Supplementary Information, Methods, Table 1, Figures 1-7 from 3-Phosphoinositide–Dependent Kinase 1 Potentiates Upstream Lesions on the Phosphatidylinositol 3-Kinase Pathway in Breast Carcinoma

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.

Abstract 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.

PURPOSE: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.MATERIALS AND METHODS: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.RESULTS: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.CONCLUSIONS: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 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. © 2013 Wiley Periodicals, Inc.

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. (Gastrointest Endosc 2012;75:74-9.)

Hairy cell leukaemia (HCL) is a rare type of B-cell non-Hodgkin lymphoma (B-NHL), which is not known to be associated with any characteristic recurrent karyotypic abnormality. A recent study that used massively parallel whole exome sequencing identified an activating V600E mutation in BRAF, which appeared specific for HCL. Here, we confirm the specificity of BRAF V600E for HCL among low and intermediate grade B-NHL and describe a real-time polymerase chain reaction method for detecting this mutation in cases with low tumour burden. The V600E mutation does not appear to be associated with microsatellite instability, unlike the case in colorectal cancer. Thus, in conjunction with prior data, our results suggest incorporation of BRAF V600E mutation analysis in the diagnostic workup of HCL cases. Additionally, targeting the Ras-Raf-Mek-Erk-Map kinase pathway should be investigated as a potential therapeutic strategy for patients with this disease.

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.

PCDH10 has 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 the PCDH10 gene by methylation‐specific PCR in 215 cases of various subsets of myeloid‐ and lymphoid‐lineage leukemias. We found that PCDH10 promoter 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%). PCDH10 expression was downregulated via promoter hypermethylation in primary ALL samples ( N = 4) and leukemia cell lines ( N = 11). The transcriptional repression caused by PCDH10 methylation could be restored by pharmacologic inhibition of DNA methyltransferases. ALL cell lines harboring methylation‐mediated inactivation of PCDH10 were less sensitive to commonly used leukemia‐specific drugs suggesting that PCDH10 methylation might serve as a biomarker of chemotherapy response. Our results demonstrate that PCDH10 is 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. © 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 (C) 2010 John Wiley & Sons, Ltd.

1Institute for Cancer Genetics, Departments of Pathology and Genetics & Development, and the Herbert Irving Comprehensive Cancer Center, Columbia University, New York, NY; 2Laboratory of Experimental Oncology and Lymphoma Unit, Oncology Institute of Southern Switzerland (IOSI), Bellinzona, Switzerland; 3Istituto Dalle Molle di Studi sull’Intelligenza Artificiale, Manno, Switzerland; and 4Division of Hematology, Department of Clinical and Experimental Medicine, Amedeo Avogadro University of Eastern Piedmont, Novara, Italy

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.

Purpose: Cholangiocarcinoma (CCA) diagnosis has been limited by poor sensitivity of biliary brush cytology. Fluorescent in situ hybridization allows early detection of chromosomal aneuploidy or deletions in biliary cells and has the potential to improve diagnostic sensitivity compared to standard cytology. Methods: All patients referred for ERCP of indeterminate biliary strictures who underwent biliary brushing between 2/2008 and 4/2009 at our institution were included. We limited the analysis of test performance to those cases where final pathology was available or where follow-up data after a 6 month interval was obtained. A cytology result was considered positive if read as “suspicious” or “malignant” by a cytopathologist. FISH was performed using CEP 3, CEP 7, and CEP 17 probes for chromosome enumeration and 9p21 (p16) for deletion. FISH result was considered positive if aneuploidy or deletion was detected with these probes. Statistical comparison between the techniques was performed with area-under-ROC-curve (AUC) analysis. Results: 26 patients underwent ERCP, cytology, and FISH during this period. 18 underwent surgery or reached a final diagnosis and 6 month follow-up was available for 8. 12 cholangiocarcinomas, 2 pancreatic adenocarcinomas, 3 primary sclerosing cholangitis, and 1 benign stricture were diagnosed. Cytology had a sensitivity and specificity of 21% and 100%, respectively, for diagnosing malignancy. FISH had a sensitivity and specificity of 86% and 100%, respectively. Cytology and FISH (considered positive when either were positive) combined to yield a sensitivity of 93%, specificity of 100%, positive predictive value (PPV) of 100% and negative predictive value (NPV) of 92%. The AUC for the combination of cytology and FISH was significantly better than for cytology alone (p<0.001). Conclusion: FISH, when added to biliary brush cytology, significantly improved the diagnostic accuracy of detecting malignancy in indeterminate strictures. This study further validates previous findings that FISH should be employed as a standard analysis for all biliary brush specimens in patients with indeterminate biliary strictures.Table

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. Copyright © 2009 John Wiley & Sons, Ltd.

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-kappa B, 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-kappa B activation. (Blood. 2009;113:4918-4921)

Abstract 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]

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, Plen(+/-) mice were crossed with Irs2(+/-) mice. Deletion of Irs2 did not affect the initiation of neoplasia found in Plen(+/-) 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 fmdings provide evidence that the PI3K-activating adaptor Irs2 contributes to tumor progression in Pten(+/-) mice by stimulating both Myc and DNA synthesis. (Am J Pathol 2009, 174:276-286. DOI: 10.2353/ajpath.2009.080086)

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) analyses. 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 = 0.05) with persistence or progression to invasive cancer. We identified a total of 26 overexpressed genes as consequence of 20q gain (N = 14), as targets of amplicon 1 (N = 9; two genes also commonly expressed with 20q gain) and amplicon 2 (N = 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. © 2008 Wiley‐Liss, Inc.

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.