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From www.bloodjournal.org by guest on April 28, 2017. For personal use only. MYC Rearrangement and Translocations Involving Band 8q24 in Diffuse Large Cell Lymphomas By Marc Ladanyi, Kenneth Offit, Suresh C. Jhanwar, Daniel A. Filippa, and R.S.K. Chaganti The configuration of the MYC gene in diffuse large cell lymphomas (DLCL) with translocations involving band 8q24 [t(8q24)] has not been systematically studied. We collected cytogenetic and clinical data on 171 consecutive cases of DLCL, including cleaved, noncleaved, and immunoblastic types, of which 96 had DNA available and 124 had abnormal karyotypes. The cases with DNA available were evaluated for MYC rearrangement (MYC-R) by Southern hybridization of EcoRI-digested tumor DNA using an exon-1 probe, a combination of probe and enzyme known t o detect over 85% of breaks in sporadic Burkitt’s lymphoma. In cases studied at diagnosis, MYC-R, t(8;14)(q24;q32), or other t(8q24) were not prognostically significant. Among the 124 cases with karyotypic abnormalities, seropositivity for human immunodeficiency virus was significantly more common in cases with a t(8q24) (72%) than in cases without it (9%) (P < .05). Of the four cases with an MYC -R, two had a t(8;14), one had a t(7;8;14)(~15;q24;932), and one had a t(8;?)(q24;?) and a T HE t(8;14)(q24;q32) translocation, or one of its variants t(2;8)(p12;q24) and t(8;22)(q24;q32), is classically seen in Burkitt’s lymphoma (BL), but is also found in some diffuse non-Burkitt’s non-Hodgkin’s lymphomas (NHL).’-4 The t(8;14) translocation has been shown to juxtapose the MYC oncogene, located at 8q24, to the immunoglobulin heavy chain (IGH) gene, located at 14q32 resulting in deregulated expression of MYC. Similarly, the variant translocations involve the light chain IG genes, IGK at 2p12, and IGL at 22qll.’ Recent studies have shown the prognostic significance of certain cytogenetic findings within histologically defined subgroups of NHL.6-8 Because the clinical significance of t(8;14) in non-Burkitt’s NHL has not been defined, we determined the incidence and clinical correlations of a cytogenetic 8q24 translocation break [t(8q24)] in a consecutive series of 171 diffuse large cell lymphomas (DLCL), of which 124 had an abnormal karyotype. In endemic BL (eBL) with t(8;14), the translocation breaks usually involve the V-DJ regions and sequences far 5’ of MYC, while in sporadic BL (sBL) and acquired immunodeficiency syndrome (AIDS)-associated BL with t(8;14), the switch regions of ZGH and MYC sequences in the first intron-first exon or regions immediately 5’ are involved?.’ In all variant translocations the breaks are . ~ the precise mapping of the breaks located 3‘ o ~ M Y CThus, in the MYC gene in numerous BL specimens and cell lines has demonstrated considerable heterogeneity and some clinical correlations. However, MYC rearrangements have not been extensively studied in non-Burkitt’s NHL. We present here the results of breakpoint mapping studies in 15 cases with a t(8q24), taken from the subset of 76 cases of DLCL where both an abnormal karyotype was documented and material for molecular studies was available. In addition, we have evaluated the same 15 cases for point mutations in the PvuII site in the first exon of MYC, because such mutations at this site have been suggested to contribute to transcriptional deregulation of MYC. lo Blood, Vol77, No 5 (March 1). 1991: pp 1057-1063 de1(8)(q24). In the three previous cases with translocations involving 8q24 and 14q32, comigration of the rearranged MYC band with either the J region or the switch-p region of the lg heavy chain gene could not be demonstrated, leaving the 14q32 breakpoint undefined at the molecular level. Among the remaining 72 cases where both an abnormal karyotype and molecular data were available, 11 had a t(8q24). either t(8;14) or t(8;22)(q24;qll), in the absence of an MYC-R. In these cases, the 8q24 break was presumably located outside of the EcoRl MYC fragment. All 15 cases with a t(8q24) were also screened for point mutations in the bull site in the first exon of MYC; two cases that were not MYC-R showed loss of this restriction site. These results indicate that in most DLCL with t(8;14) or other t(8q24). the 8q24 breakpoint lies away from the MYC gene; in a minority of these cases, point mutations in regulatory noncoding regions were detected. o 1991by The American Society of Hematology. MATERIALS AND METHODS The present series of 171 cases of DLCL is a subset of the series of 434 specimens of NHL consecutively ascertained over a 5-year period (1984 through 1988) at the Memorial Sloan-Kettering Cancer Center (New York, NY),which has been defined in more detail elsewhere! In these 171 DLCL, DNA for Southern blotting analysis was available in 96, and an abnormal karyotype was obtained in 124; 76 cases had both available, 48 had only abnormal karyotypes available, 20 had only DNA results available, and 27 cases had neither type of data available. The DLCL were classified pathologically according to the International Working Formulation,” and included cleaved, noncleaved, and immunoblastic cell types. There was no histologic component of BL in any of the cases. The cell surface IG determination and the cytogenetic analysis, which consisted of short-term culture followed by G- andlor Q-banding, were performed as previously described.’.’.’’ The karyotypes are described according to ISCN.” Comparison of proportions of cases with specific cytogenetic or molecular genetic features were performed using the method of inference from proportion^,'^ and actuarial survival rates were compared using the method of Kaplan and Meier” and the logrank test for significance. Immunoperoxidase studies were performed on B3-fixed (B3: 3% mercuric chloride in 4% formaldehyde) paraffin-embedded sec- From the Laboratory of Cancer Genetics, Sloan-Kettering Institute and the Departments of Pathology (Cytogenetics and Surgical Pathology Services) and Medicine, Memorial Hospital, New York, Ny. Submitted August 20,1990; accepted October 24, 1990. Supported by Grant Nos. CA-34775 and CA-20194 from the National Institutes of Health, Bethesda, MD. Presented in part at the 81st Annual Meeting of the American Association for Cancer Research in Washington, DC, May 25, 1990. Address reprint requests to Marc Ladanyi, MD, Cytogenetics Service, Department of Pathology, Memorial Sloan-Kettering Cancer Center, 1275 YorkAve, New York, NY10021. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 0 I991 by The American Society of Hematology. 0006-4971I91/7705-OO23$3.00l0 1057 From www.bloodjournal.org by guest on April 28, 2017. For personal use only. LADANYI ET AL 1058 tions using an avidin-biotin peroxidase complex method.16 The L26” and UCHL-1’8monoclonal antibodies, specific for B cells and T cells, respectively, were obtained from Dakopatts (Santa Barbara, CA). Gene rearrangements were studied by Southern blotting of DNA extracted from snap-frozen tissue, using an Oncor ProbeTech vacuum blotting device, as previously described: The following combinations of probes and enzymes were used. For the IGH gene, DNA digested with Hind111 or EcoRI was probed with a 5.5-kb BamHI-Hind111 fragment encompassing the entire J region (JH), a 1.3-kbEcoRI fragment of the constant-p,region (Ck), and a 2-kb Sst I fragment including all but the 3’ end of the switch-lr. (SF) region.19For analysis of the configuration of the MYC gene, DNA digested with EcoRI, HindIII, PstI, PvuII, SmaI, or Xba I restriction enzymes was probed with anXho I-Xba I genomic fragment of MYC that includes all but the 5’ end of the first exon.” For the PvuII site in exon-1 of MYC, a double digestion with PvuII and Xho I restriction enzymes was performed as described:’ followed by hybridization with the same probe as described above. The ZGH and MYC gene probes used in this study were gifts from J. Ravetch and W. Hayward, respectively. RESULTS Cytogenetics. Abnormal karyotypes were obtained in 124of 171cases. Of these, 17 (14%) had a classic t(8;14)(q24; q32) translocation, four (3%) had a t(8;22)(q24;qll) translocation, and two had other 8q24 translocations, including a t(7;8;14)(plS;q24;q32) in case 408 and a t(8;?)(q24;?) and de1(8)(q24) in case 230 (Fig 1).In all, 23 cases (19%) had a translocation involving 8q24. In addition to t(8q24), other notable karyotypic abnormalities were present in two cases listed in Table 1: case 591 also showed a t(3;22)(q27;qll) and was included in a previous report of a series of cases with this translocation,2* while in case 143 a t(8;22)(q24;qll) and a t(14;18)(q32;q21) were present together. The latter is the only case with involvement of 18q21 and 8q24 in the same specimen in this series; in all, 24 of the 124 cases of DLCL with karyotypic data showed translocations involving 18q21, most often t(14;18)(q32;q21). Gene rearrangements. DNA was available for Southern blot analysis in 96 cases, including 15 cases with a t(8q24) (Table 1).EcoRI-digested DNA from these specimens was probed with the MYC exon-1 probe; this combination of probe and enzyme is known to detect over 85% of breaks in sBL.” Four cases (4%) displayed an MYC rearrangement (cases 230,348,408, and 705). Mapping of the breakpoints was performed by studying the pattern of rearrangements obtained with additional enzymes, including HindIII, PstI, PvuII, SmaI, and XbaI (Fig 2). A clustering of breaks was observed within exon-llintron-1 for cases 348,408, and 705, and a more downstream break site was detected in case 230; these results are schematically represented in Fig 3. None of the 20 cases with molecular data alone were found to be rearranged for MYC. The 15 cases with a t(8q24) were evaluated for exon-1 MYC mutations at the PvuII restriction enzyme site (Table 1). In cases 348, 408, and 705, the PvuII fragment was rearranged; in cases 540 and 591, thePvuII site at the 3’ end of exon-1 was lost, indicating a small deletion or a point mutation at this site (Fig 4). The remaining cases showed no alteration of the PvuII fragments. Hence, 2 of 11 cases (18%) of DLCL with an 8q24 break remote from the MYC gene showed point mutation at this site. Rehybridization of blots with probes for Sp, Cp, JH, and MYC were performed in cases 348,408, and 705 to detect comigrating bands. Two rearranged JH bands were present in each of these cases (data not shown). Case 705 also showed a rearrangement of Sp, in the presence of a germline C p band, whereas in cases 348 and 408 both Sp and C p were in germline configuration (data not shown). However, in none of the three cases was comigration of rearranged Sp or JH bands with the rearranged MYC bands demonstrated. Zmmunophenoqpes. Surface IG results on fresh tumor cell suspensions were available in 11of the 15 cases in Table 1. Three cases expressed only ZGM, five cases also expressed other heavy chains, while the remaining three cases expressed no surface IG. Six cases, including one studied in frozen section only (case 295), expressed ZGK light chains, while three expressed ZGL light chains. Case 143 had an ZGK phenotype in the presence of a t(8;22). The cell Chromosome 7 a 14 la 22 143 I 230 Patient Fig 1. Q-banded partial karyotypes of cases 143, 230, and 348. In case 143, the t(8;22)(q24;qll) and the t(14;18)(q32;q21) are illustrated. In case 408, the complext(7;8;14)(p15;q24;q32) resulting in translocation of distal 7p material to 8q. of distal 8q to 14q. and of distal 14q to 7p is shown. In case 230, the de1(8)(q24) and the t(8;7)(q24;7) are shown. Arrows indicate breakpoints. From www.bloodjournal.org by guest on April 28, 2017. For personal use only. 1059 MYC REARRANGEMENTS IN t(8;14) LYMPHOMAS Table 1. Clinical, Pathologic, Cytogenetic, and Molecular Data on the 15 DLCL With t(8q24) UTN Age/ Sex Pathology Site Surface IG MYC No. of Pvull Treatment A. DLCL with t(8q24) and MYC rearrangement R G 230 24/M IMB-D LN None Cells Pre 6 14 1 29 21 32 5 5 4 348 291M LNCC-D SB None R R Pre 408 35lM IMB-D 705 38/F IMB-D LN IGG-K CW ND R R R R Post Pre 2 B. DLCL with t(8:14) withoutMYC rearrangement 70 23lF IMB-D LN None G G 147 60/F LNCC-D 178 28lM LNCC-D Post 28 19 Sp IGG-K G G Post LI IGM-L G G Pre 4 22 295 42lM LNCC-D LN K G G Post 4 23 534 SP ND G G Post 1 16 536 31/F LNCC-D 591 37lM IMB-D SP LN IGM-K IGG-L G G G D Pre Pre 626 60/F LNCC-D LN IGM-L G G Post 19 9 7 18 656 41/F LNCC-D BN IGG G G Pre 3 33 71/F LCC-D Karyotype 46,XY 47.X.-Y,+ 1,del(l)(p32),de1(8)(q24),t(2;5)(p23;q35)t(4;?)(pl6:?),t(8:?) (q24;?),+mar 46,XY 46,XY,t(8; 14)(q24;q32) 47,XY,+5,t(7;8; 14)(pl5;q24;q32) 46,X,der(X)t(X;l)(q26;q21),t(8: 14)(q24:q32),dup(ll)(q13+q23) 47,X,aer(X)t(X;l)(q26;q21),+7,t(8;14)(q24;q32),dup(l l)(q13-+q23) 47,X,der(X)t(X;l)(q26:q21),+7,t(8;14)(q24;q32),dup(l l)(q13+q23) 46,X,der(X)t(X; l)(q26:q21),t(8; 14)(q24;q32),der(4)t(4:?) (q35:?),dup(l l)(q13+q23) 46,XX,t(8: 14)(q24:q32),d up(11)(q13+q23) 50,XX,+ 11,+12,+ 14,t(1;14)(p31;q32),t(8:14)(q24:q32),der(9) (qter-cen: :?),der(g)(qter+cen::?), +de1(9)(q22+ter) 48,XX,+1,+7,+9,-20,t(1:6; 1 l)(p31~q23~q13),t(l~13)(q25~q22),t(8;14) (q24;q32),de1(6)(q21-*ter),del(ll )(pl3+ter) 46,XY 47,XY.- 13,+ 12,t(8;14)(q24;q32),+der(13)t(13;2)(q34;pl5) 46,XX 55-57,XX.6, +7,+22,t(8: 14)(q24:q32),2Xdel(lO)(pl1+ter), der(l3) t(13;?)(p12;?),+marl,2 46,XX 48,XX. - 15,+6,t(8; 14)(q24:q32).+der(X)t(l;X)(q21;q27),der(2) (qter+cen::?),+mar 48,XX,+5,t( 1;12)(q12;q13),t(8: 14)(q24:q32),i(6p),+der( 12)t(1; 12)(q12:q13) 47,XY,t(3; 22)(q27;q 11),t(8; 14)(q24;q32) 47,XY,t(3;22)(q27;ql l),t(8;14)(q24:q32),del(2)(q31),+r 51,XX,+3,+ 18,+ 19,t(8; 14)(q24;q32),del(6)(q21+ter),der(3)t( 13;3) (PI 1;pl l),+del(l)(p22-+ter),+dup(7)(ql l+q36) 98,XX,4n+/- of the clone above 85,XX,+/-,-2,-4,-6,-6,-10,-13,-15,-16,-20,-21,+7,t(8~14) (q24;q23),+del(l)(pl2-*ter),+der(6)t(6;?:6)(ql3;?;pl3),+marl C. DLCL with other translocations involving 8q24, without MYC rearrangement 48,X,-X,+7,+8,t(8:22)(q24;ql l),t(14:18)(q32;q21),del(l)(q22+ter), 143 58/F IMB-D LN IGG-K G G Post 27 +der(l)(qter+q21: :?),der(9)(qter+pl4: :9ql3+ter) 540 74lF IMB-D LN ND G D Pre 6 46,XX 21 45,X,-X,t(8:22)(q24;qIl) ~~~ ~~~~ Abbreviations: UTN. unique tumor number: LN, lymph node: SM, small bowel; CW, chest wall: SP, spleen: LI, liver; BN, bone: IMB-D, immunoblastic diffuse:LNCC-D, large noncleaved cell diffuse: LCC-D, large cleaved cell diffuse; G, germline; R, rearranged; D, deleted; hull, h u l l restriction enzyme site in MYC exon-1: Pre, studies performed on pretreatment material; Post, studies performed posttreatment material: ND, not done. lineages of the three cases lacking surface immunoglobulin (cases 70,230, and 348) and of the four cases that were not studied in cell suspension (cases 295, 534, 540, and 705) were determined using the L26 and UCHLl monoclonal antibodies in paraffin sections. Case 230 was a Ki-1 positive peripheral T-cell lymphoma; its phenotype and genotype have been reported in detail elsewhere.' The remaining cases were positive for L26 and negative for UCHL1, confirming their B-cell lineage. Clinical correlations. Of the 144 cases with an abnormal karyotype or DNA available for molecular analysis, 102 were studied before cytotoxic treatment. The median survival of the 14 patients with a t(8q24) or a rearrangement of MYC was no different from the remaining 88 patients (P = .23). Serotesting for human immunodeficiency virus (HIV) was performed in 29 of the 144 cases; of the seven HIV-positive patients, five had a t(8q24) (71%), including cases 230,408, and 591 in Table 1, compared with only two of the remaining 22 patients (9%); this difference was statistically significant (P < .05). The salient clinical features of the final study group of 15 cases of DLCL with t(8;14) or other t(8q24) translocations and DNA available for blotting are summarized in Table 1. These 15 patients ranged in age from 23 to 76 years, and there were nine women and six men. The mean age of the MYC-rearranged group (cases 230, 348, 408, and 705) was 39.5 years compared with 48.4 years for the rest of the group (P = .lo). The DLCL were classified as large non- From www.bloodjournal.org by guest on April 28, 2017. For personal use only. IADANYI ET AL 1060 408 1 I E H P S X = "&Wb" 705 230 1 I E H P b b b I 4 * i ; cleaved cell in seven cases, immunoblastic in seven cases, and cleaved cell in one case. In none of the cases was a component of BL observed. Three of the 15 specimens in Table 1were obtained from extranodal sites: small bowel in case 348, chest wall in case 705, and bone in case 656. Of the 15 cases in Table 1, cytogenetic and molecular studies were performed before cytotoxic chemotherapy in eight cases. Among these, the median survival of the three patients with rearrangement ofMYC was no different than that of the five patients with a t(8q24) but witka germline configuration of MYC(P > SO). 5' 3' Fig 3. Restriction map of the MYC gene showing the three exons (hatched boxes, from left t o right), and the regions where the breakpoints in cases 348,408,705, and 230 are located according t o the results of the Southern blotting (Fig 2). TheXho I-XbaI fragment of exon-1 used as the probe is also indicated(open rectangle). Abbreviations: EcoRI, E; Hindlll, H; Pstl, Ps; Arull, Pv;Smal, S; Xhol, Xh; Xba 1, Xb. S X Fig 2. Southern blot analysis of the MYC gene in cases 348,408,705, and 230. The DNA samples were digested with EcoRl (E), Hindlll (H), Pstl (P), Smal (S), and Xba I(X), yielding germline fragments of approximately 11, 10, 2.7, 2.0, and 6.6 kb, respectively. The rearranged fragments are indicated by arrowheads. The sizes of the rearranged fragments are, from left to right, case 348: 14,10,2.6, and 5.6 kb; case 408: 12,14, 3.5,11, and 12 kb; case 705: 12, 10.5,3.3,10.5, and 9.7 kb; case 230: 11.5 and 8 kb. The comigration of the rearrangedHindlll fragment with the germline band in case 348 was confirmed by digestion with C/al (results not shown). The DNA in case 348 could not be satisfactorily digested with Xba 1. DISCUSSION Up to 20% of DLCL show a t(8;14) translocation?"' In our series, 14% showed this translocation, and another 5% showed other translocations involving band 8q24. The incidence of t(8;14) appears to be roughly the same in the three histologic subgroups of DLCL, namely the cleaved, noncleaved, and immunoblastic. Recent long-term follow-up studies of the DLCL group have failed to demonstrate any major differences in survival between these s~btypes?~ Hence, from both the cytogenetic and clinical viewpoint, grouping the three histologic subtypes for the purposes of this analysis appears justified. Two of the cases in Table 1also displayed other characteristic translocations. Case 591 showed a t(3;22)(q27;qll), a recently identified translocation associated with diffise NHL." In case 143, the t(8;22) was present in association with a t(14;18)(q32;q21). Coexistent 18q21 and 8q24 rearrangements, involving BCL2 and MYC, have been described in rare cases of high-grade NHL or acute lymphoblastic leukemia.2s-"Our patient 143 had an immunoblastic lymphoma and died of disease 7 months after the initial diagnosis, despite aggressive chemotherapy. However, case 143 is the only case out of 24 with a t(18q21) in our series to From www.bloodjournal.org by guest on April 28, 2017. For personal use only. MYC REARRANGEMENTSIN t(8;14) LYMPHOMAS 10 1.3 - 0.4 - . . ~ .- kb Fig 4. Southern blot analysis of the MYC gene for point mutation at the h u l l restriction enzyme site in exon-1 using double digestion with hull and Xho 1. In cases 540 and 591, the hull site was lost, presumably by point mutation, producing a new fragment of the combined size of thetwogermline fragments (1.3kb = 0.9kb + 0.4kb). Case 348 shows a rearrangement within the bull fragment, producing a new band larger than 1.3 kb. Cases 408 and 705 also showed rearrangement of the h u l l fragments (not illustrated).The remaining cases showed no alteration of the h u l l fragments. also display a t(8q24), suggesting that this is not a common event in the transformation of low-gradeNHLwith t(18q21). We found MYC exon-llintron-1 breaks in cases 348,408, and 705. This region is also typically involved in the t(8;14) sBL.9s2’With the exception of case 230, the lack of detectable MYC rearrangement in the remaining cases indicates that the 8q24 break in these cases must lie outside of the EcoRI fragment containing the MYC gene; this is usually the case in eBL with t(8;14) and in the variant t(2;8) and t(8;22) translocations:.” Most breaks in eBL appear to be located at least 90 kb upstream of the MYC first exon,21 whereas the breaks in the variant translocations occur at least 10 kb distal to the MYC gene,” beyond the 3’ Hind111 site (Fig 3). Molecular studies of the MYC gene in cases of non-AIDS DLCL have been limited. Chenevix-Trench et aI2’ studied 18 DLCL and found a rearrangement of MYC in one case, located immediately 5’to exon-1; unfortunately, no karyotype data were available on this tumor, or on most of their other cases. Sozzi et alMreported a case of DLCL with a t(8;14) and a rearrangement of MYC. Delia et al” studied the configuration of the MYC gene in five primary cutaneous B-cell DLCL and found it to be germline in all five; unfortunately, no cytogenetic data were provided. The presence of Epstein-Barr virus (EBV) within the lymphoma cells, which is thought to be involved in the pathogenesis of at least some AIDS-associated NHL, was not studied in the present series. However, it should be pointed out that no consistent relationship between EBV infection and the type 1061 of cytogenetic or molecular lesion has so far been shown in these case^?^" About 75% of AIDS-associated NHL, both BL and DLCL, show MYC rearrangement, usually with breaks similar to those in S B L . ~ ~Accordingly, ” two of the three HIV-positive cases in our group of 15 DLCL with t(8q24) had a rearrangement in MYC. On the other hand, some AIDS-associated cases show eBL-type breaks in the ZGH and MYCMgenes. In our series of DLCL, HIV positivitywas significantly more common in cases with a t(8q24) (71%) than in cases without it (9%) (P < .05). Our case 230 was a T-cell Ki-1 positive NHL* and had cytogenetic involvement of the 8q24 band on both homologues [de1(8)(q24) and t(8;?)(q24;?)], while the two chromosomes 14 appeared normal. One MYC allele, most likely the one involved in the t(8;?)(q24;?), showed a rearrangement in the 3’ region or immediately downstream; it was impossible to determine whether the other MYC allele, presumably the one in the de1(8)(q24) chromosome, was deleted or germline, due to the high content of nonneoplastic cells in this specimen. However, our data do allow the exclusion of a rearrangement of this allele. Translocations involving band 8q24, typically t(8;14)(q24; qll), have been well described in other T-cell neoplasms; the break at 8q24 has been localized to the region 3’ of MYC in several T-cell leukemia cell lines?s36 Possible mechanisms of MYC activation in these translocations include: (1) transcriptional activation by adjacent ZG enhancers; (2) transcriptional activation by distant ZG enhancers; (3) mutational inactivation of 5‘ regulatory sequences by ZG-related hypermutational activity; and (4) altered half-life and kinetics of a mutated MYC Whether MYC activation is manifested by absolute overexpression or merely inappropriately sustained expression at normal levels is still not entirely clear. In relation to the third mechanism mentioned above, cases of BL with distant MYC breakpoints, most commonly endemic cases with t(8;14) or cases with variant translocations, have been found to have a high incidence of point mutation within and upstream of exon-1 and within intronl?’~’~ The most commonly affected area is the PvuII restriction site at the 3‘ end of exon-1, which is mutated in over 50% of cases, eliminating the restriction site and resulting in transcriptional deregulation of MYC. Of the 15 cases with a t(8q24) in the present series, two cases (540 and 591) showed evidence of point mutation at the PvuII site; neither of these two cases was MYC-R. Hence, they appear to be analogous to eBL with distant MYC breakpoints, although the incidence of point mutation appears to be significantly lower (15%). Although the PvuII site is the most common restriction enzyme site to undergo mutation in this setting, mutations in exon-1 outside of this site are also numerOUS,Z’*’~ and may be present in at least some other cases in our series. The t(8;14) breakpoints within the ZGH locus differs according to the form of BL? in sBL and AIDS-associated BL, the breaks usually are in the Sp region, whereas in eBL they most commonly are located in the JH region. We From www.bloodjournal.org by guest on April 28, 2017. For personal use only. 1062 LADANYI ET AL studied cases 348, 408, and 705 for comigration of rearranged MYC and I G H bands. Case 705 showed rearrangement of both JH and Sp, in the presence of a germline C p band, indicating that the lymphoma cells had undergone class switching with deletion of the C p region. In cases 348 and 408, JH was rearranged, but Sp and C p were in a germline configuration. No surface IG could be demonstrated in case 348; however, monoclonal surface IGG was present in case 408, suggesting that the class switching that occurred must have involved the 3’ end of the Sp region, as previously described in some cases? with deletion of the remainder of Sp, including the entire region covered by our Sp probe. In none of the three cases was comigration of the rearranged S k or JH bands with the rearranged MYC band demonstrated. Thus, in these cases the 14q32 breakpoint remains undefined at the molecular level. Occasionally, the breakpoints in BL have been shown to occur in the D H region or Sa or ST region^,^^^^' which we did not study. Cases of BL with the variant translocations t(8;22) and t(2;8) typically express ZGL and IGK light chains, respectively, because of the sequential rearrangement of the light chain genes.42Interestingly, however, our case 143 had an IGKphenotype in the presence of a t(8;22); this exceptional combination has also been reported in a case of BL.43 Thus, of the 15 DLCL with a t(8q24), three (20%) had breaks in the 5’ region of MYC, similar to sBL, and another two cases (13%) showed evidence of point mutation within exon-1, a regulatoly noncoding region, analogous to some cases of eBL. One T-cell DLCL had a more 3‘ break in MYC, as described in other T-cell lymphomas. The molecular events affecting the MYC gene in the other nine cases (60%)remain to be clarified. It is possible that the t(8;14) in at least some of these cases is ineffectual in terms of MYC deregulation, relative to its counterpart in BL, which would be consistent with its relative lack of impact on survival in DLCL in the present study, despite the over-representation of HIV-related cases in the t(8q24) group. In a separate study, we are currently assessing MYC expression in this series of DLCL by in situ hybridization. Thus, these studies show that cases of DLCL with t(8;14) provide another useful experimental setting for the analysis of the mechanisms of MYC deregulation. Finally, the localization of 8q24 breakpoints at the molecular level in cases of DLCL may also have future therapeutic implications, because rearrangements within the MYC gene result in abnormal MYC transcripts that may be targeted in a tumor-specific fashion by antisense oligonucleotides.@ REFERENCES 1. 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For personal use only. 1991 77: 1057-1063 MYC rearrangement and translocations involving band 8q24 in diffuse large cell lymphomas M Ladanyi, K Offit, SC Jhanwar, DA Filippa and RS Chaganti Updated information and services can be found at: http://www.bloodjournal.org/content/77/5/1057.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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