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Analysis in 5 mixed ccA/ccB tumors including 15 regions showed that CNVs were more frequent among ccB reagions(p=0.014).

Classical PTP expression, H2O2 sensitivity and irreversible oxidation in FH-deficient PRCC cell lines.

PTPN12 binds tyrosine-phosphorylated ABL1 independent of PSTPIP1 and PSTPIP2.

PCA map showed within-sample variation in a) ccA/ccB gene set and b) CCP gene set. Each color of dot represents individual tumor. Small tumors were indicated as round dot. Large tumors were indicated as triangular dot.

Profile of PTP substrate-trapping mutants in HEK293 cells.

Figure S3 shows heat map of samples-sample distance for entire gene set.

Minimal correlation of gene expression was decreasing with increasing in subclonal CNV events. (R2=0.431, p

Purpose: Tumor heterogeneity may represent a barrier to preoperative genomic characterization by needle biopsy in clear cell renal cell carcinoma (ccRCC). The extent of heterogeneity in small renal tumors remains unknown. Therefore, we set out to evaluate heterogeneity in resected large and small renal tumors.Experimental Design: We conducted a study from 2013 to 2016 that evaluated 47 consecutive ccRCC tumors resected during radical or partial nephrectomy. Cases were designated as small (7 cm) tumors. Each tumor had three regions sampled. Copy-number variation (CNV) was assessed and gene expression analysis was performed to characterize the clear-cell A and B (ccA/ccB) profile and the cell-cycle progression (CCP) score. Genomic heterogeneity between three regions was evaluated using CNV subclonal events, regional expression profiles, and correlation between gene expression.Results: Twenty-three small and 24 large tumors were analyzed. Total CNVs and subclonal CNVs events were less frequent in small tumors (P < 0.001). Significant gene expression heterogeneity was observed for both CCP scores and ccA/ccB classifications. Larger tumors had more variance in CCP scores (P = 0.026). The distribution of ccA/ccB differed between small and large tumors with mixed ccA/ccB tumors occurring more frequently in the larger tumors (P = 0.024). Analysis of five mixed tumors (with both ccA/ccB regions) demonstrated the more aggressive ccB phenotype had greater CNV events (P = 0.014).Conclusions: Small renal tumors have much less genomic complexity and fewer subclonal events. Pretreatment genomic characterization with single-needle biopsy in small tumors may be useful to assess biologic potential and may influence therapy. Clin Cancer Res; 24(17); 4137–44. ©2018 AACR.

Determinants of leukocyte telomere length among controls

PTPN12 regulates ABL1 phosphorylation.

H2O2 induces reversible oxidation of classical PTPs.

Background: It has been hypothesized that genomic instability related to telomere dysfunction may contribute to carcinogenesis. There is some evidence from case–control studies suggesting that short leukocyte telomere length may be associated with an increased risk of renal cell carcinoma (RCC); however, this association has not been investigated prospectively.Methods: We conducted a nested case–control study (209 cases, 410 controls) of RCC risk in relation to prediagnostic leukocyte telomere length in the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial. ORs and 95% confidence intervals (CI) were estimated using conditional logistic regression.Results: Leukocyte telomere length was not significantly associated with future risk of RCC (highest quartile vs. lowest: OR, 0.8; 95% CI, 0.5–1.5; Ptrend = 0.6). Analyses stratified by sex, age, and time from blood collection to RCC diagnosis were similarly null.Conclusions: The results of this study, to our knowledge the first prospective investigation of its kind, do not support an association between prediagnostic leukocyte telomere length and risk of RCC.Impact: In contrast to some earlier reports, our findings add to the evidence that leukocyte telomere length is not a biomarker of risk related to the etiology of RCC. Cancer Epidemiol Biomarkers Prev; 22(5); 997–1000. ©2013 AACR.

Supplementary materials, methods, and figure legends.

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is an inherited cancer syndrome associated with a highly aggressive form of type 2 papillary renal cell carcinoma (PRCC). Germline inactivating alterations in fumarate hydratase (FH) cause HLRCC and result in elevated levels of reactive oxygen species (ROS). Recent work indicates that FH−/− PRCC cells have increased activation of ABL1, which promotes tumor growth, but how ABL1 is activated remains unclear. Given that oxidation can regulate protein-tyrosine phosphatase (PTP) catalytic activity, inactivation of an ABL-directed PTP by ROS might account for ABL1 activation in this malignancy. Our group previously developed “q-oxPTPome,” a method that globally monitors the oxidation of classical PTPs. In this study, we present a refined q-oxPTPome, increasing its sensitivity by >10×. Applying q-oxPTPome to FH-deficient cell models showed that multiple PTPs were either highly oxidized (including PTPN12) or overexpressed. Highly oxidized PTPs were those with relatively high sensitivity to exogenous H2O2. Most PTP oxidation in FH-deficient cells was reversible, although nearly 40% of PTPN13 was irreversibly oxidized to the sulfonic acid state. Using substrate-trapping mutants, we mapped PTPs to their putative substrates and found that only PTPN12 could target ABL1. Furthermore, knockdown experiments identified PTPN12 as the major ABL1 phosphatase, and overexpression of PTPN12 inhibited ABL1 phosphorylation and HLRCC cell growth. These results show that ROS-induced oxidation of PTPN12 accounts for ABL1 phosphorylation in HLRCC-associated PRCC, revealing a novel mechanism for inactivating a tumor suppressor gene product and establishing a direct link between pathologic PTP oxidation and neoplastic disease.Significance:This work identifies a novel mechanism of activation of the oncogenic kinase ABL1 via ROS-induced, oxidation-mediated inactivation of cognate protein tyrosine phosphatases.

Purpose: Tumor heterogeneity may represent a barrier to preoperative genomic characterization by needle biopsy in clear cell renal cell carcinoma (ccRCC). The extent of heterogeneity in small renal tumors remains unknown. Therefore, we set out to evaluate heterogeneity in resected large and small renal tumors. Experimental Design: We conducted a study from 2013 to 2016 that evaluated 47 consecutive ccRCC tumors resected during radical or partial nephrectomy. Cases were designated as small (7 cm) tumors. Each tumor had three regions sampled. Copy-number variation (CNV) was assessed and gene expression analysis was performed to characterize the clear-cell A and B (ccA/ccB) profile and the cell-cycle progression (CCP) score. Genomic heterogeneity between three regions was evaluated using CNV subclonal events, regional expression profiles, and correlation between gene expression. Results: Twenty-three small and 24 large tumors were analyzed. Total CNVs and subclonal CNVs events were less frequent in small tumors (P < 0.001). Significant gene expression heterogeneity was observed for both CCP scores and ccA/ccB classifications. Larger tumors had more variance in CCP scores (P = 0.026). The distribution of ccA/ccB differed between small and large tumors with mixed ccA/ccB tumors occurring more frequently in the larger tumors (P = 0.024). Analysis of five mixed tumors (with both ccA/ccB regions) demonstrated the more aggressive ccB phenotype had greater CNV events (P = 0.014). Conclusions: Small renal tumors have much less genomic complexity and fewer subclonal events. Pretreatment genomic characterization with single-needle biopsy in small tumors may be useful to assess biologic potential and may influence therapy. Clin Cancer Res; 24(17); 4137–44. ©2018 AACR.

Elevated levels of the metabolites, 2-hydroxyglutarate (2HG), fumarate, and succinate, can occur in human malignancies due to somatic mutations in the isocitrate dehydrogenase 1/2 (IDH1/2) genes or germline mutations in fumarate hydratase (FH) and succinate dehydrogenase (SDH) genes, respectively. Mutations in IDH1 and IDH2 are found in a large number of human malignancies, most notably gliomas and acute myelogenous leukemias, along with cholangiocarcinomas, chondrosarcomas, and melanomas. Inherited mutations in FH and SDH, are linked to the familial cancer predisposition syndromes, Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC) and Hereditary Paraganglioma and Pheochromocytoma (SDH PGL/PCC), respectively. These structurally related metabolites inhibit α-ketoglutarate-dependent enzymes, including dioxygenases that modify chromatin. We have shown that they consequently suppress the pathway of homology-dependent DNA repair (HDR), conferring an exquisite sensitivity to PARP inhibitors that is currently being tested in clinical trials. In this presentation, we will discuss our recent work elucidating the mechanistic basis for this suppression of DNA repair. Rather than acting indirectly through epigenetic regulation of gene expression, we find that these metabolites act directly by inhibiting HDR factor recruitment to DNA double-strand breaks (DSBs). The metabolites inhibit the lysine demethylases, KDM4A/B, causing aberrant hypermethylation of H3K9 and disrupting signaling at the break, thereby delaying the stepwise recruitment of key HDR factors. These results define a novel mechanism of decreased DNA repair in oncometabolite producing human tumors and may provide the basis for the rational design of novel therapeutic strategies for these malignancies. Citation Format: Parker S. Sulkowski, Sebstian Oeck, Jing Li, Brian Shuch, Megan C. King, Ranjit S. Bindra, Peter M. Glazer. Oncometabolites suppress homologous recombination DNA repair by inhibition of chromatin remodeling at the DNA double-strand break [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr SY21-02.

Background: It has been hypothesized that genomic instability related to telomere dysfunction may contribute to carcinogenesis. There is some evidence from case–control studies suggesting that short leukocyte telomere length may be associated with an increased risk of renal cell carcinoma (RCC); however, this association has not been investigated prospectively. Methods: We conducted a nested case–control study (209 cases, 410 controls) of RCC risk in relation to prediagnostic leukocyte telomere length in the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial. ORs and 95% confidence intervals (CI) were estimated using conditional logistic regression. Results: Leukocyte telomere length was not significantly associated with future risk of RCC (highest quartile vs. lowest: OR, 0.8; 95% CI, 0.5–1.5; Ptrend = 0.6). Analyses stratified by sex, age, and time from blood collection to RCC diagnosis were similarly null. Conclusions: The results of this study, to our knowledge the first prospective investigation of its kind, do not support an association between prediagnostic leukocyte telomere length and risk of RCC. Impact: In contrast to some earlier reports, our findings add to the evidence that leukocyte telomere length is not a biomarker of risk related to the etiology of RCC. Cancer Epidemiol Biomarkers Prev; 22(5); 997–1000. ©2013 AACR.

INTRODUCTION: Genetic profiling of tumors used to identify treatment choices for cancer patients focus on kinase mutations and well-defined translocations; multiple clinical trials have been initiated for specific kinase-targeted agents. About 15-20% of patients have actionable mutations. HDR have been found in ~ 20 % of patient tumors molecularly profiled at the Yale Cancer Center. Mutations in HDR related genes often occur in addition to other mutations in signaling pathways. Specific targeted treatment options for these patients are limited, as breast and ovarian cancer mutations are the only indication for the HDR pathway targeted PARP inhibitors. Additional drugs are in development, but few clinical studies based on HDR other than BRCA1/2 mutations are open. METHODS: The OLAPCO study targets HDR in patients irrespective of tumor origin. The protocol includes four arms to which patients can be enrolled based on tumor profiling results: olaparib monotherapy (PARP inhibitor) for HDR; olaparib & AZD5363 ( AKT inhibitor) for PIK3CA/AKT/PTEN pathway dysregulation and ARID1A mutations; olaparib & AZD1775 ( wee1 inhibitor) for TP53 mutations and olaparib & AZD6738 (ATR inhibitor) for HDR and olaparib pre-treated patients. The clinical endpoints are to determine tumor overall response rate (ORR) and clinical benefit rate (CBR) by RECIST after 16 weeks of treatment. RESULTS: Arms a and b are open to patient accrual. Together, 34 pts have been treated, 12 remain on treatment, with no new or > Gr 2 (CTCAE4) treatment-related toxicities. TABLE 1Olaparib total = 23; median # cycles= 2, range 1-10+ ; 7 ongoingMutationATMBRCA1IDH1/2FHPALB2CHK2BAP1ARID 1A#52821212ORR1 PRCBR10201000TABLE 2Olaparib & AZD 5363 total = 12; median # cycles = 2, range 1-8+; 5 ongoingMutationAKT1AKT3AKT2 & PALB2ARID 1AARID 1A & PIK3CAPTEN#321411ORR1CBR10101 DISCUSSION: HDR defects are actionable mutations, increasing the number of patients for whom tumor profiling can identify a therapeutic option. This exploratory trial is expanding the therapeutic spectrum of olaparib alone and in combination, to include heretofore non-canonical alterations in HDR, beyond BRCA1/2. This trial design allows additional molecular targets, agents & combinations to be rapidly evaluated. AstraZeneca support is gratefully acknowledged. Citation Format: Joseph P. Eder, Geoffrey I. Shapiro, James M. Cleary, Vickie L. Keedy, Vickie L. Keedy, Ranjit Bindra, Brian Shuch, Jeffrey Sklar, Khanh Do, Juliane Juergensmeier. Olaparib combinations (OLAPCO)for homology-directed DNA repairdefects (HDR) [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 CT159.

Purpose: The goal of this study was to evaluate immunohistochemical and cytogenetic features and their prognostic value in papillary renal cell carcinoma (PRCC) subtypes. Experimental Design: One hundred fifty-eight cases of PRCC were identified and reclassified by subtype. Tumoral expression of 29 molecular markers was determined by immunohistochemistry. Cytogenetic analyses were done on a prospective series of 65 patients. Associations with clinicopathologic information and disease-specific survival were assessed. Results: Fifty-one patients (32%) had type 1 and 107 (68%) type 2 PRCC. Type 2 patients had worse Eastern Cooperative Oncology Group performance status, higher T stages, nodal and distant metastases, higher grades, and a higher frequency of necrosis, collecting system invasion and sarcomatoid features. Type 2 showed greater expression of vascular endothelial growth factor (VEGF)-R2 in the tumor epithelium, and of VEGF-R3 in both tumor epithelium and endothelium. Loss of chromosome 1p, loss of 3p, and gain of 5q were exclusively observed in type 2, whereas type 1 more frequently had trisomy 17. Type 2 PRCC was associated with worse survival than type 1, but type was not retained as an independent prognostic factor. Lower PTEN, lower EpCAM, lower gelsolin, higher CAIX, and higher VEGF-R2 and VEGF-R3 expression, loss of 1p, 3p, or 9p, and absence trisomy 17 were all associated with poorer prognosis. Conclusions: Type 2 PRCC is associated with more aggressive clinicopathologic features and worse outcome. Molecular and chromosomal alterations can distinguish between PRCC subtypes and influence their prognosis. The effect of 3p loss on survival in PRCC is opposite to the relationship seen in clear cell RCC.

BACKGROUND: Renal cell carcinoma (RCC) is made up of several histological subtypes, the most common of which include clear cell, papillary and chromophobe RCC. These subtypes possess distinct genetic and clinical characteristics, and may also differ in etiology. To better understand whether the associations with established RCC risk factors [body mass index (BMI), smoking, hypertension, family history of kidney cancer] differ across subtypes, we conducted analyses in two large case-control studies conducted in the U.S. (1,217 cases, 1,235 controls) and Central and Eastern Europe (1,097 cases, 1,476 controls). METHODS: Histology was ascertained for 706 U.S. cases (58% of participating cases) and 891 European cases (81%) through a central pathology review conducted by a single pathologist. For cases not included in the central review, histology was abstracted from the original diagnostic pathology report. Case-only analyses were performed to compute odds ratios (OR) and 95% confidence intervals (CI) summarizing subtype differences by age, sex, and race. Case-control analyses were performed to compute subtype-specific ORs for other risk factors using polytomous regression. RESULTS: In case-only analyses, papillary cases (N=237) were older (OR=1.2, 95% CI=1.1-1.4 per 10-year increase), less likely to be female (OR=0.5, 95 % CI=0.4-0.8) and more likely to be black (OR=2.6, 95% CI=1.8-3.9) compared to clear cell cases (N=1,524). In case-control analyses, BMI was associated with clear cell (OR=1.5, 95% CI=1.3-1.7 per 10 kg/m2 increase) and chromophobe RCC (N=80; OR=1.5, 95% CI=1.1-2.0), but not papillary RCC (OR=1.1, 95% CI 0.9-1.5; heterogeneity test vs. clear cell, P=0.006). No differences across subtypes were observed for other risk factors. Analyses stratified by study and restricted to cases included in the central pathology review yielded virtually identical findings. CONCLUSIONS: Our results from this analysis, to our knowledge the most comprehensive investigation of etiologic heterogeneity across RCC histologic subtypes conducted to date, support the existence of distinct age, sex and racial distributions for RCC subtypes, and suggest that the obesity-RCC association differs by histology. These findings underscore the importance of accounting for histologic subtype in investigations of RCC etiology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5515. doi:1538-7445.AM2012-5515