Leukemia (1999) 13, 85–91  1999 Stockton Press All rights reserved 0887-6924/99 $12.00 http://www.stockton-press.co.uk/leu Allelic loss of 11q13 as detected by MEN1-FISH is not associated with mutation of the MEN1 gene in lymphoid neoplasms C Thieblemont, S Pack, A Sakai, M Beaty, E Pak, AO Vortmeyer, A Wellmann, Z Zhuang, ES Jaffe and M Raffeld

Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Building 10, Room 2N110, Bethesda, MD 20892, USA

Deletions and rearrangements involving the long arm of chro- of the long arm of chromosome 11 are not uncommon in mosome 11 are not infrequent occurrences in the non-Hodg- NHL, and include several reports involving the 11q13 kin’s lymphomas. Recently, a tumor suppressor gene, the mul- 22–28 tiple endocrine neoplasia type 1 gene (MEN1) was cloned and region. This may indicate the presence of a tumor sup- mapped to chromosome 11q13. To assess the potential pressor gene at this locus which may be a target of inacti- involvement of this gene in lymphomagenesis, we examined 94 vation during lymphomagenesis. primary cases of lymphoma and 12 cell lines by a combination Over the past decade, the region 11q13 has been the focus of fluorescent in situ hybridization and PCR-SSCP analysis. In of interest of mapping studies and linkage analysis,29–35 in the our initial analysis of 41 primary B or T lymphomas, MEN1 FISH analysis revealed allelic deletions in 15 cases (three of four B search for the gene responsible for the multiple endocrine cell chronic lymphocytic leukemias, six of 15 follicular lym- neoplasia type 1 (MEN1) syndrome. The MEN1 syndrome is phomas, three of nine diffuse large B cell lymphomas, two of an autosomal, dominantly inherited disorder characterized by five mantle cell lymphomas, one of four Burkitt’s lymphoma). the development of multiple endocrine tumors in target organs To discern whether the MEN1 gene was in fact the target of the such as parathyroid, endocrine pancreas and anterior pituitary deletions, we assessed 20 of these 41 cases and an additional 36 74 primary lymphomas and 12 cell lines for MEN1 gene gland. The gene responsible for the MEN1 syndrome was 37 mutations using PCR-SSCP analysis. Abnormal SSCP patterns identified recently by positional cloning and was mapped to were found in exon 2 in two of the primary lymphoma cases chromosome 11q13. Patients with the MEN1 syndrome pos- and in one of the cell lines, but not in any of the original cases sess germline mutations in one MEN1 allele. A tumor sup- that showed MEN1 deletions by FISH. Furthermore, sequencing pressor function has been proposed based on the observation analysis revealed that the abnormal SSCP patterns in exon 2 were the result of a previously described genetic polymor- that loss of the wild-type MEN1 allele occurs in tumors that 38,39 phism (S145S: AGC → ACT), and in one sample, the result of arise in affected individuals. The function of the protein this S145S polymorphism associated with a second nucleotide translated from the MEN1 gene is still unknown. substitution at position 498 which left the encoded amino acid Allelic (heterozygous) deletions associated with germline unchanged. Our study indicates that the 11q13 locus is a fre- mutations of the MEN1 gene have been reported in 63–100% quent target of deletion in lymphoid neoplasms, but that there 40–44 are no associated mutations of the MEN1 gene. This suggests of MEN1-associated tumors. MEN1 gene mutations have that the 11q deletions either target another gene in lymphomas, also been reported in sporadic non-MEN1-associated tumors or that the MEN1 gene is inactivated through means other such as parathyroid tumors, gastrinomas, pancreatic endo- than mutation. crine tumors and pituitary tumors.45–48 Fifteen to 35% of spor- Keywords: MEN1; non-Hodgkin’s lymphoma; tumor suppressor adic parathyroid tumors have somatic mutations of MEN1 gene; chromosomal deletions gene.45–47 In sporadic pancreatic tumors and gastrinomas, loss of heterozygosity at 11q13 has been detected in 44 and 19%, respectively, of the 95 tumors analyzed.40 In sporadic pituitary Introduction gland tumors, somatic MEN1 deletions were detected in two of 39 (5%) cases analyzed.48 Although these tumors are Non-random chromosomal translocations are the most not associated with the MEN1 syndrome, all arise from characteristic cytogenetic abnormalities occurring in the non- endocrine tissue. Hodgkin’s lymphomas (NHL), and have been linked to distinct subtypes of lymphomas.1–4 Nonetheless, other types of abnor- We undertook this study to determine whether the MEN1 malities, particularly partial or complete chromosomal gene could be the target of the 11q deletions found in lymph- deletions, are also frequent.5 Loss of genetic material from 6q, oid tumors and to establish if this gene could also play a role 9p and 17p are among the most frequent deleted regions and contribute to tumor formation in a non-endocrine tissue reported in lymphomas,6–10 but other less well known such as lymphoid tissue. Several tumor suppressor genes such chromosomal losses have also been reported. Recurring as RB1, p16 or p53 were first described in inherited cancer 49 50 chromosomal deletions, which occur in nearly all types of syndromes (familial retinoblastoma, familial melanoma or 51 neoplasia as well as hematologic malignancies, are almost Li–Fraumeni syndrome respectively), and have now been always associated with the loss of a tumor suppressor gene shown to be involved in a substantial fraction of many differ- located within the deleted locus.11–13 For instance, chromo- ent types of sporadic cancers, including lymphomas.52–55 some 9p deletions are associated with the loss of a cyclin- We initially evaluated a subgroup of 41 lymphoid tumors dependent kinase inhibitor p16 (CDKN2),14 while deletions of for allelic loss of the MEN1 gene by fluorescent in situ chromosome 17p are associated with loss of the p53 tumor hybridization (FISH) using a cosmid that spans the MEN1 suppressor gene.15 coding region. We followed up this analysis using PCR-SSCP Among the lesser known chromosomal deletions in lym- to identify potential mutations in the MEN1 coding sequences phomas are those involving chromosome 11.16–21 Deletions in a series of 94 B and T lymphomas and 12 lymphoma/ leukemia cell lines representing a variety of different histo- logic subtypes. Correspondence: M Raffeld; Fax: 301 402 2415 Received 16 January 1998; accepted 3 August 1998 Allelic loss of 11q13 is not associated with mutation of MEN1 gene C Thieblemont et al 86 Materials and methods Table 1 Primer sets used for MEN1 gene PCR-SSCP analysis

Cases and cell lines studied Exon Sequence Expected PCR product size (bp) One hundred and fifteen patient samples consisting of lymph node, peripheral blood, spleen or other tissue diagnosed histo- 2A Forward: 5Ј-ttgccttgcaggccgccgcc-3Ј 201 logically as B or T NHL, were selected from case material Reverse: 5Ј-tggtagggatgacgcggttg-3Ј referred to the Hematopathology Section of the Laboratory of 2B Forward: 5Ј-ggcttcgtggagcattttct-3Ј 202 Ј Ј Pathology, National Cancer Institute (National Institutes of Reverse: 5 -ctcgaggatagagggacagg-3 2C Forward: 5Ј-ttcaccgcccagatccgagg-3Ј 297 Health, Bethesda, MD, USA). All cases were characterized Reverse: 5Ј-taagattcccacctactggg-3Ј using a panel of antibodies by paraffin section immunohisto- 3 Forward: 5Ј-attacctcccccttccaca-3Ј 249 chemistry. In addition, all samples contained at least 30% Reverse: 5Ј-taagattcccacctactggg-3Ј tumor cells, well within the sensitivity limits for the FISH and 4 Forward: 5Ј-cataatgatctcatcccccc-3Ј 171 PCR-SSCP analyses described below. Reverse: 5Ј-attggctcagccctcacctg-3Ј Ј Ј Twelve human B and T hematopoietic cell lines were also 5 Forward: 5 -gttccgtggctcataactct-3 98 Reverse: 5Ј-tggccacttccctctactga-3Ј studied. These included two EBV-transformed lymphoblastoid 6 Forward: 5Ј-ggactccttgggatcttcctgtg-3Ј 142 cell lines (SB and RPMI-8392), four high-grade lymphoma cell Reverse: 5Ј-ttctgcaccctccttagatg-3Ј lines (SUDHL6, SUDHL7, SUDHL9 and NUDHL1), one Burk- 7 Forward: 5Ј-ggactccttgggatcttcctgtg-3Ј 183 itt’s lymphoma cell line (Raji), two T acute leukemia/ Reverse: 5Ј-atcctcactcctggatgacagtg-3Ј lymphoma cell lines (Jurkat, MOLT4), one null-acute 8 Forward: 5Ј-cagagaccccactgctctca-3Ј 181 Reverse: 5Ј-ggctggagctccagcctttc-3Ј leukemia/lymphoma cell line (Nall-1) and two non-lymphoid Ј Ј leukemia cell lines (U937 and K562). 9 Forward: 5 -ctgctaaggggtgagtaagagac-3 225 Reverse: 5Ј-gtctgacaagcccgtggctgctg-3Ј 10A Forward: 5Ј-tcaccttgctctccccactg-3Ј 189 Reverse: 5Ј-ccaggcccttgtccagtgct-3Ј Ј Ј MEN1 allelic deletion analysis 10B Forward: 5 -ccaagaagccagcactggac-3 183 Reverse: 5Ј-cactctggaaagtgagcact-3Ј 10C Forward: 5Ј-ctgaaggtggcagcacggct-3Ј 219 Touch preps were made from frozen tumor specimens. After Reverse: 5Ј-gtagtcactaggggtggaca-3Ј fixation in methanol-acetic acid (3:1) for 20 min, slides were air-dried, equilibrated in 2 × SSC solution and dehydrated in ethanol series of 70, 80, 90, 100%. In situ hybridization was performed using cosmid clone c10B11 containing the MEN1 gene as a probe. The DNA was labeled with digoxigenin-11- dUTP by nick translation (Boehringer Mannheim, Indiana- PCR-SSCP analysis polis, IN, USA) and ethanol precipitated in the presence of 50× herring sperm DNA and 50× Cot-1 fraction of human Mutation screening was performed by SSCP analysis56 of the DNA. The DNA pellet was resuspended in hybrisol solution entire MEN1 coding region using PCR primers designed to (50% deionized formamide/10% dextran sulphate/2 × SSC) amplify 13 overlapping products from DNA (Table 1). The pri- with the final concentration of 25 ng/ml. Slides were mers were all purchased from Biosynthesis (Lewisville, TX, denatured in 70% formamide/2 × SSC at 72°C for 2 min with USA). 100 ng of genomic DNA was amplified in a 10 ␮l vol- the following incubation in cold (−20°C) ethanol series of 70, ume reaction containing 50 pm concentrations of each sense 80, 90, 100% for 2 min each and air-dried. Probes were and anti-sense primers, 1 × PCR buffer II (Perkin Elmer, Nor- ° ␮ denatured at 78 C for 10 min and then incubated for 30 min walk, CT, USA), 2.5 MgCl2 (Perkin-Elmer), 50 m of dATP, at 37°C for preannealing. A total amount of 250 ␮g DNA dGTP and dTTP each, 2 ␮Ci of ␣-32P dCTP (specific activity, probe was applied on the slide. Overnight hybridization was 6000 Ci/mmol; Dupont NEN, Boston, MA, USA), and 0.5 units done in a humidified chamber at 37°C. of AmpliTaq DNA (Perkin-Elmer). To increase the sensitivity Post-hybridization washes were performed at 45°C in 50% and the specificity of the PCR amplification, 1.4 ␮m of formamide/2 × SSC (5 min × 3), 1 × SSC (5 min × 2) and TaqStart Antibody (Clontech Laboratories, Palo Alto, CA, USA) 0.1 × SSC (5 min × 2). Detection was achieved using avidin- was included in the reaction. The amplification was perfor- FITC and anti-digoxigenin rhodamine (40 min at 37°C) fol- med in a Perkin-Elmer DNA 480 Thermal Cycler for 35 cycles lowed by washing in 4 × SSC/0.1% Tween 20 solution at 45°C consisting of 1 min of denaturation at 94°C, 1 min of (2 min × 3 times) and counterstaining with DAPI-antifade annealing at variable temperature depending on the pairs of (0.25 mg/ml). primers (Table 1), and 1 min of extension at 72°C, after an Hybridization signals were scored using a Zeiss Axiophot initial denaturation at 95°C for 5 min. PCR products were epifluorescence microscope (Thornwood, NY, USA) and two- diluted 1:10 in a denaturating loading buffer (95% formamide, color images were captured on a Photometrics CCD camera 12 mm EDTA, 0.2% bromophenol blue and 0.2% xylene (Photometrics, Tucson, AZ, USA) using IP Lab image software cyanol). The resulting mixture was heated at 94°C for 5 min (Signal Analytics Corporation, Vienna, VA, USA). At least 100 for denaturation, and chilled in an ice-water bath for 5 min. interphases with strong hybridization signals were scored. The A 2.5 ␮l sample of the product was immediately loaded onto presence of more than 15% cells with one MEN1 signal was a SSCP gel ((0.5 × Mutation Detection Enhancement solution interpreted as an allelic loss. In previous studies with these (FMC Bioproducts, Rockland, ME, USA)) to be subjected to probes on normal control tissues, we found a maximum of electrophoresis in 0.5 × TBE buffer at 8 W for 16 h at room 5% monoallelic deletions.48 In the present study, our normal temperature. Gels were transferred onto filter paper (3MM controls consisted of two reactive tonsils, each of which Whatman, Maidstone, UK), dried and exposed at −70°CtoX- showed 3% of cells with one MEN1 signal. OMAT film (Kodak, Rochester, NY) for 2–8 h. Allelic loss of 11q13 is not associated with mutation of MEN1 gene C Thieblemont et al 87 Subcloning and DNA sequencing strategy Table 2 Incidence of MEN1 gene alterations in human lymphoid malignancies For sequence analysis, variant and wild-type bands were excised from SSCP gels following autoradiography and resus- Classification No. of Deletion Mutational cases analysis analysis pended in 60 ␮l of water. One ␮l of the eluted DNA sample Lineage Tumor type (FISH (PCR-SSCP was used as a template for PCR amplification under conditions analysis) analysis) similar to those described above for SSCP analysis, except that deleted cases deoxynucleotide triphosphates were at 200 ␮m and radiolab- elled dCTP was omitted. One ␮l of each PCR product was Cases electrophoresed in an agarose gel (SaeKem GTG; FMC Bio- B B-CLL 14 3/4 0 product, Rockland, ME, USA), and visualized with ethidium MALT 9 — 0 a bromide. One ␮l of the PCR product was ligated into a pCR2. FL 16 6/15 1 MCL 13 2/5 1b 1 vector (Original TA Cloning Kit; InVitrogen, San Diego, DLBCL 25 3/9 0 USA). The ligated product was used to transform competent BL 12 1/4 0 bacteria (DH5␣; Gibco BRL, Gaithersburg, MD, USA) and the T PTCL 12 0/2 0 transformants were selected for ampicillin resistance. Recom- T-LBL 10 0/2 0 binant clones were grown up and screened to verify the cor- ALCL 4 — 0 rect sized insert by agarose gel electrophoresis. To exclude mutations due to the Taq polymerase, 10 clones Cell lines containing the correct-sized insert for each variant band were B Lymphoblastoid 2 — 0 selected, pooled, and automatically sequenced using the M13 high grade L 4 — 0 BL 1 — 0 forward sequencing primer. The resultant sequences were compared to the genomic DNA MEN1 sequence using T/null LBL 2 — 1b MacVector software. null-ALL 1 — 0 others myeloid 2 — 0

a Abnormal conformers due to known polymorphism at nucleotide Results 546, previously reported as S145S at the codon 145 (AGC → ACT) and a second silent nucleotide alteration at position 498, codon 129 (CTC → CTG). MEN1 allelic deletion analysis b Abnormal conformers due to known polymorphism at nucleotide 546 as above. FISH analysis to detect allelic deletions of the MEN1 gene was performed on an initial group of 41 cases including four B cell chronic lymphocytic lymphomas (CLL), 15 follicular lym- lymphomas (ALCL)). Twelve leukemia/lymphoma cell lines phomas (FL), five mantle cell lymphomas (MCL), nine diffuse were also screened. large B cell lymphomas (DLBCL), four Burkitt’s lymphoma Abnormal SSCP conformers were detected in only two (BL), two peripheral T cell lymphomas (PTCL) and two lym- cases (one FL and one MCL) (Figure 2). Neither of these cases phoblastic lymphomas of T cell phenotype (T-LBL). As sum- showed allelic loss of the MEN1 gene in the FISH analysis. In marized in Table 2, loss of one allele occurred in 15 of these the interphases examined, the FL case showed a normal pat- 41 cases (36.5%). These included three CLL, six FL, three tern with two rhodamine signals per cell, while the MCL case DLBCL, two MCL and one BL cases. Allelic deletions were displayed polyploidy with four rhodamine signals in the detected in 30–81% of the interphases examined for each majority of the interphases. One T-LBL cell line (Jurkat) had case. an altered SSCP mobility band (Figure 2). In all three samples, Allelic deletions were not detected in the other lymphoid the abnormal conformer was found for the primer set 2c PCR tumors studied. Three cases (one FL, one MCL and one product which spans the 5Ј region of MEN1 exon 2. No alter- DLBCL) showed polyploidy with three or four rhodamine ations in exons 3 to 10 were detected in the 94 cases and the MEN1 signals in the majority of the interphases. Figure 1 12 cell lines (data not shown). None of the five cases with shows a normal pattern with the presence of two rhodamine mono-allelic MEN1 deletions detected by FISH analysis signals per cell in panel a, and a case with polyploidy in panel showed an altered mobility band in the PCR-SSCP gel electro- b. Panels c and d show representative cases with allelic loss phoresis. of MEN1 gene, each showing between 30 and 40% of nuclei The abnormal conformers identified in the two cases and with allelic deletion. The number of nuclei showing allelic one cell line were subjected to sequencing analysis. All three deletion was roughly equivalent to the number of tumor cells tumors showed the same mutation at nucleotide 546 in the biopsied lymph nodes. (AGC→ AGT) corresponding to a previously described poly- morphism S145S at the codon 145 (Zhuang, personal communication). In addition, the follicular lymphoma case PCR-SSCP and sequence analysis showed a second mutation at nucleotide 498 (CTC → CTG). This mutation left the encoded amino acid unchanged at The above results encouraged us to look further for mutation codon 129 (leucine → leucine). within the MEN1 gene. A total of 94 cases including 20 of the 41 previous cases that were analyzed by FISH were selected. This included 68 cases of B cell malignancies (10 B-CLL, nine Discussion mucosa-associated lymphoid tissue lymphomas (MALT), 10 FL, 10 MCL, 20 DLBCL, nine BL), and 26 cases of T cell malig- We analyzed a total of 127 hematologic malignancies includ- nancies (12 PTCL, 10 T-LBL, and four anaplastic large cell ing 115 primary NHL cases representing the most common Allelic loss of 11q13 is not associated with mutation of MEN1 gene C Thieblemont et al 88

Figure 1 Representative FISH analyses with 11q13 (MEN1 gene) probe. (a) Lymphoblastic lymphoma with no evidence of allelic loss. Most nuclei contain two rhodamine signals. (b) Follicular lymphoma showing tetraploidy, but no evidence of allelic deletion. Most nuclei have four signals. (c) and (d) Two follicular lymphoma cases with allelic deletion of the MEN1 gene. 5/12 (42%) and 4/13 (31%) of nuclei, respectively, have only one rhodamine signal.

histological subtypes of B and T cell NHL and 12 hematopo- deletion and mutation of the remaining allele.55 In MEN1 syn- ietic cell lines, for loss and mutation of a new tumor sup- drome families, inactivation of the MEN1 gene occurs through pressor gene, the MEN1 gene, located at chromosome 11q13. hemizygous deletion and mutation of the alternate allele in For this purpose we used a combination of FISH analysis, 85% or more of the tumors arising in these patients (Zhuang, PCR-SSCP analysis and automated sequencing. Our FISH personal communication and Ref. 58). Whether promoter analysis revealed 15/41 (36.5%) primary cases with monoal- methylation or mutation of non-coding regulatory regions lelic deletions involving the MEN1 gene locus. However, we plays a role in the minority of cases where mutations or homo- failed to identify mutations in any of 127 neoplasms assayed zygous deletions are not found, is currently not known; how- by PCR-SSCP, including five of the 15 cases with mono-allelic ever these instances are likely to account for only a small per- MEN1 deletions detected by FISH. centage of cases. If the potential abnormalities of MEN1 in The failure to detect mutations in these cases is unlikely to lymphomas follow the pattern seen in tumors from MEN1 syn- be due to the inability of our primer sets to identify potential drome patients, our study would suggest that the MEN1 gene mutations in our cases. The primer sets used in the current is probably not the target of 11q13 deletions detected using study are identical to those published by Zhuang (personal the MEN1 FISH probe. Nonetheless, we have not formally communication) which have been shown to identify the ruled out the possibility that the MEN1 gene could have been majority (Ͼ85%) of mutations that occur in MEN1 families. inactivated through a combination of mono-allelic deletion Therefore, our failure to identify mutations in the MEN1 gene, and promoter methylation, or mutation of noncoding regulat- despite a relatively high frequency of mono-allelic deletion of ory elements, in the lymphoid neoplasms investigated. the MEN1 locus by FISH analysis, suggests that either the Deletions of the long arm of chromosome 11 have been MEN1 gene is not the targeted gene in the deletion, or that reported in several cytogenetic studies of NHL.16–28,59,60 The the remaining MEN1 allele is inactivated by means other than most common region of loss is between 11q14 and 11q24, mutation and deletion. and represents a common aberration detected in chronic lym- Although we have not ruled out all possible mechanisms of phocytic leukemia.16,59 It has been suggested that this deletion tumor suppressor gene inactivation, our study has excluded could be an important prognostic factor since patients with the two most common mechanisms, ie gene inactivation via CLL and 11q deletion exhibited a more rapid disease pro- homozygous deletion, and gene inactivation via hemizygous gression, a shorter survival time and a shorter treatment-free Allelic loss of 11q13 is not associated with mutation of MEN1 gene C Thieblemont et al 89 References 1 Yunis JJ, Oken MM, Kaplan ME, Ensrud KM, Howe RR, Theolog- ides A. Distinctive chromosomal abnormalities in histologic sub- types of non-Hodgkin’s lymphoma. New Engl J Med 1982; 307: 1231–1236. 2 Bloomfield CD, Arthur DC, Frizzera G, Levine EG, Peterson BA, Gajl-Peczalska KJ. Nonrandom chromosome abnormalities in lym- phoma. Cancer Res 1983; 43: 2975–2984. 3 Croce CM, Nowell PM. Molecular basis of human B cell neo- plasia. Blood 1985; 65: 1–7. 4 Chaganti RSK, Doucette LA, Offit K, Filippa DA, Allen GJ, Condon MR, Jhanwar SC, Clarkson BD, Lieberman PH. Specific translo- cations in non-Hodgkin’s lymphoma: incidence, molecular detec- tion, and histological and clinical correlations. Cancer Cells 1989; 7: 33–36. 5 Yunis JJ, Frizzera G, Oken MM, McKenna J, Theologides A, Arn- esen M. Multiple recurrent genomic defects in follicular lym- phoma. A possible model for cancer. New Engl J Med 1987; 316: 79–84. 6 Gaidano G, Hauptschein RS, Parsa NZ, Offit K, Rao PH, Lenoir G, Knowles DM, Chaganti RS, Dalla-Favera R. Deletions involving two distinct regions of 6q in B-cell non-Hodgkin lymphoma. Blood 1992; 80: 1781–1787. 7 Maseki N, Hayashi Y, Kaneko Y, Sakurai M. Partial loss of the short arm of chromosome 9 in leukemia and lymphoma. Cancer Figure 2 Single-stranded conformational polymorphism analysis Genet Cytogenet 1986; 22: 89–91. for exon 2. Aberrant band patterns, distinct from the germ line pattern 8 Pollak C, Hagemeijer A. Abnormalities of the short arm of chromo- (GL), are shown in lanes 1 and 3 (FL case and MCL case, respectively). some 9 with partial loss of material in hematological disorders. Lanes 2 and 4 are cases with germline pattern. Lane 5 (Jurkat cell line) Leukemia 1987; 7: 541–548. was run on a separate gel and shows a slightly different migration 9 Fernandez-Piqueras J, Santos J, Perez de Castro I, Melendez B, pattern. Martinez B, Robledo M, Rivas C, Benitez J. Frequent allelic losses of 9p21 markers and low incidence of mutations at p16(CDKN2) gene in non-Hodgkin lymphomas of B-cell lineage. Cancer Genet Cytogenet 1997; 98: 63–68. 20,21,60 10 Rodriguez MA, Ford RJ, Goodacre A, Selvanayagam P, Cabanillas interval than the patients without clonal 11q deletion. − A second area of 11q loss is represented by loss of the 11q13 F, Deisseroth AB. Chromosome 17 and p53 changes in lym- phoma. Br J Haematol 1991; 79: 575–582. region. Deletions involving 11q13 are reported less frequently 11 Ponder B. Gene losses in human tumors. Nature 1988; 335: than those involving 11q14-11q24, but occur in a variety of 400–402. lymphoma subtypes.22–28 These include small lymphocytic 12 Stanbridge EJ. Human tumor suppressor genes. Annu Rev Genet lymphoma,24,25 follicular lymphoma26,27 and lymphoblastic 1990; 24: 615–657. lymphoma.28 In the present study we detected allelic loss at 13 Marshall CJ. Tumor suppressor genes. Cell 1991; 64: 313–326. 11q13 in 15 of 41 (36.5%) lymphomas, primarily of B cell 14 Kamb A, Shattuck-Eidens D, Eeles R, Liu Q, Gruis NA, Ding W, Hussey C, Tran T, Miki Y, Weaver-Feldhaus J, McClure M, Aitken origin. These included both indolent lymphoid tumors such JF, Anderson DE, Bergman W, Frants R, Goldgar DE, Green A, as chronic lymphocytic leukemia and follicular lymphoma as MacLennan R, Martin NG, Meyer LJ, Youl P, Zone JJ, Skolnick well as aggressive diffuse large B cell lymphomas. The surpris- MH, Cannon-Albright LA. Analysis of the p16 gene (CDKN2) as ingly high percentage of cases containing 11q13 deletions in a candidate for the chromosome 9p melanoma susceptibility our study indicates that loss of genetic material at 11q13 in locus. Nat Genet 1994; 8: 23–26. lymphoid tumors is more frequent than previously reported, 15 Isobe M, Emanuel BS, Givol D, Oren M, Croce CM. Localization of gene for human p53 tumour antigen to band 17p13. Nature and is consistent with the ability of FISH to detect small inter- 1986; 320: 84–85. stitial deletions that may not be detectable in classical cyto- 16 Kobayashi H, Espinosa R III, Fernald AA, Begy C, Diaz MO, genetic analysis. Le Beau MM, Rowley JD. Analysis of deletions of the long arm of Interestingly, 11q13 deletions occur in a variety of other chromosome 11 in hematologic malignancies with fluorescence neoplastic tumors including breast cancer, colon cancer, in situ hybridization. Genes Chromosom Cancer 1993; 8: 246– ovary cancer and melanoma.61–64 However, to date, MEN1 252. 17 Slavutsky I, Labal de Vinuesa M, Este´vez ME, Sen L, Dupont J, gene mutations have not been identified in any of these neo- Larripa I. Chromosome studies in human hematologic diseases: plasms (Zhuang, personal communication and Ref. 65) and non-Hodgkin’s lymphomas. Haematologica 1987; 72: 29–37. appear to be confined to neoplasms arising in the MEN1 syn- 18 Juliusson G, Gahrton G. Chromosome aberrations in B-cell drome setting and to certain types of sporadic endocrine chronic lymphocytic leukemia. Pathogenetic and clinical impli- tumors. These studies in solid tumors and ours in lymphomas cations. Cancer Genet Cytogenet 1990; 45: 143–160. underscore the possibility that the 11q13 region may contain 19 Juliusson G, Oscier DG, Fitchett M, Ross FM, Stockdill G, Mackie MJ, Parker AC, Castoldi GL, Guneo A, Knuutila S, Elonen E, one or more tumor suppressor genes other than MEN1. Gahrton G. Prognostic subgroups in B-cell chronic lymphocytic leukemia defined by specific chromosomal abnormalities. New Engl J Med 1990; 323: 720–724. Acknowledgements 20 Hernandez JM, Mecucci C, Creil A, Meeus P, Michaux L, Van Hoof A, Verhoef G, Louwagie A, Scheiff J-M, Michaux J-L, Booga- We wish to thank Dr KSJ Elenitoba-Johnson for his critical erts M, Van den Berghe H. Cytogenetic analysis of B cell chronic lymphoid leukemias classified according to morphologic and review of the manuscript. CT was supported by a grant immunophenotypic (FAB) criteria. Leukemia 1995; 9: 2140–2146. founded by the Association Michel Clavel (Lyon, France) and 21 Fegan C, Robinson H, Thompson P, Whittaker JA, White D. Kary- by the National Cancer Institute. otypic evolution in CLL. Identification of a new sub-group of Allelic loss of 11q13 is not associated with mutation of MEN1 gene C Thieblemont et al 90 patients with deletions of 11q and advanced of progressive dis- AM, Burns AL, Marx SJ. Positional cloning of the gene for multiple ease. Leukemia 1995; 9: 2003–2008. endocrine neoplasia-type 1. Science 1997; 276: 404–407. 22 Mark J, Ekedahl C, Dahlenfors R. Characteristics of the banding 38 Larsson C, Weber G, Teh BT, Lagercrantz J. Genetics of multiple patterns in non-Hodgkin and non-Burkitt lymphomas. Hereditas endocrine neoplasia type 1. Ann NY Acad Sci 1994; 733: 453– 1978; 88: 229–242. 463. 23 Palka G, Fioritone G, Lombardo M, Donti E, Calabrese G, Stuppia 39 Thakker RV, Bouloux P, Wooding C, Chotai K, Broad PM, Spurr L, Di Marco G, Franchi PG, Parruti G, Geraci L. Chromosome NK, Besser GM, O’Riordan J. Association of parathyroid tumors abnormalities in PHA-stimulated and non stimulated bone marrow in multiple endocrine neoplasia type 1 with loss of alleles of chro- cultures from patients with non-Hodgkin lymphoma. Haematolog- mosome 11. New Engl J Med 1989; 321: 218–224. ica 1988; 73: 489–494. 40 Debelenko LV, Zhuang Z, Emmert-Buck MR, Chandrasekharappa 24 Yunis JJ. Recurrent chromosomal defects are found in most SC, Manickam P, Guru SC, Marx SJ, Skarulis MC, Spiegel AM, patients with acute nonlymphocytic leukemia. Cancer Genet Collins FS, Jensen RT, Liotta LA, Lubensky IA. Allelic deletions Cytogenet 1984; 11: 125–137. on chromosome 11q13 in multiple endocrine neoplasia type-1 25 Koduru PR, Filippa DA, Richardson ME, Jhanwar SC, Chaganti SR, associated and sporadic gastrinomas and pancreatic endocrine Koziner B, Clarkson BD, Lieberman PH, Chaganti RS. Cytogenetic tumors. Cancer Res 1997; 57: 2238–2243. and histologic correlations in malignant lymphomas. Blood 1987; 41 Lubensky IA, Debelenko LV, Zhuang Z, Emmert-Buck MR, Dong 69: 97–102. Q, Chandrasekharappa SC, Guru SC, Manickam P, Olufemi S-E, 26 Nishida K, Taniwaki M, Misawa S, Abe T. Nonrandom rearrange- Marx SJ, Spiegel AM, Collins FS, Liotta LA. Allelic deletions on ment of chromosome 14 at band q32.33 in human lymphoid chromosome 11q13 in multiple tumors from individual MEN1 malignancies with mature B-cell phenotype. Cancer Res 1989; 49: patients. Cancer Res 1996; 56: 5272–5278. 1275–1281. 42 Beckers A, Abs R, Reyniers E, De Boulle K, Steavenaert A, Heller 27 Takechi M, Tanaka K, Hashimoto T, Asaoku H, Dohy H, Kikuchi FR, Klo¨ppel G, Meurisse M, Willems PJ. Variable regions of chro- M, Kamada N. Cytogenetic, molecular biological and clinical mosome 11 loss in different pathological tissues of a patient with study of B cell lymphomas with 14;18 translocation in Japanese the multiple endocrine neoplasia type 1 syndrome. J Clin Endocri- patients. Leukemia 1991; 5: 1069–1075. nol Metab 1994; 79: 1498–1502. 28 Kaneko Y, Frizzera G, Shikano T, Kobayashi H, Maseki N, Sakurai 43 Radford DM, Ashley SW, Wells SA Jr, Gerhard DS. Loss of hetero- M. Chromosomal and immunophenotypic patterns in T cell acute zygosity of markers on chromosome 11 in tumors from patients lymphoblastic leukemia (T ALL) and lymphoblastic lymphoma with multiple endocrine neoplasia syndrome type 1. Cancer Res (LBL). Leukemia 1989; 3: 886–892. 1990; 50: 6529–6533. 29 Larsson C, Skogseid B, O¨ berg K, Nakamura Y, Nordenskjo¨ld M. 44 Bale AE, Norton JA, Wong EL, Fryburg JS, Maton PN, Oldfield Multiple endocrine neoplasia type 1 gene maps to chromosome EH, Streeten E, Aurbach GD, Brandi ML, Friedman E, Spiegel AM, 11 and is lost in insulinoma. Nature 1988; 332: 85–87. Taggart RT, Marx SJ. Allelic loss on chromosome 11 in hereditary 30 Bale SJ, Bale AE, Stewart K, Dachowski L, McBride OW, Glaser and sporadic tumors related to familial multiple endocrine type T, Green SE III, Mulvihill JJ, Brandi ML, Sakaguchi K, Aurbach 1. Cancer Res 1991; 51: 1154–1157. GD, Marx SJ. Linkage analysis of multiple endocrine neoplasia 45 Heppner C, Kester MB, Agarwal SK, Debelenko LV, Emmert-Buck type 1 with INT2 and other markers on chromosome 11. Geno- MR, Guru SC, Manickam P, Olufemi S-E, Skarulis MC, Doppman mics 1989; 4: 320–322. JL, Alexander RH, Kim YS, Saggar SK, Lubensky IA, Zhuang Z, 31 Bystrom CC, Larsson C, Blomberg K, Sandelin U, Falkmer B, Skog- Liotta LA, Chandrasekharappa SC, Collins FS, Spiegel AM, Burns seid B, O¨ berg K, Wermer S, Nordenskjo¨ld M. Localization of the AL, Marx SJ. Somatic mutation of the MEN1 gene in parathyroid MEN1 gene to a small region within the chromosome 11q13 by tumors. Nat Genet 1997; 16: 375–378. deletion mapping in tumors. Proc Natl Acad Sci USA 1990; 87: 46 Friedman E, De Marco L, Gejman PV, Norton JA, Bale AE, Aur- bach GD, Spiegel AM, Marx SJ. Allelic loss from chromosome 11 1968–1972. in parathyroid tumors. Cancer Res 1992; 52: 6804–6809. 32 Thakker RV, Wooding C, Pang JT, Farren B, Harding B, Anderson 47 Iwasaki H. A possible tumor suppressor gene for parathyroid aden- DC, Besser GM, Bouloux P, Brenton DP, Buchanan KD, Edwards omas. Int Surg 1996; 81: 71–76. CR, Heath DA, Jackson CE, Jansen S, Lips K, Norum RA, Sampson 48 Zhuang Z, Ezzat SZ, Vortmeyer AO, Weil R, Oldfield EH, Park J, Shalet SM, Taggart RT, Tailor D, Wheeler MH, Woollard PM, W-S, Pack S, Huang S, Agarwa SK, Guru SC, Manickam P, Debe- Yates J. Linkage analysis of 7 polymorphic markers at chromosome lenko LV, Kester MB, Olufemi S-E, Heppner C, Crabtree JS, Burns 11p11.2-11q13 in 27 multiple endocrine neoplasia type 1 famil- AL, Spiegel AM, Marx SJ, Chandrasekharappa SC, Collins FS, ies. Ann Hum Genet 1993; 57: 17–25. Emmert-Buck MR, Liotta LA, Asa SL, Lubensky IA. Mutations of 33 Courseaux A, Grosgeorge J, Gaudray P, Pannett AA, Forbes SA, the MEN1 tumor suppressor gene in pituitary tumors. Cancer Res Williamson C, Bassett D, Thakker RV, Teh BT, Farnebo F, 1997; 57: 5446–5451. Shepherd J, Skogseid B, Larsson C, Giraud S, Zhang CX, Salandre 49 Friend SH, Bernards R, Rogelj S, Weinberg RA, Rapaport JM, J, Calender A. Definition of the minimal MEN1 candidate area Albert DM, Dryja TP. A human DNA segment with properties of based on a 5-Mb integrated map of proximal 11q13. Genomics the gene that predisposes to retinoblastoma and osteosarcoma. 1996; 37: 354–365. Nature 1986; 323: 643–646. 34 Smith CM, Wells SA, Gerhard DS. Mapping eight new polymor- 50 Hussussian CJ, Struewing JP, Goldstein AM, Higgins PA, Ally DS, phism in 11q13 in the vicinity of the multiple endocrine neoplasia Sheahan MD, Clark WH Jr, Tucker MA, Dracopoli NC. Germline type 1: identification of a new distal recombinant. Hum Genet p16 mutations in familial melanoma. Nat Genet 1994; 8: 15–21. 1995; 96: 377–387. 51 Srivastava S, Zou ZQ, Pirollo K, Blattner W, Chang EH. Germ-line 35 Debelenko LV, Emmert-Buck MR, Manickam P, Kester M-B, Guru transmission of a mutated p53 gene in a cancer-prone family with SC, Difranco EM, Olufemi S-E, Agarwal S, Lubensky IA, Zhuang Li–Fraumeni syndrome. Nature 1990; 348: 747–749. Z, Burns AL, Speigel AM, Liotta LA, Collins FS, Marx SJ, Chandra- 52 Ginsberg AM, Raffeld M, Cossman J. Inactivation of the retinoblas- sekharappa SC. Haplotype analysis defines a new minimal interval toma gene in human lymphoid neoplasms. Blood 1991; 77: for multiple endocrine neoplasia type 1 (MEN1) gene. Cancer Res 833–840. 1997; 57: 1039–1042. 53 Imamura J, Miyoshi I, Koeffler HP. p53 in hematologic malig- 36 Metz DC, Jensen RT, Bale A, Skarulis MC, Eastman R, Nieman L, nancies. Blood 1994; 84: 2412–2421. Norton JA, Friedman E, Larsson C, Amorosi A, Brandi ML, Marx 54 Prokocimer M, Rotter V. Structure and function of p53 in normal SJ. Multiple endocrine neoplasia type 1: clinical features and man- cells and their aberrations in cancer cells: projection on the hema- agement. In: Bileezekian JP, Levine MA, Marcus R (eds). The Para- tologic cell lineages. Blood 1994; 84: 2391–2411. thyroids. Raven Press: New York; 1994, pp 591–646. 55 Otsuki T, Clark HM, Wellmann A, Jaffe ES, Raffeld M. Involvement 37 Chandrasekharappa SC, Guru SC, Manickam P, Olufemi S-E, Col- of CDKN2 (p16INK4A/MTS1) and p15INK4B/MTS2 in human leuke- lins FS, Emmert-Buck MR, Debelenko LV, Zhuang Z, Lubensky IA, mias and lymphomas. Cancer Res 1995; 55: 1436–1440. Liotta LA, Crabtree JS, Wang Y, Roe BA, Weisemann J, Boguski 56 Orita M, Iwahana H, Kanazawa H, Hayashi K, Sekiya T. Detection MS, Agarwal SK, Kester MB, Kim YS, Heppner C, Dong Q, Spiegel of polymorphisms of human DNA by gel electrophoresis as single- Allelic loss of 11q13 is not associated with mutation of MEN1 gene C Thieblemont et al 91 strand conformation polymorphisms. Proc Natl Acad Sci USA 61 Tomlinson IPM, Nicolai H, Solomon E, Bodmer WF. The fre- 1989; 86: 2766–2770. quency and mechanism of loss of heterozygosity on chromosome 57 Fearon ER. Human cancer syndromes: clues to the origin and nat- 11q in breast cancer. J Pathol 1996; 180: 38–43. ure of cancer. Science 1997; 278: 1043–1050. 62 Gustafson CE, Young J, Leggett B, Searle J, Chevenix-Trench G. 58 Basset JHD, Forbes SA, Pannett AAJ, Lloyd SE, Christie PT, Wood- Loss of heterozygosity on the long arm of chromosome 11 in col- ing C, Harding B, Besser GM, Edwards CR, Monson JP, Sampson orectal tumours. Br J Cancer 1994; 94: 395–397. J, Wass JAH, Wheeler MH, Thakker RV. Characterization of 63 Healy E, Rehman I, Angus B, Rees JL. Loss of heterozygosity in mutations in patients with multiple endocrine neoplasia type 1. sporadic cutaneous melanoma. Genes Chromosom Cancer 1995; Am J Hum Genet 1998; 62: 232–244. 12: 152–156. 59 Karhu R, Knuutila S, Kallioniemi O-P, Siitonen S, Aine R, Vilpo 64 Foulkes WD, Campbell IG, Stamp GWH, Trowsdale J. Loss of het- L, Vilpo J. Frequent loss of the 11q14-24 region in chronic lym- erozygosity and amplification on chromosome 11q in human phocytic leukemia: a study by comparative genomic hybridiz- ovarian cancer. Br J Cancer 1993; 67: 268–273. ation. Genes Chromosom Cancer 1997; 19: 286–290. 65 Bo¨ni R, Vortmeyer AO, Huang S, Burg G, Hofbauer G, Zhuang 60 Do¨hner H, Stilgenbauer S, James MR, Benner A, Weilguni T, Bentz Z. Mutation analysis of the MEN1 tumor suppressor gene in malig- M, Fisher K, Hunstein W, Lichter P. 11q deletions identify a new nant melanoma (submitted). subset of B-cell chronic lymphocytic leukemia characterized by extensive nodal involvement and inferior prognosis. Blood 1997; 89: 2516–2522.