BM cells (Table 1). As this clinical condition. Serum immunoelectrophoresis revealed an transcript was cloned by amplifying mRNA of IgM (m) type and IgA paraprotein and BJ-k proteinuria. Skeletal survey showed the vast majority of MM undergo class switch, it was postulated multiple osteolytic lesions and diffuse osteopenia. BM biopsy as derived from the B-CLL clone present in both PB and BM displayed 30% B-CLL and 15% CD38- and CD138-positive cells. The same transcript was detected with a low frequency in mPCs. A diagnosis of B-CLL (stage Rai II) and IgA k-MM CD19 þ selected PB cells in December 2007, when the patient (IIIA by S&D) was made (Supplementary Table 1). A mutated had stable MM and B-CLL diseases. LCs expressed by the B-CLL IgVH status and biallelic deletion of chromosome 13q14 were

Leukemia Letters to the Editor 886 Table 1 Immunological and molecular features of concomitant CLL and MM patients

Patients CLL Specimen description IgVH family/ SNP-array copy number results Specimen FISH Clonality vs used for molecular mutation used for MM analysis status FISH analysis

#1 CLL DNA from CD19+ VH-4-39/ No aberration n.d. Not CLL/MM PB cells unmutated resolved MM DNA from unselected VH-4-39/ No aberration n.d. BM cells unmutated #2 CLL cDNA and DNA from VH 3-15/ del 13q14 (5.8 Mb) n.d. Biclonal CLL/MM unselected PB cells mutated MM s.n.o. s.n.o n.d. BM smears (del 13q14 and and cytospins del 13q34) #3 CLL DNA and cDNA from VH 3-72/ Bi-allelic del 13q14 (5.6/2.7 Mb) n.d Biclonal CLL-MM CD19+ PB cells mutated in CD19 + cells MM s.n.o. n.d. n.d. BM smears Trisomy 11 #4 CLL DNA and cDNA from VH 4-34/ No aberration in CD19 +cells n.d. Biclonal CLL-MM CD19+ PB cells mutated MM DNA from CD138+ VH 1-18/ Complex aberrations: gain 4p (19.9 Mb), n.d. BM cells mutated del10q (6.6 Mb), gain 11q(53.4 Mb), del 12p (15.8 Mb), del 17p (8.1 Mb) #5 CLL DNA and cDNA from VH 3-9 (cDNA) del 11q23 (15.4 Mb), del 11q24 (5.5 Mb) Unselected del 11q23 Biclonal CLL-MM unselected BM CLL cells VH 3-15 (DNA)/ in unselected BM and PB cells BM cells unmutated MM DNA from unselected VH 3-9 (DNA) n.d. Unselected t(11;14), BM MM cells VH 3-15 (DNA)/ BM/BM hyperdiplody unmutated smears of chr. 11 Summary CLL IgVH mut:unmut 3:2 CLL SNParray CLL FISH bc: n ¼ 4 MM IgVH mut:unmut 1:2 abnor:nor 3:2 abnor:nor 1:0 MM SNParray MM FISH abnor:nor 1:1 abnor:nor 3:0 Abbreviations: bc, biclonal; BM, bone marrow; CLL, cell chronic lymphocytic leukemia; FISH, fluorescence in situ hybridization; MM, multiple myeloma; PB, peripheral blood; n.d., not done; SNP, single-nucleotide polymorphism; s.n.o., sample not obtained.

detected in CD19 þ selected PB (B-CLL) cells. Moreover, the showed 50% B-CLL cells and 40% coexisting monoclonal two deleted alleles were 5.62 and 2.70 Mb in size, both k-expressing mPCs. Differently mutated clonal IgVH transcripts, overlapping micro RNA genes MIRNA15A/MIRN16-1 (Table 1). VH4-34 and VH1-18, were present in CD19 þ PB and We performed FISH analyses on BM smears on 50 mPCs and CD138 þ BM cells, respectively, strongly implying a biclonal all showed two signals for the chromosomal region 13q14, origin of both diseases (Table 1). Copy number analysis with indicating the absence of del13q14; moreover, one MM 250K NspI SNP arrays was also performed on CD19 þ selected subclone (40% of all evaluated mPCs) showed a clonal PB cells and CD138 þ selected BM cells. No aberrations were evolution with three signals for the centromere region of detected in B-CLL cells (Figure 1c, left panel), but a complex chromosome 11, implying trisomy 11 (Figure 1b). Two different molecular karyotype in mPCs (including gain on 4pter-p15.2 genomic aberrations in different clonal cells indicated a biclonal and 11q13.1-24.1, and deletions on 10q24.2-25.2, 12pter- origin of B-CLL and MM (Table 1). High-dose dexamethasone p12.3 and 17p13.1-p12; Figure 1c, right panel), verified the was followed by two cycles of EVC. As the patient declined to biclonal origin of B-CLL and MM. As both B-CLL and MM receive an auto-PBSC transplantation, MP consolidation diseases were aimed to be treated and fludarabine response had was performed, followed by thalidomide plus prednisone been disappointing, cyclophosphamide, thalidomide and maintenance for 12 months. Owing to these anti-MM therapies, dexamethasone was given and the patient transferred to our the patient’s IgA paraprotein significantly decreased and his department in February 2008. A skeletal survey showed multiple general condition improved (Eastern Cooperative osteolytic lesions and BM revealed dense B-CLL persistence, Group (ECOG) 3-1). BM biopsy showed few mPCs (10%), and whereas mPCs remained stable. Because of a loss of CD5 no persisting B-CLL infiltrates. From October 2007 until July coexpression on B-CLL cells, occurrence of Richter transforma- 2008, the patient received dexamethasone maintenance, tion was suggested. -cyclophosphamide, doxorubicin, stabilizing his MM and leading to persistent complete remission vincristine, prednisone (R-CHOP) was given and due of his B-CLL. In July 2008, MM progression progressive disease to persistence of pancytopenia and dense B-CLL-infiltrates, (PD) occurred and the patient died of PD in October 2008 R-bendamustin thereafter. This not only led to a marked (Supplementary Table 2). decrease of BM B-CLL and mPC infiltrates, but also to prolonged The fourth patient was a 68-year-old male diagnosed with BM aplasia. As the patient’s general condition decreased (ECOG B-CLL in July 2003 (Supplementary Table 1). Because of stage 2-3) and BM aplasia prohibited chemotherapy continuation, Rai 0 B-CLL, no B-CLL-specific therapy was initiated. In he was discharged home with best supportive care measures. November 2007, the patient developed Rai stage IV B-CLL He deceased 3 months thereafter with B-CLL progression cells (Rai IV) and fludarabine chemotherapy was begun. As a (Supplementary Table 2). subsequent complication, the patient acquired recurrent septic The fifth patient was a 60-year-old male diagnosed with infections due to pronounced granulocytopenia. BM reanalysis B-CLL in September 2003 (Supplementary Table 1). In February

Leukemia Letters to the Editor 887

ATM ATM ATM t(11:14) t(11:14) t(11:14) t(11:14) FISH probe 1x 2x 3x+4x 2R2G 3R3G 4R3G 6R4G

% of interphase nuclei 89.3 8.08 2.57 94.00 0.85 4.00 1.17

Figure 1 FISH, SNP, histology section and May–Gru¨nwald–Giemsa (MGG)-stained BM smears analyses in four patients with CLL and MM. (a) Cytospin slides from BM sample of patient #2 were stained with MGG (left panel). FISH on the same field (right panel) with DNA probes for 13q14 (Spectrum orange, SO) and 13q34 (Spectrum aqua, SA). One red and one blue signal indicated a monosomy 13 in one plasma cell (arrow; MGG staining). (b)MGGstained PC subclone in patient #3 showed two signals for the chromosomal region 13q14 (SO) and three signals (Spectrum green, SG) for the centromere of 11 (arrow). (c) Copy number analyses of patient #4 using 250K NspI SNP mapping arrays. The data analysis was performed with the Affymetrix Genotyping Console 2.1. software, from CD19 positively selected PB cells (left panel) and from CD138 positively selected BM cells (right panel). Black circles label tumor-associated chromosomal aberrations; blue and red dots present genomic gains and losses, respectively, which overlap with entries in the database of genomic variants (http://projects.tcag.ca/variation/) and thus most likely present copy number variation regions also present in the germ line. (d) Histological section with focal CD20 þ B-CLL infiltrates in patient #5 (time period of the disease: February 2005; Â 10 magnification; CD20 þ CLL infiltrates in red). (e) MGG-stained BM smear with both coexisting B-CLL (black arrow) and PCs (red arrow) in patient #5. PCs show a basophilic cytoplasm, excentric nuclei, paranuclear halo and mono- as well as polyploid cells (time period of the disease: July 2005). (f) l-LC restriction of polynucleated PCs (red arrows), whereas B-CLL cells are l-negative (patient #5). (g) Bizarre, multinucleated giant PC, surrounded by more typical PCs ( Â 100 magnification) in patient #5. (h)Results of FISH analyses (with LSI ATM/CEP11 and LSI IGH/CCDN1 t(11;14) translocation probes) at the beginning of the disease, February 2005 (patient #5). (i) One hyperdiploid nucleus shows four ATM (SO) and four centromere 11 signals (SG; patient #5). (j) One interphase nucleus shows two signals of CCND1 (SO), one signal of IGH (SG) and two fusion signals (patient #5).

Leukemia Letters to the Editor 888 2005, white blood cell increased to 107 Â 109/l and the BM concomitant B-CLL and MM seemed to have a more unfavor- showed 90% CD20 þ B-CLL infiltrates (Figure 1d). After 2 able MM course, showing adverse prognostic features and early months, the patient presented with increased splenomegaly, death due to myeloma progression.1 As one promising anemia and thrombocytopenia. Owing to assumed B-CLL therapeutic approach for both diseases, high-dose chemo- progression, chemotherapy with chlorambucil and prednisone therapy and auto-PBSC transplantation have been performed was initiated, but did not improve the patient’s condition in selected patients, which also proved helpful in our patients #1 (ECOG 3). In July 2005, urine immunoelectrophoresis and and #5. Another important finding is, that under certain serum protein electrophoresis revealed monoclonal l-SFLCs circumstances, a third neoplasia or CLL transformation may (serum free light chain), and the typical MM phenotype with occur (AML in patient #2 and Richter’s syndrome in patient #4). l-LC-expressing mPCs (Figures 1e–g). A skeletal survey showed In most previously reported patients, MM developed during the multiple lytic lesions, and renal impairment became apparent. course of B-CLL (in 3/5 of our patients), arguing for mechanisms On the basis of increasing serum and urine l-LCs, atypical BM of a causal relationship. Although it has been described that mPCs (Figures 1e–g), lytic bone lesions and renal insufficiency, B-CLL cells can differentiate further and acquire mPC char- symptomatic stage IIIB MM was diagnosed. Molecular analyses acteristics, our findings strongly argue against this possibility. of PB and BM cells identified two unmutated IgVH gene The largest published group of B-CLL/MM patients supports our rearrangements (Table 1). The VH3-9 transcript was presumably conclusion showing a coexistence of two distinct clonal derived from his B-CLL, as it was amplified from cDNA expansions in six patients based on a different Ig LC in B-CLL of PB MNCs. An additional VH3-15 rearrangement could be and MM cells.1 Our results also show that the simultaneous amplified only from genomic DNA, which was not possible occurrence of MM and B-CLL is a rare phenomenon, but that for the VH3-9 rearrangement. Sequence analysis of both other oligoclonal malignancies are associated with B-CLL. The clonal rearrangements revealed a point mutation in the family- occurrence of AML has been described in a previously untreated specific VH3 primer binding site of the VH3-9 rearrangement as B-CLL patient, although we are not aware of AML with well as a stop codon in the CDR3 region of the VH3-15 concomitant B-CLL and MM in any patient as yet.7 Other rearrangement. Whereas the first mutation was likely to abolish hematological malignancies observed in conjunction with the amplification from DNA, the latter could lead to nonsense- B-CLL are prolymphocytic leukemia, Hodgkin’s lymphoma, mediated mRNA decay, which may explain the absence myelodysplastic syndrome and non-Hodgkin’s .1–5,7 of the VH3-15 transcript and the missing heavy chain protein Our calculation of the prevalence of concomitant B-CLL and in the immunofluorescence assay (Table 1). As both clonal MM within 348 B-CLL and 374 MM patients treated within in rearrangements were identified in PB cells, they were most our institution between 2005–08 was 1.44 and 1.34%, likely derived from the B-CLL clone lacking allelic exclusion. respectively, suggesting that concomitant B-CLL and MM is Copy number analysis with 50K SNP arrays identified two indeed a rare phenomenon, but may be discovered more deletions on chromosome 11 (11q23 and 11q24) in unselected frequently today with the use of better diagnostic options for BM and PB cells (Table 1). FISH with an ATM probe confirmed both diseases. the deletion of chromosome 11q23 in 89% of unselected During early lymphoid differentiation, the Ig gene rearrange- BM cells, thus representing the B-CLL clone given the frequency ments generally follow a hierarchical order.8 Ig heavy chain of 90% of B-CLL cells in the BM at the time of analysis gene (IGH) rearrangement precedes Ig LC gene rearrangement, (Figure 1h). Moreover, a small fraction of BM cells (2.5%) and DH to JH joining precedes VH to DJH joining.9 The process showed tetrasomy of chromosome 11 (four signals for ATM and of allelic exclusion ensures that once a functional VDJH four signals for centromere 11 (Figures 1h and i). An expression rearrangement has been achieved, the other IGH allele is array analysis with this sample showed high cyclin D1 generally excluded from further recombination attempts.10 After expression in unselected BM cells (data not shown), indicative this process, the Ig LC loci proceed to rearrange; initial attempts for an aberrant expression due to a translocation event. We occur at the Ig k locus (IGK), and if a functional IGK performed an additional FISH with a probe for t(11;14) in rearrangement is not achieved, the Ig l locus (IGL) undergoes unselected BM cells and detected a small percentage of recombination. Without further molecular analyses, a biclonal interphase nuclei (4%) with the t(11;14), together with origin solely based on different LC types may mistakenly be one additional signal for cyclin D1 (Figures 1h and j). This assumed.1 Most previous reports have indeed focused on heavy- correlated with giant, multinucleated mPCs by May–Gru¨nwald– and LC similarities vs discrepancies to explain the mono- vs Giemsa staining (Figure 1g), which were hyperdiploid for biclonal origin, respectively: in 13 of 23 (56%) published chromosome 11 (data not shown). This mPC fraction had a B-CLL/MM cases, B-CLL and MM cells shared different LCs, different genomic aberration than B-CLL cells, confirming the suggesting a biclonal origin of both diseases.1–5,7 However, biclonal origin of B-CLL and MM in this patient also. identical LCs do not necessarily support monoclonality in B-cell Chemotherapy with cyclophosphamide was initiated, but BM malignancies, because of inconclusive immunophenotyping mPC infiltrates increased to 90% and l-SFLCs remained (as observed in patient #1), or demonstration through genotype elevated, whereas B-CLL infiltrates completely disappeared. analyses that both B-CLL and MM are biclonal, despite Two cycles of VAD were given and the patient underwent auto- identical LCs (as observed in patient #3). Additional analyses PBSC transplantation in October 2005. Thereafter, his general have been used to resolve the clonal origin of B cells in B-CLL/ condition improved (ECOG 3-2) and l-SFLCs decreased MM patients. Most of them are based on the detection of substantially. Bortezomib maintenance therapy was initiated, genomic abnormalities in B-CLL and MM cells. We used SNP but in February 2006 MM relapse occurred and the patient died arrays as a new screening method in the detection of of myeloma progression and severe pneumonia 2 months later, chromosomal imbalances of selected B-CLL and MM cells. As whereby of note, his previous B-CLL remained in complete this technology depends on around 30% aberrant cells for an remission (Supplementary Table 2). imbalance to be detected, highly enriched cell populations As explicitly demonstrated in these five patients, the clinical allow the assignment of identified aberrations to the respective picture of patients with B-CLL and MM was the combination of cell compartment, as could be demonstrated in patient #4. typical features of both diseases. Of note, patients with However, when only one tumor cell type can be enriched, or

Leukemia Letters to the Editor 889 B-CLL and MM cells cannot be separated because of limited Conflict of interest cells or sample availability (as in patients #2, #3 and #5), identical aberrations have to be related to the two cell The authors declare no conflict of interest. compartments to prove clonality. Our rationale in those patients was to use SNP mapping arrays from CD19 positively selected PB cells for the screening of genomic aberrations in B-CLL and Acknowledgements subsequent FISH on BM smears to confirm the presence of these aberrations in mPCs. The SNP arrays from unselected and We thank the anonymous internal and external reviewers for CD19 þ selected PB cells were highly informative in four valuable comments and Dr Anke Spoo and Professor Dr Dr h.c. patients. One mono- and one biallelic deletion of chromosome Roland Mertelsmann for fruitful discussions and continuous 13q14 was detected in patients #2 and #3, respectively. support. This article is dedicated to Professor Dr Norbert P Nevertheless, as del13q14 is the most frequent chromosomal Su¨dkamp, Department of Orthopedic Surgery and Traumatology, abnormality in both B-CLL (in 450%)11 and MM (via Freiburg University Hospital, for his exceptional support, medical FISH in 40–50%), additional FISH with probes from 13q34 enthusiasm and thrive to best and innovatively treat his and our outside the deleted 5.8 Mb region clearly showed that both joint myeloma patients. regions were deleted in almost all analyzed mPCs. Therefore, M Pantic1, P Schroettner2, D Pfeifer1, J Rawluk2, U Denz2, two different chromosomal aberrations (del13q14 in PB cells A Schmitt-Gra¨ff3, H Veelken2,RWa¨sch2 and M Engelhardt2 and monosomy 13 in mPCs) strongly indicated a biclonal 1Core facility Genomics, Department of Hematology and origin of B-CLL and MM also in patient #2. The biallelic Oncology, University Medical Center Freiburg, Freiburg, deletion of 13q14 in CD19 þ cells from patient #3 with two Germany; differently sized alleles (5.6 and 2.7 Mb) was not detected in 2Department of Hematology and Oncology, University Medical Center Freiburg, Freiburg, Germany and mPCs on BM slides. Instead, a trisomy of chromosome 11 was 3 discovered in 40% of mPCs, disclosing a biclonal origin Pathology Department, University Medical Center Freiburg, of both cell populations. Both aberrations have an intermediate Freiburg, Germany E-mail: [email protected] prognosis12 and explain the achievement of stable disease of both B-CLL and MM in this patient. The two large 11q deletions (15.4 and 5.5 Mb) detected in B-CLL cells of patient #5 span the ATM and ADAMTS 8/15 genes, respectively. More- References over, we suggest that t(11;14) detected by FISH at B-CLL diagnosed in a small fraction of unselected BM cells (o5%), 1 Brouet JC, Fermand JP, Laurent G, Grange MJ, Chevalier A, accompanied by a high cyclin D1 expression, led to the Jacquillat C et al. The association of chronic lymphocytic expansion of the mPC clone. The genomic abnormalities leukaemia and multiple myeloma: a study of eleven patients. Br J Haematol 1985; 59: 55–66. discovered in mPCs of patient #4 were recurrent for MM; 2 Chang H, Wechalekar A, Li L, Reece D. Molecular cytogenetic however, the complex karyotype with gain on 11q and 4p, abnormalities in patients with concurrent chronic lymphocytic together with losses on 10q, 12p and 17p (including p53), leukemia and multiple myeloma shown by interphase fluorescence assigned patient #4 as high risk.13 in situ hybridization: evidence of distinct clonal origin. Our study bears the limitation that analyzed consecutive Cancer Genet Cytogenet 2004; 148: 44–48. MM/CLL patients were retrieved from a single institution rather 3 Hoffman KD, Rudders RA. Multiple myeloma and chronic lymphocytic leukemia in a single individual. Arch Intern Med than from registry data from many centers. Our analysis, 1977; 137: 232–235. however, also has many strengths, such as: (1) use of the 4 Saltman DL, Ross JA, Banks RE, Ross FM, Ford AM, Mackie MJ. combined Affymetrix SNP mapping array and FISH analyses on Molecular evidence for a single clonal origin in biphenotypic BM smears, whereby SNP revealed recurrent and new chromo- concomitant chronic lymphocytic leukemia and multiple somal aberrations in B-CLL and MM, and FISH connected myeloma. Blood 1989; 74: 2062–2065. specific chromosomal aberrations to different cell compart- 5 Shpilberg O, Mark Z, Biniaminov M, Rosner E, Rosenthal E, Gipsh N et al. 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Blood 2002; 100: 1425–1429. condition of the BM microenvironment. An intriguing possibility 8 Gonzalez D, van der Burg M, Garcia-Sanz R, Fenton JA, Langerak would be that leukemic B cells may secrete relevant factors that AW, Gonzalez M et al. Immunoglobulin gene rearrangements ‘trigger’ other B cells or mPCs.1 The presence of monoclonal and the pathogenesis of multiple myeloma. Blood 2007; 110: B-cell populations recently discovered in the PB of apparently 3112–3121. 14 9 Chang Y, Bosma MJ, Bosma GC. Extended duration of DH-JH healthy individuals support the ‘multi-hit’ hypothesis of rearrangement in immunoglobulin heavy chain transgenic mice: oncogenesis and suggest that the monoclonal ‘B-CLL-pheno- implications for regulation of allelic exclusion. J Exp Med 1999; type’ cells could be a starting point in the cascade of different 189: 1295–1305. events responsible for leukemogenesis. The mechanisms 10 Hieter PA, Korsmeyer SJ, Waldmann TA, Leder P. Human involved in B-cell are unknown at present, but it is immunoglobulin kappa light-chain genes are deleted or rearranged tempting to speculate that infectious agents associated with in lambda-producing B cells. Nature 1981; 290: 368–372. 11 Dohner H, Stilgenbauer S, Benner A, Leupolt E, Krober A, certain susceptibility genes may trigger polyclonal B-cell Bullinger L et al. Genomic aberrations and survival proliferation, with the subsequent evolution of cytogenetically in chronic lymphocytic leukemia. N Engl J Med 2000; 343: distinct clones. 1910–1916.

Leukemia Letters to the Editor 890 12 Avet-Loiseau H, Attal M, Moreau P, Charbonnel C, Garban F, of TP53 loss and the identification of potential novel transcrip- Hulin C et al. Genetic abnormalities and survival in multiple tional targets of TP53 in multiple myeloma. Blood 2008; 112: myeloma: the experience of the Intergroupe Francophone du 4235–4246. Myelome. Blood 2007; 109: 3489–3495. 14 Rachel JM, Zucker ML, Fox CM, Plapp FV, Menitove JE, Abbasi F 13 Xiong W, Wu X, Starnes S, Johnson SK, Haessler J, Wang S et al. Monoclonal B-cell lymphocytosis in blood donors. Br J et al. An analysis of the clinical and biologic significance Haematol 2007; 139: 832–836.

Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)

The use of low-dose alemtuzumab in pretreated B-cell chronic lymphocytic leukemia

Leukemia (2010) 24, 890–891; doi:10.1038/leu.2009.297; a second malignancy. Assessment of response, according to published online 21 January 2010 NCI-WG criteria and including a BM biopsy in all responding patients, was performed 2 months after the end of the treatment. We read with interest the article by Cortelezzi et al.1 published Overall survival, time to progression and time to retreatment in a recent issue of this journal. They report their experience were calculated from the start of alemtuzumab treatment to the with low-dose subcutaneous alemtuzumab (10 mg three event. times per week for 18 weeks) in 49 patients with pretreated Three patients (16%) obtained a complete response (CR) and B-cell chronic lymphocytic leukemia (B-CLL). The rationale for 6 (31%) obtained a partial response (PR), with an overall the use of subcutaneous low-dose alemtuzumab was to reduce response rate of 47% (Table 1). Two of the remaining patients the profound lymphopenia and high risk of infections when experienced progressive disease shortly after the end of the using the conventional intravenous schedule (30 mg three times treatment, whereas 5 patients had stable disease. Of the 5 a week for 12 weeks), which are serious problems particularly in patients with 17p-karyotype, 1 obtained a complete response older chronic lymphocytic leukemia patients and those already and another a partial response, whereas only 1 of the 8 severely immunocompromised by advanced disease and earlier fludarabine-refractory patients achieved a partial response. The therapies. The authors show interesting data regarding the response rate according to other clinical and biological features efficacy of low-dose alemtuzumab in terms of response rate, is reported in Table 1. progression-free survival, time to retreatment, and a favorable Except for grade IV neutropenia requiring G-CSF in 4 patients toxicity profile in a group of poor prognosis patients. and grade IV anemia requiring red blood cell transfusions in We now report our clinical experience with low-dose 2 patients, therapy was well tolerated both by patients who alemtuzumab in 19 heavily pretreated chronic lymphocytic received subcutaneous and patients who received intra- leukemia patients. Twelve of these patients, who have been venous alemtuzumab. Other hematological and extra-haemato- previously reported, received intravenous low-dose alemtuzu- logical side effects were relatively mild and consisted of mab (10 mg three times a week) in ten weeks.2 Seven additional fever, itching, rigor, headache and hypotension. No episode of patients were subsequently treated with the same schedule, febrile neutropenia or bacterial/fungal infection occurred during except that alemtuzumab was administered subcutaneously to treatment. Nine patients (47%) showed cytomegalovirus (CMV) reduce infusion related toxicity.3,4 reactivation after a median of 6 weeks of treatment, when the All patients had progressive disease according to NCI-WG median lymphocyte counts were 140/mmc. Treatment was criteria at the time when treatment with low-dose alemtuzumab temporarily interrupted and patients were successfully treated was started. Eighteen patients had previously received alkylating with oral ganciclovir for a median of 14 days. agents-based therapy, 15 fludarabine-containing regimen, 4 After a median follow-up of 33 months, 10 patients (52%) rituximab-containing regimen and 2 autologous PBSCT (median died with a median overall survival of 29 months. Only 2 of number of previous treatments 2, range 1–6). Eight patients were these patients responded to the treatment. The other 7 fludarabine refractory. Median age of the patients was 62 years responding patients experienced progression of the disease. (range 50–78 years), 13 were male and 6 female. The median progression-free survival was 11 months (range Sixteen patients completed the planned treatment. Two 5–45 months). Thirteen patients, 5 responders and 8 non- patients discontinued the treatment prematurely for documented responders, received further treatment. The median treatment- progressive disease and 1 because of the development of free survival was 20 months (range 6–70 months).

Table 1 Patients’ clinical and biological characteristics and response to therapy

Characteristics OR CR PR SD PD

Evaluable patients (n ¼ 16) 93652 Binet/Rai stage (n ¼ B/II, 9FC/III, 1FC/IV, 6) 7-1-1 3-0-0 4-1-1 2-0-5 0-0-2 FISH karyotype (n ¼ 17p-, 4/normal or 13q-, 9) 2/5 1/2 1/3 2/5 0/1 IgVH mutational status (U, 9/M, 5) 4/3 1/2 3/1 4/1 1/1 Zap-70+ (n ¼ 8) 00251 CD38+ (n ¼ 10) 11432 CR, complete response; M, mutated; n, number; OR, odds ratio; PD, progressive disease; PR, partial response; SD, stable disease; U, unmutated; FISH, Fluorescence In Situ Hybridization.

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