Marston Linehan

Molecular analysis of the short arm of chromosome 3 in small-cell and non-small-cell carcinoma of the lung.

Previous studies have suggested that the loss of DNA sequences on the short arm of chromosome 3 (3p) is associated with small-cell lung carcinoma. We therefore looked for loss of 3p alleles in tumor tissue from 42 patients with either small-cell or non-small-cell lung carcinoma. All 13 patients with small-cell lung carcinoma who were heterozygous for one or more alleles at 3p in normal tissue had the loss of at least one codominant allele in the tumor tissue. Cell lines of small-cell lung carcinoma from an additional eight patients were homozygous for 3p alleles; this result was significantly different from the predicted frequency of homozygosity. The tumor tissue studied included cell lines of small-cell lung carcinoma obtained from biopsy specimens, an autopsy sample, and an excised lymph node containing tumor cells. Loss of alleles at 3p was observed in tumor samples obtained before and after chemotherapy. Four of 15 evaluable patients with non-small-cell carcinoma of the lung had loss of 3p alleles. We conclude that loss of alleles at 3p is a change found consistently in small-cell lung carcinoma and occasionally in non-small-cell lung carcinoma.

Von Hippel-Lindau disease: identification of deletion mutations by pulsed-field gel electrophoresis

Von Hippel-Lindau disease (VHL) is an inherited multisystem neoplastic disorder. We prepared a 2.5-megabase (Mb) restriction map of the region surrounding the VHL gene and identified and characterized overlapping deletions in three unrelated patients affected with VHL. The smallest nested deletion (100 kb) was located within a 510-kb NruI fragment detected by 19–63′. The rearrangements detected will be useful in isolating and evaluating candidate cDNAs for the VHL gene. The detailed physical map will be useful in studying the organization and structure of genes in the VHL region.

Loss of Heterozygosity Occurs Centromeric to RB Without Associated Abnormalities in the Retinoblastoma Gene in Tumors from Patients with Metastatic Renal Cell Carcinoma

Tumor suppressor genes have been found to have loss of function in a number of malignancies. This loss of function is believed to contribute to malignant transformation or metastatic spread. In the present study, expression of the retinoblastoma (RB) tumor suppressor gene was examined in cell lines and tumor tissue obtained from primary renal and metastatic sites in patients with metastatic renal cell carcinoma. Three of fifteen (20%) of informative renal carcinoma cell lines had loss of heterozygosity (LOH) in the RB gene (intron 20) detected by polymerase chain reaction analysis. Using restriction fragment length polymorphism (RFLP) analysis, 7 of 22 (32%) informative cell lines had LOH centromeric to the RB gene at the D13S1 locus. No LOH (0 of 7) was seen telomeric to the RB gene at the D13S2 locus. None of the 28 cell lines examined had decreased RB mRNA expression compared with short-term cultures of proximal renal tubular cells. Western blotting demonstrated phosphorylated and unphosphorylated forms of RB protein of expected molecular weight in all 41 cell lines (33 primary and 8 metastatic) examined. Twenty-nine primary cell lines and 6 metastatic cell lines all demonstrated normal immunohistochemical staining. Loss of RB immunohistochemical staining in paraffin-embedded tissue was detected in none of the primary tumors (0 of 30) or metastatic tumors (0 of 12). The absence of abnormalities of RB expression detected in these renal cell carcinomas suggests that abnormalities of the RB gene are not central to malignant transformation or progression in this tumor type; however, another tumor suppressor gene centromeric to the RB locus may be important in renal cell carcinogenesis.

Pathological Oxidation of PTPN12 Underlies ABL1 Phosphorylation in HLRCC

Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC) is an inherited cancer syndrome associated with a highly aggressive form of type 2 papillary renal cell carcinoma (PRCC). Germ line 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 ABL1 activation, which promotes tumor growth, but how ABL1 is activated remained unclear. Oxidation can regulate protein-tyrosine phosphatase (PTP) catalytic activity; conceivably, ROS-catalyzed inactivation of an ABL-directed PTP might account for ABL1 activation in this malignancy. Previously, our group developed “q-oxPTPome,” a method that can globally monitor the oxidation of classical PTPs. We have now refined the q-oxPTPome approach, increasing its sensitivity by >10X. Applying q-oxPTPome to FH-deficient cell models shows that multiple PTPs are either highly oxidized (including PTPN12) or overexpressed. In general, highly oxidized PTPs were those that have relatively high sensitivity to exogenous H2O2. Most PTP oxidation in FH-deficient cells is reversible, although nearly 40% of PTPN13 is oxidized irreversibly 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 identify PTPN12 as the major ABL1 phosphatase in HLRCC. Overall, our results show that ROS-induced PTPN12 oxidation accounts for ABL1 phosphorylation in HLRCC-associated PRCC, reveal a novel mechanism for inactivating a tumor suppressor gene product, and establish a direct link between pathological PTP oxidation and neoplastic disease.

MP50-20 INCREASED THYROID CANCER RISK IN PATIENTS WITH KIDNEY CANCERS

INTRODUCTION AND OBJECTIVES: Intermittent hypoxia, a characteristic feature of obstructive sleep apnea (OSA), is becoming a topic of great interest in cancer research. Recently, population-based studies have suggested that OSA is associated with an increased incidence of cancer. At the molecular level, the upregulated hypoxia inducible factor subunit 1 a (HIF-1a) pathway seen in OSA is known to play an important role in the pathogenesis of clear cell renal cell carcinoma (ccRCC). We aimed to assess the association between OSA and clinical markers of tumor biology, including Fuhrman grade and tumor size, in patients with ccRCC, and to evaluate whether OSA is a predictor of lower recurrence-free survival (RFS) or cancer-specific survival (CSS). METHODS: Our study population comprised 2,579 patients who underwent radical or partial nephrectomy for ccRCC at a US (19912014; 2,322 patients) and a European (1999-2012; 257 patients) tertiary care center. The prevalence of self-reported OSA was 6%. A linear regression model, adjusted for smoking history, age, sex, BMI, ASA score and tumor grade, was used to assess the association of OSA with tumor size, and a logistic regression model, adjusted for smoking history, age, sex, BMI, ASA score and tumor size, was used to assess the association with Fuhrman grade, which was categorized as low (Fuhrman grade 1, 2 or low) or intermediate/high (Fuhrman grade 3, 4, intermediate or high). We compared RFS and CSS between patients with and without OSA using the Kaplan-Meier method. A Cox proportional hazards model was used to determine OSA association with RFS and CSS, controlling for tumor size and grade. RESULTS: OSA was associated with intermediate/high Fuhrman grade on both univariate (OR 1.57, 95% CI 1.15-2.14, p 1⁄4 0.004) and multivariable (OR 1.47, 95% CI 1.06-2.03, p 1⁄4 0.021) analyses. However, OSA was not found to be significantly associated with tumor size. We found no significant difference in RFS (p 1⁄4 0.3) or CSS (p 1⁄4 0.3) between patients with and without OSA. In multivariable analyses, OSA was not an independent predictor of RFS (HR 0.81, 95% CI 0.451.52; p 1⁄4 0.5) and CSS (HR 0.61, 95% CI 0.19-1.93; p 1⁄4 0.4). CONCLUSIONS: Patients with OSA undergoing surgery for ccRCC are more likely to present with higher grade disease. Our study offers a clinical correlate to molecular studies highlighting hypoxiarelated pathways in the pathogenesis of ccRCC. Further prospective studies are warranted to confirm our findings and to further investigate OSA impact on survival.

Can Mutations in the BAP1 Gene be Detected by Immunohisto- chemistry in Hereditary Kidney Cancers?

Background : Hereditary renal cell carcinoma (RCC) constitutes about 5% of all RCCs. The most common and well studied syndromes include, VHL, HLRCC, BHD, Familial Oncocytoma, RCC Papillary Type 1, TSC, RCC associated with Succinate dehydrogenase B (SHDB) mutations and others. Several genes, including VHL , MET , FLCN , FH and genes encoding the succinate dehydrogenase (SDH) subunits B/C/D have been identified as causative. However, the genetic basis of a significant percentage of familial RCC, some with clear cell morphology remain unknown. BAP1 (BRCA1 associated protein-1), a tumor suppressor gene that encodes a nuclear deubiquitinase, is inactivated in 15% of sporadic clear cell RCCs and its loss was associated with high tumor grade and poor prognosis. In this study, we investigated the possible role of this gene in the spectrum of RCC part of hereditary syndromes. Materials and Methods : To elucidate the role of BAP1 in all the spectrum of hereditary RCC, we studied by IHC a panel of RCCs which covers the spectrum of kidney cancers and included 10 VHL tumors, 6 HLRCCs, 8 chromophobe, 5 Hereditary Papillary Type 1, 6 Oncocytomas, 3 BHD (hybrid), and 24 sporadic clear cell RCCs. To analyze the BAP1 expression in these tumors, formalin fixed paraffin embedded (FFPE) tissues were immunostained with mouse monoclonal anti-human BAP1 antibody (Clone C-4, Santa Cruz). Results : We found that all the tumors except two showed positive nuclear staining for BAP1. The two negative cases that were negative for BAP1 were Clear cell type and belonged to two siblings. Molecular analysis in a prepublished study showed both patients harboring the p.L14H mutation. Conclusion : Our study supports the hypothesis that BAP1 mutations can play a role in hereditary syndromes predominantly in clear cell tumors. Staining for BAP1 should be done when there is no definite known mutation in a clear cell cancer but the patient gives history of familial kidney cancer. The two related patients who had similar mutations had aggressive, metastatic disease, which suggests that probably BAP1 does play a role in hereditary RCC clear cell type.

Abstract LB-113: Genome-wide mapping of TFE3-fusion target genes.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Xp11.2 translocation renal cell carcinoma (RCC), also known as TFE3-fusion associated RCC, is a recently classified distinct subtype of RCC. It is defined by several different translocations involving chromosome Xp11.2, all resulting in gene fusions involving the Transcription Factor Binding to IGHM Enhancer 3 ( TFE3 ) gene. However, the detailed function of TFE3-fusion proteins in TFE3-fusion associated RCC still remains unknown, and there is no effective treatment for patients with this RCC subtype. In this study we sought to investigate the detailed regulatory network of TFE3-fusion proteins by using doxycycline-inducible HA-tagged TFE3-fusion cell lines and chromatin immunoprecipitation (ChIP)-sequencing. Doxycycline-inducible cell lines that express HA-tagged TFE3-fusion (PRCC-TFE3, PSF-TFE3 and NONO-TFE3) and TFE3 wild-type proteins were established in the HK2 cell line. Genome-wide screening of the binding characteristics of TFE3-fusion proteins and TFE3 wild-type protein in doxycycline-induced cell lines was performed using a chromatin immunoprecipitation sequencing (ChIP-seq) assay. Hierarchical clustering was used to analyze the genes regulated by each type of TFE3-fusion protein compared with TFE3 wild-type protein. The total number of CHIP-seq reads in TFE3 wild-type cell line was 18552874 and enrichment of the promoter region was 1.2. In contrast, the total number of CHIP-seq reads in PRCC-TFE3, PSF-TFE3 and NONO-TFE3 cell lines were 15332969, 20426159 and 17639467, respectively. Enrichment of promoter region in each of the TFE3-fusion cell line was 1.5, 2.4 and 2.2, respectively. There were 80 motifs enriched (Z score >1.5) by expression of PRCC-TFE3 protein compared to TFE3-wild type protein, 104 motifs enriched by PSF-TFE3 protein and 39 motifs by NONO-TFE3 protein expression. Work is ongoing to identify the target genes that are co-regulated in TFE3-fusion cell lines relative to the TFE3 wild-type cell line. Our aim is to identify activated pathways in common among the different TFE3-fusion cell lines compared to the TFE3 wild-type cell line. We anticipate that this study will further our understanding of mechanisms that activate TFE3-fusion proteins facilitating the identification of therapeutic targets for effective treatment of TFE3-fusion associated RCC. Citation Format: Ying Huang, Masaya Baba, Hisashi Hasumi, Yukiko Hasumi, Laura Schmidt, Yoshi Wakabayashi, Jun Zhu, Wenjing Yang, Marston Linehan. Genome-wide mapping of TFE3-fusion target genes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-113. doi:10.1158/1538-7445.AM2013-LB-113

Abstract LB-107: Mitochondrial DNA mutations are diagnostic markers of renal oncocytomas.

Oncocytomas are mostly benign tumors of epithelial origin, characterized by dense accumulation of defective mitochondria. To date, no nuclear gene has been found to be responsible for these tumors, but a correlation of the oncocytic phenotype with the occurrence of disruptive mitochondrial DNA (mtDNA) mutations, mostly affecting genes encoding for proteins of Complex I of the respiratory chain (NADH dehydrogenase), has been shown in sporadic oncocytomas of different organs, including renal oncocytomas (RO). Kidney cancer is made up of a number of tumors that present with a different histology and clinical course. They are caused by different genes, and therefore therapy needs to be adapted for each type of renal tumor. Imaging techniques are usually not sufficient to distinguish between benign and malignant forms of renal cell cancer (RCC). In addition, it is difficult to differentiate benign ROs from malignant forms of RCCs in samples from biopsies, because they share ultrastructural appearances, including abnormal mitochondria with altered cristae. Therefore, a reliable diagnostic tool would be invaluable to guide clinicians in the management of patients with RCC of unclear phenotype. In our current work we tested whether mtDNA mutations can be used as diagnostic tools to distinguish between benign ROs and more aggressive forms of RCC. We sequenced the entire mtDNA in 36 samples that included sporadic, bilateral and multifocal as well as familial ROs and 30 non-oncocytic RCCs of different histologies. Moreover, the mitochondrial copy number was determined in all samples by real-time PCR. Furthermore, we sequenced the mtDNA from biopsies and compared it to the mtDNA sequence of the resected tumor. We found a strong correlation between the presence of disruptive mtDNA mutations affecting complex I genes and the oncocytic phenotype in renal tumors. Additionally, oncocytomas showed significantly higher mtDNA copy number when compared to other RCCs. We also show that the mtDNA sequences from the biopsies correspond to the sequence of the tumor itself and are predictive of the phenotype. Hence, we propose that sequencing of the mitochondrial genome, supported by the analysis of the mtDNA copy number, may be used as a diagnostic tool to distinguish between benign and aggressive forms of RCCs to guide physicians in choosing the appropriate treatment for patients. Citation Format: Martin Lang, Cathy D. Vocke, Maria J. Merino, Laura S. Schmidt, Marston W. Linehan. Mitochondrial DNA mutations are diagnostic markers of renal oncocytomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-107. doi:10.1158/1538-7445.AM2013-LB-107