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The cytogenetic alterations in renal oncocytoma (RO) are poorly understood. We analyzed 130 consecutive RO for karyotypic alterations. Clonal chromosome abnormalities were identified in 63 (49%) cases, which could be categorized into three classes of mutually exclusive cytogenetic categories. Class 1 (N = 20) RO had diploid karyotypes with characteristic 11q13 rearrangement in balanced translocations with 10 or more different chromosome partners in all cases. We identified recurrent translocation partners at 5q35, 6p21, 9p24, 11p13-14, and 11q23, and confirmed that CCND1 gene rearrangement at 11q13 utilizing fluorescence in situ hybridization (FISH). Class 2 RO (N = 25) exhibited hypodiploid karyotypes with loss of chromosome 1 and/or losses of Y in males and X in females in all cases. The class 3 tumors comprising of 18 cases showed diverse types of abnormalities with the involvement of two or more chromosomes exclusive of abnormalities seen in classes 1 and 2 tumors. Furthermore, karyotypically uninformative cases were subjected to FISH analysis to identify classes 1 and 2 abnormalities. In this group, we found similar frequencies of CCND1 rearrangement, loss of chromosome 1 or Y as with karyotypically abnormal cases. We validated our results against 91 tumors from the Mitelman database. Correlation of clinical data with all the three classes of ROs showed no clear evidence of overall patient survival. Our findings support the hypothesis that RO exhibit three principal cytogenetic categories, which may have different roles in initiation and/or progression. These cytogenetic markers provide a key tool in the diagnostic evaluation of RO.

Accurate discrimination of benign oncocytoma and malignant renal cell carcinoma is useful for planning appropriate treatment strategies for patients with renal masses. Classification of renal neoplasms solely based on histopathology can be challenging, especially the distinction between chromophobe renal cell carcinoma and oncocytoma. In this study we develop and validate an algorithm based on genomic alterations for the classification of common renal neoplasms.Using TCGA renal cell carcinoma copy number profiles and the published literature, a classification algorithm was developed and scoring criteria were established for the presence of each genomic marker. As validation, 191 surgically resected formalin fixed paraffin embedded renal neoplasms were blindly submitted to targeted array comparative genomic hybridization and classified according to the algorithm. CCND1 rearrangement was assessed by fluorescence in situ hybridization.The optimal classification algorithm comprised 15 genomic markers, and involved loss of VHL, 3p21 and 8p, and chromosomes 1, 2, 6, 10 and 17, and gain of 5qter, 16p, 17q and 20q, and chromosomes 3, 7 and 12. On histological rereview (leading to the exclusion of 3 specimens) and using histology as the gold standard, 58 of 62 (93%) clear cell, 51 of 56 (91%) papillary and 33 of 34 (97%) chromophobe renal cell carcinomas were classified correctly. Of the 36 oncocytoma specimens 33 were classified as oncocytoma (17 by array comparative genomic hybridization and 10 by array comparative genomic hybridization plus fluorescence in situ hybridization) or benign (6). Overall 93% diagnostic sensitivity and 97% specificity were achieved.In a clinical diagnostic setting the implementation of genome based molecular classification could serve as an ancillary assay to assist in the histological classification of common renal neoplasms.

PCDH10 is epigenetically inactivated in multiple tumor types; however, studies in mature lymphoid malignancies are limited. Here, we have investigated the presence of promoter hypermethylation of the PCDH10 gene in a large cohort of well-characterized subsets of lymphomas. PCDH10 promoter hypermethylation was identified by methylation-specific PCR in 57 to 100% of both primary B- and T-cell lymphoma specimens and cell lines. These findings were further validated by Sequenom Mass-array analysis. Promoter hypermethylation was also identified in 28.6% cases of reactive follicular hyperplasia, more commonly occurring in states of immune deregulation and associated with rare presence of clonal karyotypic aberrations, suggesting that PCDH10 methylation occurs early in lymphomagenesis. PCDH10 expression was down regulated via promoter hypermethylation in T- and B-cell lymphoma cell lines. The transcriptional down-regulation resulting from PCDH10 methylation could be restored by pharmacologic inhibition of DNA methyltransferases in cell lines. Both Tand B-cell lymphoma cell lines harboring methylation-mediated inactivation of PCDH10 were resistant to doxorubicin treatment, suggesting that hypermethylation of this gene might contribute to chemotherapy response. V C 2013 Wiley Periodicals, Inc.

Christopher B. Anderson1, Michael Lipsky1, Subhadra V. Nandula2,3, Christopher E. Freeman2, Thomas Matthews2, Catlin Walsh2, Gen Li1, Matthias Szabolcs2, Mahesh M. Mansukhani 2, James M. McKiernan1, Vundavalli V. Murty 2 1Departments of Urology, 2Pathology and Cell Biology, Columbia University Medical Center, New York, New York; 3Cancer Genetics Inc, Rutherford, New Jersey The cytogenetic alterations in renal oncocytoma (RO) are poorly understood. We analyzed 130 consecutive RO for karyotypic alterations. Clonal chromosome abnormalities were identified in 63 (48.5%) cases, which could be categorized into 3 classes of mutually exclusive cytogenetic categories. Class 1 RO had diploid karyotypes with 11q13 rearrangement in balanced translocations with ten or more different chromosome partners in all 20 (31.7%) cases. We identified recurrent translocation partners at 5q35, 6p21, 9p24, 11p13-14 and 11q23, and confirmed that CCND1 gene rearrangement at 11q13 utilizing fluorescence in situ hybridization (FISH). Class 2 RO exhibited hypodiploid karyotypes with loss of chromosome 1 and concurrent losses of Y in males and X in females in 25 (39.7%) cases. The class 3 tumors comprising of 18 (30.2%) cases showed diverse types of abnormalities with the involvement of 2 or more chromosomes exclusive of abnormalities seen in Class 1 and 2 tumors. Furthermore, karyotypically uninformative cases were subjected to FISH analysis to identify class 1 and 2 abnormalities. In this group we found similar frequencies of CCND1 rearrangement, loss of 1 or Y as with karyotypically abnormal cases. We validated our results against 91 tumors from the Mittleman database. Correlation of clinical data with all the 3 classes of RO showed no clear evidence of overall patient survival. Our findings support the hypothesis that RO exhibit 3 principal cytogenetic categories, which may have different roles in initiation and/or progression. These cytogenetic markers provide a key tool in the diagnostic evaluation of RO. Citation Format: Christopher B. Anderson, Michael Lipsky, Subhadra V. Nandula, Freeman E. Christopher, Matthews Thomas, Caitlin E. Walsh, Gen Li, Matthias Szabolcs, Mahesh M. Mansukhani, James M. McKiernan, Murty V. Vundavalli. Cytogenetics of renal oncocytomas identify three distinct and mutually exclusive diagnostic classes of chromosome aberrations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3415.

Lesions of ERBB2, PTEN, and PIK3CA activate the phosphatidylinositol 3-kinase (PI3K) pathway during cancer development by increasing levels of phosphatidylinositol-3,4,5-triphosphate (PIP3). 3-Phosphoinositide-dependent kinase 1 (PDK1) is the first node of the PI3K signal output and is required for activation of AKT. PIP3 recruits PDK1 and AKT to the cell membrane through interactions with their pleckstrin homology domains, allowing PDK1 to activate AKT by phosphorylating it at residue threonine-308. We show that total PDK1 protein and mRNA were overexpressed in a majority of human breast cancers and that 21% of tumors had five or more copies of the gene encoding PDK1, PDPK1. We found that increased PDPK1 copy number was associated with upstream pathway lesions (ERBB2 amplification, PTEN loss, or PIK3CA mutation), as well as patient survival. Examination of an independent set of breast cancers and tumor cell lines derived from multiple forms of human cancers also found increased PDK1 protein levels associated with such upstream pathway lesions. In human mammary cells, PDK1 enhanced the ability of upstream lesions to signal to AKT, stimulate cell growth and migration, and rendered cells more resistant to PDK1 and PI3K inhibition. After orthotopic transplantation, PDK1 overexpression was not oncogenic but dramatically enhanced the ability of ERBB2 to form tumors. Our studies argue that PDK1 overexpression and increased PDPK1 copy number are common occurrences in cancer that potentiate the oncogenic effect of upstream lesions on the PI3K pathway. Therefore, we conclude that alteration of PDK1 is a critical component of oncogenic PI3K signaling in breast cancer. [Cancer Res 2009;69(15):6299–306]

Diffuse large B-cell lymphoma (DLBCL), the most common form of lymphoma in adulthood, comprises multiple biologically and clinically distinct subtypes including germinal centre B-cell-like (GCB) and activated B-cell-like (ABC) DLBCL(1). Gene expression profile studies have shown that its most aggressive subtype, ABC-DLBCL, is associated with constitutive activation of the NF-kappa B transcription complex(2). However, except for a small fraction of cases(3), it remains unclear whether NF-kappa B activation in these tumours represents an intrinsic program of the tumour cell of origin or a pathogenetic event. Here we show that >50% of ABC-DLBCL and a smaller fraction of GCB- DLBCL carry somatic mutations in multiple genes, including negative (TNFAIP3, also called A20) and positive (CARD11, TRAF2, TRAF5, MAP3K7 (TAK1) and TNFRSF11A ( RANK)) regulators of NF-kappa B. Of these, the A20 gene, which encodes a ubiquitin-modifying enzyme involved in termination of NF-kappa B responses, is most commonly affected, with similar to 30% of patients displaying biallelic inactivation by mutations and/or deletions. When reintroduced in cell lines carrying biallelic inactivation of the gene, A20 induced apoptosis and cell growth arrest, indicating a tumour suppressor role. Less frequently, missense mutations of TRAF2 and CARD11 produce molecules with significantly enhanced ability to activate NF-kappa B. Thus, our results demonstrate that NF-kappa B activation in DLBCL is caused by genetic lesions affecting multiple genes, the loss or activation of which may promote lymphomagenesis by leading to abnormally prolonged NF-kappa B responses.

Cytogenetic abnormalities in B-cell posttransplant lymphoproliferative disorders (PTLD) have not been well characterized. We thus performed cytogenetic analysis of 28 cases of B-cell PTLD, 1 infectious mononucleosis (IM)–like lesion, 9 polymorphic PTLD, 17 monomorphic PTLD, and 1 classical Hodgkin lymphoma (HL), and correlated the karyotypic findings with the phenotype, Epstein- Barr virus infection status, and clinical outcome. Karyotypes of 19 cases of posttransplant florid follicular hyperplasia (FFH) were also analyzed. Informative karyotypes were obtained in 20 (71.4%) of 28 PTLDs and 18 (94.7%) of 19 FFHs. Clonal karyotypic abnormalities were detected in 13 (65%) of 20 PTLDs, including 9 (75%) of 12 monomorphic PTLDs, 2 (33.3%) of 6 polymorphic PTLDs, 1 IM-like lesion, and 1 HL, and 2 (11.1%) of 18 FFHs. Recurrent chromosome breaks at 1q11-21 (n = 6, including 1 FFH), 14q32 (n = 3, including 1 FFH), 16p13 (n = 3), 11q23-24 (n = 2), and 8q24 (c-MYC) (n = 2); gains of chromosome 7 (n = 4), X (n = 3), 2 (n = 3), 12 (n = 2); and loss of chromosome 22 (n = 2, including 1 IM- like lesion) were identified. The presence of cytogenetic abnormalities did not correlate with PTLD phenotype, Epstein-Barr virus infection, or clinical outcome. We describe novel karyotypic aberrations in PTLD and report clonal cytogenetic abnormalities in posttransplant FFH and an IM-like lesion for the first time. Our findings provide validation of the current World Health Organization classification of PTLD and also suggest incorporation of FFH as the earliest recognizable precursor of PTLD.

Balanced reciprocal translocations involving chromosomal region 3q27, which led to identification of the BCL6 protooncogene, are one of the most common recurrent chromosomal abnormalities reported in B-cell non-Hodgkin lymphomas (B-NHL). Cloning of the breakpoints of these translocations has facilitated the identification of a number of BCL6 partners including immunoglobulin genes and more than 20 non-immunoglobulin genes on almost all human chromosomes. Fusion of BCL6 with these genes leads to deregulated BCL6 expression because of substitution of its promoter with that of the translocation partner. Despite the promiscuous nature of BCL6 translocations, intrachromosomal rearrangements of the BCL6 gene have not been well recognized. In the present study, we present evidence for intrachromosomal rearrangements, because of interstitial deletions and inversions, involving region 3q27 as an overlooked mechanism of BCL6 deregulation. These rearrangements accounted for 3/20 (15%) of all BCL6 translocations occurring in B-NHL, including follicular lymphomas, diffuse large B-cell lymphomas, and marginal zone B-cell lymphomas, diagnosed at our institute. In addition to confirming previously described partner loci on chromosome 3, we also identified a novel BCL6 partner locus (3p24) that to our knowledge has not been reported previously. Our data should help facilitate the identification of new BCL6 partner genes, which may enhance our understanding of the clinical and biological role of BCL6 in B-NHL pathogenesis.

PR domain containing 1 with zinc finger domain (PRDM1)/B lymphocyte–induced maturation protein 1 (BLIMP1) is a transcriptional repressor expressed in a subset of germinal center (GC) B cells and in all plasma cells, and required for terminal B cell differentiation. The BLIMP1 locus lies on chromosome 6q21-q22.1, a region frequently deleted in B cell lymphomas, suggesting that it may harbor a tumor suppressor gene. We report here that the BLIMP1 gene is inactivated by structural alterations in 24% (8 out of 34) activated B cell–like diffuse large cell lymphoma (ABC-DLBCL), but not in GC B cell–like (n = 0/37) or unclassified (n = 0/21) DLBCL. BLIMP1 alterations included gene truncations, nonsense mutations, frameshift deletions, and splice site mutations that generate aberrant transcripts encoding truncated BLIMP1 proteins. In all cases studied, both BLIMP1 alleles were inactivated by deletions or mutations. Furthermore, most non–GC type DLBCL cases (n = 20/26, 77%) lack BLIMP1 protein expression, despite the presence of BLIMP1 mRNA. These results indicate that a sizable fraction of ABC-DLBCL carry an inactive BLIMP1 gene, and suggest that the same gene is inactivated by epigenetic mechanisms in an additional large number of cases. These findings point to a role for BLIMP1 as a tumor suppressor gene, whose inactivation may contribute to lymphomagenesis by blocking post–GC differentiation of B cells toward plasma cells.

SummaryDiffuse large B cell lymphomas (DLBCL) derive from germinal center (GC) B cells and display chromosomal alterations deregulating the expression of BCL6, a transcriptional repressor required for GC formation. To investigate the role of BCL6 in DLBCL pathogenesis, we have engineered mice that express BCL6 constitutively in B cells by mimicking a chromosomal translocation found in human DLBCL. These mice display increased GC formation and perturbed post-GC differentiation characterized by a decreased number of post-isotype switch plasma cells. Subsequently, these mice develop a lymphoproliferative syndrome that culminates with the development of lymphomas displaying features typical of human DLBCL. These results define the oncogenic role of BCL6 in the pathogenesis of DLBCL and provide a faithful mouse model of this common disease.

Patients presenting with thrombocytosis require thorough clinical and laboratory evaluation to determine whether they suffer from essential thrombocythemia or another myeloproliferative disorder. This distinction becomes increasingly relevant as targeted agents become available to treat specific myeloproliferative diseases. Cytogenetic testing plays a major role in this analysis. This study presents a patient with Philadelphia chromosome positive (Ph + ) thrombocytosis and a cryptic der(9q)t(5;9)t(9;22) not found by conventional cytogenetics, whose disease progressed within 2 years to typical myeloblastic crisis of CML. It discusses the entity of Ph + ET, the utility of molecular cytogenetic testing in the diagnosis of this unusual disease entity and the importance of cytogenetic testing in the prognosis of ET.

Loss of chromosome arm 18q is a common event in human pancreatic, colon, and breast cancers and is often interpreted as representing loss of one or more tumor-suppressor genes. In this article, we describe two novel biallelic deletions at chromosome band 18q21.1 in a recently characterized human breast cancer cell line, HCC-1428. One lesion deletes a fragment of approximately 300 kb between SMAD4 and DCC that encodes no known genes. The second lesion is an in-frame SMAD4 deletion (amino acids 49–51) that affects the level of SMAD4 protein but not the SMAD4 message. This change accelerates 26S proteasome–mediated degradation of both endogenous and exogenous mutant SMAD4. Examination of normal DNA from the same patient demonstrated that both lesions are somatic and associated with loss of both normal alleles. These data support the concept that two independent tumor-suppressor loci exist at chromosome segment 18q21.1, one at SMAD4 and the other potentially at an enhancer of DCC or an unrelated novel gene. © 2005 Wiley-Liss, Inc.

Patients with advanced stage invasive cervical cancer (CC) exhibit highly complex genomic alterations and respond poorly to conventional treatment protocols. In our efforts to understand the molecular genetic basis of CC, we examined the role of Fanconi Anemia (FA)-BRCA pathway. Here, we show that FANCF gene is disrupted by either promoter hypermethylation and/or deregulated gene expression in a majority of CC. Inhibition of DNA methylation and histone deacetylases induces FANCF gene re-expression in CC cell lines. FANCF-deregulated CC cell lines also exhibit a chromosomal hypersensitivity phenotype after exposure to an alkylating agent, a characteristic of FA patients. We also show the involvement of BRCA1 gene by promoter hypermethylation or down-regulated expression in a small subset of CC patients. Thus, we have found inactivation of genes in the FA-BRCA pathway by epigenetic alterations in a high proportion of CC patients, suggesting a major role for this pathway in the development of cervical cancer. Thus, these results have important implications in understanding the molecular basis of CC tumorigenesis and clinical management in designing targeted experimental therapeutic protocols.

PCDH10 is epigenetically inactivated in multiple tumor types; however, studies in mature lymphoid malignancies are limited. Here, we have investigated the presence of promoter hypermethylation of the PCDH10 gene in a large cohort of well-characterized subsets of lymphomas. PCDH10 promoter hypermethylation was identified by methylation-specific PCR in 57 to 100% of both primary B- and T-cell lymphoma specimens and cell lines. These findings were further validated by Sequenom Mass-array analysis. Promoter hypermethylation was also identified in 28.6% cases of reactive follicular hyperplasia, more commonly occurring in states of immune deregulation and associated with rare presence of clonal karyotypic aberrations, suggesting that PCDH10 methylation occurs early in lymphomagenesis. PCDH10 expression was down regulated via promoter hypermethylation in T- and B-cell lymphoma cell lines. The transcriptional down-regulation resulting from PCDH10 methylation could be restored by pharmacologic inhibition of DNA methyltransferases in cell lines. Both T- and B-cell lymphoma cell lines harboring methylation-mediated inactivation of PCDH10 were resistant to doxorubicin treatment, suggesting that hypermethylation of this gene might contribute to chemotherapy response.

Background: The diagnosis of indeterminate biliary strictures is limited because of the low sensitivity of cytology. However, an accurate diagnosis of malignancy is critical in the management of patients with suspected biliary malignancy. Testing for chromosomal aneuploidy by fluorescence in situ hybridization (FISH) may increase the yield. Objective: To evaluate the diagnostic accuracy of FISH in indeterminate biliary strictures and the additional value of including deletion of 9p21 (p16) in the diagnostic criteria of malignant biliary strictures. Design: Retrospective review. Setting: Academic medical center. Patients: This study involved 76 consecutive patients who were seen for the evaluation of indeterminate strictures at our institution. These patients were screened, and 50 patients with either a final pathologic diagnosis or ≥ 12 months' conclusive follow-up were included in the analysis. Main outcome measurements: Sensitivity, specificity, and area under the curve (AUC) analysis of cytology alone compared with the presence of FISH polysomy versus FISH polysomy and 9p21 deletion. Results: The presence of increased copy numbers (polysomy) of chromosome 3, 7, or 17 by FISH increased the sensitivity of brush cytology from 21% to 58%, and when the presence of 9p21 deletion was included, the sensitivity increased to 89%. The specificity of FISH was 97% (vs 100% for cytology). The accuracy of cytology combined with FISH polysomy (AUC = 0.93) or p16 deletion was significantly greater than the accuracy of cytology alone (AUC 0.6; P < .001) or even cytology combined with FISH polysomy (AUC = 0.77; P ≤ .05). Limitations: Sample size. There is a relatively high incidence of malignant biliary strictures in the entire cohort but low incidence among primary sclerosing cholangitis patients, and the majority of cancers are cholangiocarcinomas (as opposed to pancreatic). Conclusion: FISH significantly improves the diagnostic accuracy of brush cytology in indeterminate biliary strictures. In our series, the addition of 9p21 deletion to FISH polysomy and cytology further improved sensitivity. This suggests that 9p21 deletion may be added to the diagnostic criteria in indeterminate strictures.

PCDH10has been implicated as a tumor suppressor, since epigenetic alterations of this gene have been noted in multiple tumor types. However, to date, studies regarding its role in acute and chronic leukemias are lacking. Here, we have investigated the presence of promoter hypermethylation of two CpG islands of thePCDH10gene by methylation-specific PCR in 215 cases of various subsets of myeloid- and lymphoid-lineage leukemias. We found thatPCDH10promoter hypermethylation was frequent in both B-cell (81.9%) and T-cell (80%) acute lymphoblastic leukemia (ALL), while it was present in low frequency in most subtypes of myeloid leukemias (25.9%) and rare in chronic myeloid leukemia (2.2%).PCDH10expression was downregulated via promoter hypermethylation in primary ALL samples (N!4) and leukemia cell lines (N!11). The transcriptional repression caused byPCDH10methylation could be restored by pharmacologic inhibition of DNA methyltransferases. ALL cell lines harboring methylation-mediated inactivation ofPCDH10were less sensitive to commonly used leukemia-specific drugs suggesting thatPCDH10methylation might serve as a biomarker of chemotherapy response. Our results demonstrate thatPCDH10is a target of epigenetic silencing in ALL, a phenomenon that may impact lymphoid-lineage leukemogenesis, serve as an indicator of drug resistance and may also have potential implications for targeted epigenetic therapy. V C 2011 Wiley Periodicals, Inc.

Non-random karyotypic abnormalities associated with non-Hodgkin lymphomas (NHLs) have been described in cases of reactive lymphoid hyperplasia (RLH). However, the frequency and types of cytogenetic aberrations detected and their clinical relevance are unknown. To address these questions, we undertook a retrospective analysis of a large series of RLH diagnosed at our institute over 8 years. Cytogenetic abnormalities were identified in 20 of 116 (17%) cases with informative karyotypes, comprising 14 (70%) structural and 11 (55%) numerical changes. Clonal (n = 14, 70%) and non-clonal (n = 6, 30%) abnormalities were observed. Aberrations of chromosome 14 were the most frequent (n = 8, 42%, 7 represented IgH translocations), followed by chromosome 3 (n = 4, 3 represented BCL6 translocations), and chromosome 12 (n = 4). Abnormal karyotypes were most often associated with florid follicular hyperplasia. Isolated lymphoid organ (lymph node, tonsil or spleen) enlargement (12/20, 60%) was more common, no specific etiology was identified in 10/20 (50%) cases and only 1 of 18 patients with clinical follow-up (range 2–107 months, median 60 months) developed lymphoma. In our experience, cytogenetic abnormalities involving loci associated with B-cell NHL are not infrequently detected in RLH. Their occurrence portends low risk for lymphomagenesis, however longer follow-up is prudent to further evaluate the natural history of such cases. Copyright © 2010 John Wiley & Sons, Ltd.

Primary effusion lymphoma (PEL) is a rare type of B-cell non-Hodgkin lymphoma (NHL), which predominantly occurs in HIV-infected individuals, and is pathogenetically linked with Kaposi sarcoma (KS)-associated herpes virus/human herpes virus-8 (KSHV/HHV-8) infection with or without evidence of Epstein-Barr virus (EBV) co-infection. Although uncommon, PELs have been reported in immunocompetent patients and recipients of solid organ allografts. Rare cases of KSHV(-) EBV(+) post-transplant effusion lymphomas resembling PEL have also been described, as have KSHV(-) EBV(-) effusion lymphomas, the latter including those arising in individuals with chronic liver disease. We report a unique KSHV(-) EBV(-) post-transplant effusion lymphoma associated with serum paraproteins, occurring in an HIV(-) individual, which had cytologic features and phenotype similar to PEL, and displayed a complex karyotype including isochromosome 12p and translocation t(8;22), resulting in rearrangement of c-MYC.

Unique and shared cytogenetic abnormalities have been documented for marginal zone lymphomas (MZLs) arising at different sites. Recently, homozygous deletions of the chromosomal band 6q23, involving the tumor necrosis factor alpha–induced protein 3 (TNFAIP3, A20) gene, a negative regulator of NF-κB, were described in ocular adnexal MZL, suggesting a role for A20 as a tumor suppressor in this disease. Here, we investigated inactivation of A20 by DNA mutations or deletions in a panel of extranodal MZL (EMZL), nodal MZL (NMZL), and splenic MZL (SMZL). Inactivating mutations encoding truncated A20 proteins were identified in 6 (19%) of 32 MZLs, including 2 (18%) of 11 EMZLs, 3 (33%) of 9 NMZLs, and 1 (8%) of 12 SMZLs. Two additional unmutated nonsplenic MZLs also showed monoallelic or biallelic A20 deletions by fluorescent in situ hybridization (FISH) and/or SNP-arrays. Thus, A20 inactivation by either somatic mutation and/or deletion represents a common genetic aberration across all MZL subtypes, which may contribute to lymphomagenesis by inducing constitutive NF-κB activation.

Mutations in the phosphatase and tensin homologue (PTEN)/phosphatidylinositol-3 kinase-alpha (PI3K) signaling pathway are frequently found in human cancer. In addition, Pten(+/-) mice develop tumors in multiple organs because of the activation of the PI3K signaling cascade. Because activation of PI3K signaling leads to feedback inhibition of insulin receptor substrate-2 (IRS2) expression, an upstream activator of PI3K, we therefore anticipated that IRS2 expression would be low in tumors that lack PTEN. Surprisingly, however, an elevation of IRS2 was often detected in tumor samples in which PTEN levels were compromised. To determine the potential contribution of Irs2 to tumor progression, Pten(+/-) mice were crossed with Irs2(+/-) mice. Deletion of Irs2 did not affect the initiation of neoplasia found in Pten(+/-) mice but suppressed cancer cell growth, proliferation, and invasion through the basement membrane. Deletion of Irs2 also attenuated the expression of Myc in prostatic intraepithelial neoplasia in Pten(+/-) mice. In addition, the expression levels of IRS2 and MYC were highly correlated in human prostate cancer, and IRS2 could stimulate MYC expression in cultured cells. Our findings provide evidence that the PI3K-activating adaptor Irs2 contributes to tumor progression in Pten(+/-) mice by stimulating both Myc and DNA synthesis.

Dual IgH/BCL2 and BCL6 translocations are rarely observed in B-cell non-Hodgkin lymphomas (B-NHLs). We investigated the morphologic, phenotypic, and cytogenetic spectrum of B-NHL with such dual translocations. Dual IgH/BCL2 and BCL6 translocations were detected in follicular lymphomas (FLs) and diffuse large B-cell lymphomas (DLBCLs), representing 6.1% of 132 B-NHLs in our series, including 6 (11%) of 56 FLs (grades 1, 2, and 3a) and 2 (3%) of 76 DLBCLs; 33% of FLs with dual translocations had variant morphologic features. All dual-translocation FLs were CD10+/BCL6+/BCL2+/MUM1-, and the DLBCLs demonstrated "activated" germinal center (CD10+/BCL6+/MUM1+) and non-germinal center (CD10-/BCL6+/MUM1+) phenotypes. BCL6 translocations in all cases involved nonimmunoglobulin genes/loci. Mean chromosome abnormalities in dual-translocation FLs and DLBCLs did not differ from IgH/BCL2 FLs and DLBCLs. Detection of dual translocations predominantly in low-grade FLs suggests that BCL6 abnormalities are acquired early in the histologic evolution of a subset of IgH/BCL2-associated FLs.

Cytogenetic abnormalities of chromosome 12p involving the TEL/ETV6 gene are observed in a variety of hematopoietic neoplasms including acute leukemias, myelodysplastic syndromes, and myeloproliferative disorders. Karyotypic aberrations, including rearrangements, deletions, and amplifications of chromosome 12p, have been documented in B-cell non-Hodgkin lymphoma; however, rearrangements targeting TEL have rarely been reported. Here we describe a diffuse large B- cell lymphoma that had a complex karyotype including t(9;12)(q22;p13), which was confirmed by fluorescence in situ hybridization to represent rearrangement of TEL. Additional cytogenetic abnormalities included t(3;14)(q27;q32) involving the variant, alternative breakpoint region of the BCL6 gene and del(6)(q13q23), resulting in the loss of 1 allele of BLIMP1. This case reiterates the importance of correlating morphologic and phenotypic findings with the results of cytogenetic analysis to avoid errors in diagnosing hematologic neoplasms and highlights the rare association of B-cell non- Hodgkin lymphoma with aberrations of TEL.

Cancer cells exhibit nonrandom and complex chromosome abnormalities. The role of genomic changes in cancer is well established. However, the identification of complex and cryptic chromosomal changes is beyond the resolution of conventional banding methods. The fluorescence microscopy afforded by imaging technologies, developed recently, facilitates a precise identification of these chromosome alterations in cancer. The three most commonly utilized molecular cytogenetics methods comparative genomic hybridization, spectral karyotype, and fluorescence in situ hybridization, that have already become benchmark tools in cancer cytogenetics, are described in this chapter. Comparative genomic hybridization is a powerful tool for screening copy-number changes in tumor genomes without the need for preparation of metaphases from tumor cells. Multicolor spectral karyotype permits visualization of all chromosomes in one experiment permitting identification of precise chromosomal changes on metaphases derived from tumor cells. The uses of fluorescence in situ hybridization are diverse, including mapping of alteration in single copy genes, chromosomal regions, or entire chromosomes. The opportunities to detect genetic alterations in cancer cells continue to evolve with the use of these methodologies both in diagnosis and research.

Recurrent karyotypic abnormalities are a characteristic feature of cervical cancer (CC) cells, which may result in deregulated expression of important genes that contribute to tumor initiation and progression. To examine the role of gain of the long arm of chromosome 20 (20q), one of the common chromosomal gains in CC, we evaluated CC at various stages of progression using single nucleotide polymorphism (SNP) array, gene expression profiling, and fluorescence in situ hybridization (FISH) anal- yses. This analysis revealed copy number increase (CNI) of 20q in >50% of invasive CC and identified two focal amplicons at 20q11.2 and 20q13.13 in a subset of tumors. We further demonstrate that the acquisition of 20q gain occurs at an early stage in CC development and the high-grade squamous intraepithelial lesions (HSIL) that exhibit 20q CNI are associated (P 5 0.05) with persistence or progression to invasive cancer. We identified a total of 26 overexpressed genes as consequence of 20q gain (N 5 14), as targets of amplicon 1 (N 5 9; two genes also commonly expressed with 20q gain) and amplicon 2 (N 5 6; one gene also commonly expressed with 20q gain). These include a number of functionally important genes in cell cycle regulation (E2F1, TPX2, KIF3B, PIGT, and B4GALT5), nuclear function (CSEL1), viral replication (PSMA7 and LAMA5), methylation and chromatin remodeling (ASXL1, AHCY, and C20orf20), and transcription regulation (TCEA2). Our findings implicate a role for these genes in CC tumorigenesis, represent an important step toward the development of clinically significant biomarkers, and form a framework for testing as molecular therapeutic targets.

Background and Aim. In the current WHO classification, marginal zone B-cell lymphomas (MZL) have been divided into three distinct subtypes (extranodal, EMZL; nodal, NMZL; splenic, SMZL) according to clinical, pathological and cytogenetic features, however the relationship between any of the different subtypes is still unclear. We thus performed a comprehensive analysis of genomic DNA copy number changes in a very large series of MZL cases with the aim of addressing this question. Materials and Methods. DNA extracted from frozen samples of MZL was analyzed with Affymetrix Human Mapping 250K SNP arrays. Results. Samples from 193 patients with MZL (23 NMZL, 63 EMZL, 84 SMZL and 23 not better specified MZL) from 15 Institutions were analyzed. Among EMZL, main primary sites were orbital adnexa in 28%, GI-tract in 16%, lung and salivary glands 5% each. Overall, MZL displayed more recurrent deletions than gains. Gains of 3q and 18 gains were common to all three MZL subtypes as were losses at 1p32.3-pter, 7p22.1-pter, 7q11.21–q11.23, 9q33.3-qter, 16p, 17p and 19. EMZL had significantly more gains at 3p14.2-pter, 6p and 7q31.3-qter and losses at 6q15, 6q23.3 and 14q11.2 than SMZL. Conversely, the latter had deletions of 7q31.32-qter and 14q23.3–q32.13 more frequently than EMZL. NMZL profiles showed the EMZL-specific 6q losses and the SMZL-related 7q losses at very low frequency while presented recurrent gains of chromosome 11, which was unique of NZML among the three subtypes. The 6q23.3 region contains the gene coding for TNFAIP3/A20, a negative regulator of the NFkB pathway. In SMZL a minimal deleted region was observed in 7q32.1. Regarding chromosome 3, gains targeted the whole chromosome in EMZL while, in SMZL and NMZL, the long arm was more commonly affected. Conclusions. EMZL, NMLZ and SMLZL share genetic alterations, which differ from other B-cell lymphomas. However subtype-specific genetic lesions are observed. In particular, gains of 6p and losses of 6q23.3 (TNFAIP3) are specific for EMZL, and 7q31-qter for SMZL.

Women with germ-line mutations of the BRCA1 tumor suppressor gene are highly susceptible to breast and ovarian cancer. The protein product of BRCA1 is involved in a broad spectrum of biological processes and interacts with many diverse proteins. One of these, BARD1, associates with BRCA1 to form a heterodimeric complex that is enzymatically active as an ubiquitin E3 ligase. Although the BRCA1/BARD1 heterodimer has been implicated in several aspects of BRCA1 function, its role in tumor suppression has not been evaluated. To address this question, we generated mouse strains carrying conditional alleles of either Bard1 or Brca1 and used Cre recombination to inactivate these genes in mammary epithelial cells. Significantly, the conditional Bard1 - and Brca1 -mutant mice developed breast carcinomas that are indistinguishable from each other (and from those of double conditional Bard1 / Brca1 -mutant animals) with respect to their frequency, latency, histopathology, and cytogenetic features. Reminiscent of the basal-like breast carcinomas seen in human BRCA1 mutation carriers, these tumors are “triple negative” for estrogen and progesterone receptor expression and HER2/neu amplification. They also express basal cytokeratins CK5 and CK14, have an elevated frequency of p53 lesions, and display high levels of chromosomal instability. The remarkable similarities between the mammary carcinomas of Bard1 -, Brca1 -, and Bard1 / Brca1 -mutant mice indicate that the tumor suppressor activities of both genes are mediated through the BRCA1/BARD1 heterodimer.

The BRCA1 tumor suppressor exists as a heterodimeric complex with BARD1, and this complex is thought to mediate many of the functions ascribed to BRCA1, including its role in tumor suppression. The two proteins share a common structural organization that features an N-terminal RING domain and two C-terminal BRCT motifs, whereas BARD1 alone also contains three tandem ankyrin repeats. In normal cells, the BRCA1/ BARD1 heterodimer is believed to enhance chromosome stability by promoting homology-directed repair (HDR) of double strand DNA breaks. Here we have investigated the structural requirements for BARD1 in this process by complementation of Bard1-null mouse mammary carcinoma cells. Our results demonstrate that the ankyrin and BRCT motifs of BARD1 are each essential for both chromosome stability and HDR. Tandem BRCT motifs, including those found at the C terminus of BARD1, are known to form a phosphoprotein recognition module. Nonetheless, the HDR function of BARD1 was not perturbed by synthetic mutations predicted to ablate the phospho-recognition activity of its BRCT sequences, suggesting that some functions of the BRCT domains are not dependent on their ability to bind phosphorylated ligands. Also, cancer-associated missense mutations in the BRCT domains of BARD1 (e.g. C557S, Q564H, V695L, and S761N) have been observed in patients with breast, ovarian, and endometrial tumors. However, none of these was found to affect the HDR activity of BARD1, suggesting that any increased cancer risk conferred by these mutations is not because of defects in this repair mechanism.

Cervical cancer (CC) cells exhibit complex karyotypic alterations, which is consistent with deregulation of numerous critical genes in its formation and progression. To characterize this karyotypic complexity at the molecular level, we used cDNA array comparative genomic hybridization (aCGH) to analyze 29 CC cases and identified a number of over represented and deleted genes. The aCGH analysis revealed at least 17 recurrent amplicons and six common regions of deletions. These regions contain several known tumor-associated genes, such as those involved in transcription, apoptosis, cytoskeletal remodeling, ion-transport, drug metabolism, and immune response. Using the fluorescence in situ hybridization (FISH) approach we demonstrated the presence of high-level amplifications at the 8q24.3, 11q22.2, and 20q13 regions in CC cell lines. To identify amplification-associated genes that correspond to focal amplicons, we examined one or more genes in each of the 17 amplicons by Affymetrix U133A expression arrays and semiquantitative reverse-transcription PCR (RT-PCR) in 31 CC tumors. This analysis exhibited frequent and robust upregulated expression in CC relative to normal cervix for genes EPHB2 (1p36), CDCA8 (1p34.3), AIM2 (1q22-23), RFC4, MUC4, and HRASLS (3q27-29), SKP2 (5p12-13), CENTD3 (5q31.3), PTK2, RECQL4 (8q24), MMP1 and MMP13 (11q22.2), AKT1 (14q32.3), ABCC3 (17q21-22), SMARCA4 (19p13.3) LIG1 (19q13.3), UBE2C (20q13.1), SMC1L1 (Xp11), KIF4A (Xq12), TMSNB (Xq22), and CSAG2 (Xq28). Thus, the gene dosage and expression profiles generated here have enabled the identification of focal amplicons characteristic for the CC genome and facilitated the validation of relevant genes in these amplicons. These data, thus, form an important step toward the identification of biologically relevant genes in CC pathogenesis. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.

Cancer cells exhibit nonrandom and complex chromosome abnormalities. The role of genomic changes in cancer is well established. However, the identification of complex and cryptic chromosomal changes is beyond the resolution of conventional banding methods. The fluorescence microscopy afforded by imaging technologies, developed recently, facilitates a precise identification of these chromosome alterations in cancer. The three most commonly utilized molecular cytogenetics methods comparative genomic hybridization, spectral karyotype, and fluorescence in situ hybridization, that have already become benchmark tools in cancer cytogenetics, are described in this chapter. Comparative genomic hybridization is a powerful tool for screening copy-number changes in tumor genomes without the need for preparation of metaphases from tumor cells. Multicolor spectral karyotype permits visualization of all chromosomes in one experiment permitting identification of precise chromosomal changes on metaphases derived from tumor cells. The uses of fluorescence in situ hybridization are diverse, including mapping of alteration in single copy genes, chromosomal regions, or entire chromosomes. The opportunities to detect genetic alterations in cancer cells continue to evolve with the use of these methodologies both in diagnosis and research.

Acute promyelocytic leukemia (APL) is characterized by the translocation t(15;17)(q22;q21), resulting in the promyelocytic leukemia (PML)-retinoic acid receptor alpha (RARA) fusion protein in 95% cases whereas variant translocations involving PLZF (11q23), NPM (5q35), NUMA (11q13) and STAT5b (17q23) account for the rest. Leukemias with PML-RARA translocations respond well to all-trans retinoic acid (ATRA) or arsenic trioxide (ATO) therapy whereas those with PLZF-RARA fusions respond poorly. Although primary resistance to ATRA is rare, secondary or acquired resistance is frequently observed in patients treated with ATRA alone or in combination with other chemotherapy regimens. However, molecular abnormalities mediating resistance to ATRA therapy are underexplored. Here, we report two cases of APL with RARA-PML deletions on der(17) or der(15), which displayed clinical evidence of primary and secondary resistance to therapy, respectively.

Congenital acute leukemia is a rare form of childhood leukemia, in which lineage conversion at relapse is very rarely reported. Here we describe a case of congenital B-cell acute lymphoblastic leukemia (B-ALL) with t(4;11) and t(1;6) translocations, which at relapse underwent a switch to monocytic lineage with persistence of the original cytogenetic translocations and clonal rearrangement of the JH gene. Similar to the other described cases of congenital acute leukemia with lineage conversion, our case had a MLL gene rearrangement and followed an aggressive clinical course.

Patients with advanced stage invasive cervical cancer (CC) exhibit highly complex genomic alterations and respond poorly to conventional treatment protocols. In our efforts to understand the molecular genetic basis of CC, we examined the role of Fanconi Anemia (FA)-BRCA pathway. Here, we show that FANCF gene is disrupted by either promoter hypermethylation and/or deregulated gene expression in a majority of CC. Inhibition of DNA methylation and histone deacetylases induces FANCF gene re-expression in CC cell lines. FANCF-deregulated CC cell lines also exhibit a chromosomal hypersensitivity phenotype after exposure to an alkylating agent, a characteristic of FA patients. We also show the involvement of BRCA1 gene by promoter hypermethylation or down-regulated expression in a small subset of CC patients. Thus, we have found inactivation of genes in the FA-BRCA pathway by epigenetic alterations in a high proportion of CC patients, suggesting a major role for this pathway in the development of cervical cancer. Thus, these results have important implications in understanding the molecular basis of CC tumorigenesis and clinical management in designing targeted experimental therapeutic protocols.

Mature B-cell acute lymphoblastic leukemia (ALL) is typically associated with the FAB-L3 morphology and rearrangement of the MYC gene, features characteristic of the leukemic phase of Burkitt's lymphoma. However, the term 'mature' has also been used to describe other rare cases of B-ALL with light-chain surface immunoglobulin expression. In contrast, infantile B-cell ALL is generally characterized by rearrangement of the MLL gene, an immature pro-B-cell phenotype, and CD10 negativity. We describe two unusual cases of infantile B-ALL with non-L3 morphology, expressing a mature B-cell phenotype (lambda sIg+, CD19+, CD10-, TdT-, and CD34-), and showing MLL rearrangement without MYC rearrangement at presentation. Both infants relapsed after months of morphologic and genetic remission. At relapse, the t(9;11) translocation was detected in both cases by spectral karyotyping. After the initial relapse, both cases followed a rapid and aggressive course. Literature search identified few similar cases, all expressed lambda surface immunoglobulin and showed MLL rearrangement (majority with the t(9;11) translocation). These cases show that B-ALL with MLL rearrangement, especially the t(9;11) translocation, can express a 'mature' B-cell phenotype and may represent a distinct subset. Identification of additional cases will further clarify the significance of MLL rearrangements in mature B-ALL.

Abstract 2635 BACKGROUND: Hairy cell leukemia (HCL) is a rare type of B-cell non Hodgkin lymphoma (B-NHL), which is characterized by neoplastic cells exhibiting slender cytoplasmic projections and an activated phenotype. Unlike other B-NHL, HCL lacks characteristic recurrent chromosome abnormalities and its etiology remains elusive. Recently, the BRAF V600E mutation was described to occur at a high frequency in HCL in contrast to other B-NHL, suggesting that the BRAF V600E mutation could represent a disease defining lesion and a possible therapeutic target. In this study, we determined the prevalence of the BRAF V600E mutation in a large series of low and intermediate grade B-NHL and assessed the relationship of this mutation, if any, with microsatellite instability (MSI). METHODS: DNA was extracted from various low and intermediate grade B-NHL (n=100), as defined by the 2008 World Health Organization Classification, which comprised HCL (n=10 from 6 patients), chronic lymphocytic leukemia/small lymphocytic lymphoma (n=22), mantle cell lymphoma (n=19), marginal zone lymphoma (n=22), follicular lymphoma (n=20) and lymphoplasmacytic lymphoma (n=7). Percentage of neoplastic cells was assessed by flow cytometry (n=99) or immunohistochemistry (n=1). Presence of BRAF V600E mutation was determined using a real time PCR assay using allele-specific hydrolysis (“Taqman”) probes. The detection limit of the assay was determined by diluting DNA extracted from a fresh HCL sample with normal high molecular weight DNA extracted by the same method. MSI analysis was performed using a multiplex reaction for 5 quasi monomorphic mononucleotide repeat markers (BAT-25, BAT-26, MONO-27, NR-21 and NR-24) and two highly polymorphic penta nucleotide markers (Penta C and Penta D) as sample identifiers. In the absence of normal DNA for comparison, a sample was considered MSI-H if greater than two markers showed an altered pattern, indeterminate if only two showed an alteration and MSS if no marker showed an altered allele. RESULTS: The ubiquitous occurrence and high specificity of the BRAF V600E mutation for HCL was confirmed in our series of B-NHL. The HCL samples consisted of 10 specimens from 6 patients and were the initial diagnostic specimen in 2 patients. The percentage of neoplastic cells in these samples ranged from 1% to 78.4% (median 18.2%). The BRAF V600E mutation was detected in 7 of 10 (70%) samples of HCL from 5 of 6 (83%) patients. The proportion of hairy cells was ≤5% in samples where the mutation was not detected. All 90 of the other low and intermediate grade B-NHL (>50% tumor burden) were negative for the V600E mutation. Compared to the reported sensitivity of approximately 30% tumor cells for detecting the BRAF V600E mutation by Sanger sequencing, our real time PCR assay allowed detection of the mutation in samples containing ≥9.8% tumor cells. Analysis of the HCL cases for MSI revealed that all cases had a microsatellite stable (MSS) phenotype. CONCLUSIONS: The BRAF V600E mutation appears specific for HCL among low and intermediate grade B-NHL and is not associated with microsatellite instability. These characteristics thus warrant inclusion in disease definition. Further refinements of a realtime PCR based approach for detecting the BRAF V600E mutation might be of utility in diagnosis and disease monitoring, as the neoplastic cellular yield is often limited in HCL due to underlying myelofibrosis. Disclosures: No relevant conflicts of interest to declare.

Marginal zone lymphoma (MZL) is a B-cell non-Hodgkin lymphoma (B-NHL) derived from marginal-zone B-cells. The current WHO classification recognizes three types of MZLs, which comprise extranodal MZL (EMZL), nodal MZL (NMZL), and splenic MZL (SMZL). Four recurrent and mutually exclusive chromosomal translocations: t(11;18)(q21;q21), t(1;14)(p22;q32), t(14;18)(q32;q21), and t(3;14)(p14.1;q32) have been described in EMZLs, but not in NMZLs or SMZLs, with frequencies ranging from 0–40% depending on differing anatomic sites and geographic regions. At least three of these translocations, t(11;18) API2/MALT1, t(1;14) IgH/BCL10, and t(14;18) IgH/MALT, result in constitutive activation of nuclear factor-kB (NF-kB), a transcription factor complex regulating multiple cellular processes including cell growth and survival. Approximately 25% of MZL however, lack any recognizable recurrent genetic alteration. Recently, biallelic deletions of the chromosomal region 6q23.3-q24.1 containing the tumor necrosis factor alpha-induced protein 3 (TNFAIP3, A20), which is a negative regulator of NF-kB, were detected in ocular adnexal MZLs, suggesting a role for A20 as a tumor suppressor in this disease entity. Here, we investigated a panel of 32 cases representing all three types of MZLs by PCR amplification and sequencing of the A20 coding exons. FISH was also performed using locus specific probes for A20 and Blimp1. Five 1–2 bp deletions and a single bp insertion, all leading to premature stop codons, were identified in 5/20 (25%) non-splenic MZLs and 1/12 (8.3%) SMZLs. The somatic nature of the mutations was confirmed by analyzing matched normal DNA in 4 cases. All A20 mutations were predictive of severely truncated polypeptides lacking functionally relevant domains. Genetic loss of A20 was detected by FISH in 4/20 (20%) non-splenic MZLs, including 1 with biallelic loss, but not in SMZLs. Two of the four A20 deletions seen by FISH were also observed by array-CGH. Deletions encompassing Blimp1 were not detected in cases with A20 deletions. No evidence for epigenetic silencing of A20 via promoter methylation of CpG islands was found in 10 MZLs analyzed. Thus, biallelic inactivation of A20, either via deletion of both alleles or frameshift mutations of one allele and loss of the second allele was identified in 3/20 (15%) non-splenic MZLs, while an additional 4/20 (20%) non-splenic MZLs displayed monoallelic A20 inactivation by mutations (N=3) or deletion (N=1). When we determined the relationship between A20 inactivation and trisomy 18 or the translocations associated with constitutive activation of NF-kB, A20 inactivation appeared mutually exclusive with these cytogenetic aberrations, with the exception of one case that had trisomy 18. In summary, our results indicate that A20 is mono- or biallelically inactivated in approximately one third of MZLs, arising in both nodal (3/9, 33.3%) and extranodal sites (4/11, 36.4%), and in a minority of SMZLs (1/12, 8.3%). Evidence of a classic two-hit mechanism was observed in 3/20 (15%) non-splenic MZL. In addition, detection of MZLs with monoallelic inactivation suggests a haploinsufficient role of A20 as a tumor suppressor. Thus, A20 inactivation may represent a common mechanism for constitutive activation of the NF-kB pathway, which, in turn, may contribute to lymphomagenesis by stimulating cell proliferation and survival. The identification of genetic lesions in MZLs that deregulate NF-kB activity offers the possibility of targeted therapeutic intervention.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease comprising multiple biologically and clinically distinct subgroups, including germinal center B cell-like (GCB) and activated B cell-like (ABC) DLBCL. Gene expression profile studies have shown that a key feature of its most aggressive subtype, ABC-DLBCL, is the constitutive activation of the NF-kB transcription complex. However, except for a small fraction of cases (Lenz et al., Science 2008), it remains unclear whether NF-kB activation in these tumors reflects an intrinsic program of the cell of origin or represents a primary pathogenetic event. To address this question, we first characterized 165 DLBCL samples (18 cell lines and 147 primary biopsies, including 26 ABC, 28 GCB, 10 unclassified and 83 not profiled) for the presence of active, nuclear NF-kB complexes by using immunohistochemical/immunofluorescence staining of NFKB1 p105/p50 (as a readout for the canonical pathway) and NFKB2 p100/p52 (as a readout for the non-canonical pathway). Nuclear localization of NF-kB, indicative of constitutive activity, was observed in 14/26 (54%) ABC-DLBCL and 8/28 (28%) GCB-DLBCL primary biopsies, as well as in 3/10 (30%) unclassified and 48/83 (58%) non-profiled cases, and correlated with significant enrichment in expression of NF-kB target genes, as assessed by gene set enrichment analysis (GSEA) of transcriptionally profiled cases. In addition, the more sensitive GSEA approach detected a gene expression signature of NF-kB activity in >90% ABC-DLBCLs and 53% GCB-DLBCLs, indicating that constitutive activation of this key signaling pathway is a common feature of ABC-DLBCL but can also be observed in a smaller fraction of GCB-DLBCL. To investigate whether NF-kB activity represents a primary pathogenetic event, we then screened for mutations the complete coding sequences of 31 genes encoding for NF-kB pathway components in a panel of 14 ABC-DLBCLs, which was expanded to 48 samples (12 ABC, 26 GCB and 10 not profiled) for validation of the mutated genes. The results showed that >50% of ABC-DLBCL (n=15/26) and a smaller fraction of GCB-DLBCL (n=8/26, 31%) carry somatic mutations in multiple genes, including negative (TNFAIP3/A20) and positive (CARD11, TRAF2, TRAF5, MAP3K7/TAK1 and TNFRSF11A/RANK) regulators of NF-kB. Of these, the A20 gene, which encodes for a ubiquitin-editing enzyme involved in termination of NF-kB responses, is the most commonly affected, with ~27% ABC-DLBCLs and 25% (8/32) immunohistochemically classified non-GC DLBCL displaying biallelic A20 inactivation by somatic mutations and/or deletions. Sequence changes include premature nonsense mutations, frameshift deletions/insertions and splice site mutations, leading to severely truncated proteins that lack functionally relevant domains and have thus lost their enzymatic activity. In virtually all mutated cases, FISH analysis revealed loss of the second allele, while 4 additional samples showed biallelic deletion of the gene. Thus, A20 is inactivated by a classic “two-hit” mechanism, suggesting a tumor suppressor role. Less frequently, missense mutations of CARD11 (10%) and TRAF2 (4%) produce molecules with significantly enhanced ability to activate NF-kB in transient transfection/reporter gene assays. Our results demonstrate that NF-kB activation in DLBCL is caused by genetic lesions affecting multiple genes, whose loss or activation may promote lymphomagenesis by leading to abnormally prolonged NF-kB responses. These findings provide the rationale and the assays for the identification of patients amenable to NF-kB targeted therapeutic intervention.