Faculty Bibliography

BACKGROUND: Historically, VHL was the only frequently mutated gene in clear cell renal cell carcinoma (ccRCC), with conflicting clinical relevance. Recent sequencing efforts have identified several novel frequent mutations of histone modifying and chromatin remodeling genes in ccRCC including PBRM1, SETD2, BAP1, and KDM5C. PBRM1, SETD2, and BAP1 are located in close proximity to VHL within a commonly lost (approximately 90%) 3p locus. To date, the clinical and pathologic significance of mutations in these novel candidate tumor suppressors is unknown. OBJECTIVE: To determine the frequency of and render the first clinical and pathologic outcome associated with mutations of these novel candidate tumor suppressors in ccRCC. DESIGN, SETTING, AND PARTICIPANTS: Targeted sequencing was performed in 185 ccRCCs and matched normal tissues from a single institution. Pathologic features, baseline patient characteristics, and follow-up data were recorded. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The linkage between mutations and clinical and pathologic outcomes was interrogated with the Fisher exact test (for stage and Fuhrman nuclear grade) and the permutation log-rank test (for cancer-specific survival [CSS]). RESULTS AND LIMITATIONS: PBRM1, BAP1, SETD2, and KDM5C are mutated at 29%, 6%, 8%, and 8%, respectively. Tumors with mutations in PBRM1 or any of BAP1, SETD2, or KDM5C (19%) are more likely to present with stage III disease or higher (p=0.01 and p=0.001, respectively). Small tumors (<4cm) with PBRM1 mutations are more likely to exhibit stage III pathologic features (odds ratio: 6.4; p=0.001). BAP1 mutations tend to occur in Fuhrman grade III-IV tumors (p=0.052) and are associated with worse CSS (p=0.01). Clinical outcome data are limited by the number of events. CONCLUSIONS: Most mutations of chromatin modulators discovered in ccRCC are loss of function, associated with advanced stage, grade, and possibly worse CSS. Further studies validating the clinical impact of these novel mutations and future development of therapeutics remedying these tumor suppressors are warranted.

Non-small cell lung cancer (NSCLC) often expresses mutant KRAS together with tumor-associated mutations of the CDKN2A locus, which are associated with aggressive, therapy-resistant tumors. Here, we unravel specific requirements for the maintenance of NSCLC that carries this genotype. We establish that the extracellular signal-regulated kinase (ERK)/RHOA/focal adhesion kinase (FAK) network is deregulated in high-grade lung tumors. Suppression of RHOA or FAK induces cell death selectively in mutant KRAS;INK4A/ARF-deficient lung cancer cells. Furthermore, pharmacologic inhibition of FAK caused tumor regression specifically in the high-grade lung cancer that developed in mutant Kras;Cdkn2a-null mice. These findings provide a rationale for the rapid implementation of genotype-specific targeted therapies using FAK inhibitors in patients with cancer.

BACKGROUND: In this phase II trial, we investigated the efficacy of a metronomic temozolomide schedule in the treatment of recurrent malignant gliomas (MGs). METHODS: Eligible patients received daily temozolomide (50 mg/m2) continuously until progression. The primary endpoint was progression-free survival rate at 6 months in the glioblastoma cohort (N = 37). In an exploratory analysis, 10 additional recurrent grade III MG patients were enrolled. Correlative studies included evaluation of 76 frequent mutations in glioblastoma (iPLEX assay, Sequenom) aiming at establishing the frequency of potentially "drugable" mutations in patients entering recurrent MG clinical trials. RESULTS: Among glioblastoma patients, median age was 56 y; median Karnofsky performance score (KPS) was 80; 62% of patients had been treated for >/=2 recurrences, including 49% of patients having failed bevacizumab. Treatment was well tolerated; clinical benefit (complete response + partial response + stable disease) was seen in 10 (36%) patients. Progression-free survival rate at 6 months was 19% and median overall survival was 7 months. Patients with previous bevacizumab exposure survived significantly less than bevacizumab-naive patients (median overall survival: 4.3 mo vs 13 mo; hazard ratio = 3.2; P = .001), but those patients had lower KPS (P = .04) and higher number of recurrences (P < .0001). Mutations were found in 13 of the 38 MGs tested, including mutations of EGFR (N = 10), IDH1 (N = 5), and ERBB2 (N = 1). CONCLUSIONS: In spite of a heavily pretreated population, including nearly half of patients having failed bevacizumab, the primary endpoint was met, suggesting that this regimen deserves further investigation. Results in bevacizumab-naive patients seemed particularly favorable, while results in bevacizumab-failing patients highlight the need to develop further treatment strategies for advanced MG. Clinical trials.gov identifier NCT00498927 (available at http://clinicaltrials.gov/ct2/show/NCT00498927).

Inactivation of the von Hippel-Lindau tumor suppressor gene, VHL, is an archetypical tumor-initiating event in clear cell renal carcinoma (ccRCC) that leads to the activation of hypoxia-inducible transcription factors (HIFs). However, VHL mutation status in ccRCC is not correlated with clinical outcome. Here we show that during ccRCC progression, cancer cells exploit diverse epigenetic alterations to empower a branch of the VHL-HIF pathway for metastasis, and the strength of this activation is associated with poor clinical outcome. By analyzing metastatic subpopulations of VHL-deficient ccRCC cells, we discovered an epigenetically altered VHL-HIF response that is specific to metastatic ccRCC. Focusing on the two most prominent pro-metastatic VHL-HIF target genes, we show that loss of Polycomb repressive complex 2 (PRC2)-dependent histone H3 Lys27 trimethylation (H3K27me3) activates HIF-driven chemokine (C-X-C motif) receptor 4 (CXCR4) expression in support of chemotactic cell invasion, whereas loss of DNA methylation enables HIF-driven cytohesin 1 interacting protein (CYTIP) expression to protect cancer cells from death cytokine signals. Thus, metastasis in ccRCC is based on an epigenetically expanded output of the tumor-initiating pathway.

PURPOSE: HER2-amplified breast cancer is sometimes clinically insensitive to HER2-targeted treatment with trastuzumab. Laboratory models of resistance have causally implicated changes in HER2 expression and activation of the phosphoinositide 3-kinase (PI3K)-AKT pathway. We conducted a prospective tissue acquisition study to determine if there is evidence for these lesions in metastatic tumors that have progressed on trastuzumab-containing therapy. EXPERIMENTAL DESIGN: From 2/2007 to 11/2011, 63 patients with HER2-amplified breast cancer with recurrence of disease after adjuvant trastuzumab therapy or World Health Organization-defined progression of metastatic disease on a trastuzumab-containing regimen were prospectively enrolled and underwent tumor biopsy. Specimens were analyzed for activating mutations in PIK3CA and HER2 by Sequenom and analyzed for HER2 and PTEN status by immunohistochemistry. RESULTS: In 53/60 cases (88%, 3 cases not evaluable for HER2), HER2 overexpression persisted in the metastatic tumor following trastuzumab exposure. Among the 7 cases lacking HER2 overexpression, repeat analysis of the pretreatment tumor failed to confirm HER2 overexpression in five cases. Among cases evaluable for PTEN (56) or PI3K mutation (45), absent or significantly diminished PTEN expression was noted in 33 (59%) and activating mutations in PIK3CA in 13 (29%). The combined rate of PTEN loss and PIK3CA mutation in the trastuzumab-refractory tumors was 71% compared with 44% (P = 0.007) in an unexposed cohort of 73 HER2-amplified tumors. CONCLUSIONS: In this series of prospectively collected trastuzumab-refractory human breast cancers, loss of HER2 overexpression was rare, whereas activation of the PI3K-AKT pathway through loss of PTEN or PIK3CA mutation was frequently observed.

Aging is characterized by clonal expansion of myeloid-biased hematopoietic stem cells and by increased risk of myeloid malignancies. Exome sequencing of three elderly females with clonal hematopoiesis, demonstrated by X-inactivation analysis, identified somatic TET2 mutations. Recurrence testing identified TET2 mutations in 10 out of 182 individuals with X-inactivation skewing. TET2 mutations were specific to individuals with clonal hematopoiesis without hematological malignancies and were associated with alterations in DNA methylation.

Cancer drugs often induce dramatic responses in a small minority of patients. We used whole-genome sequencing to investigate the genetic basis of a durable remission of metastatic bladder cancer in a patient treated with everolimus, a drug that inhibits the mTOR (mammalian target of rapamycin) signaling pathway. Among the somatic mutations was a loss-of-function mutation in TSC1 (tuberous sclerosis complex 1), a regulator of mTOR pathway activation. Targeted sequencing revealed TSC1 mutations in about 8% of 109 additional bladder cancers examined, and TSC1 mutation correlated with everolimus sensitivity. These results demonstrate the feasibility of using whole-genome sequencing in the clinical setting to identify previously occult biomarkers of drug sensitivity that can aid in the identification of patients most likely to respond to targeted anticancer drugs.

The molecular foundations of lower-grade gliomas (LGGs)-astrocytoma, oligodendroglioma, and oligoastrocytoma-remain less well characterized than those of their fully malignant counterpart, glioblastoma. Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) likely represent initiating pathogenic events. However, while IDH mutations appear to dramatically alter cellular epigenomic landscapes, definitive downstream transformative mechanisms have not been characterized. It remains likely, therefore, that additional genomic abnormalities collaborate with IDH mutation to drive oncogenesis in LGG. We performed whole exome sequencing in 4 LGGs, followed by focused resequencing in an additional 28, and found a high incidence of mutations in the ATRX gene (alpha thalassemia/mental retardation syndrome X-linked). ATRX forms a core component of a chromatin remodeling complex active in telomere biology. Mutations in ATRX have been identified in multiple tumor types and appear to cause alternative lengthening of telomeres (ALT), a presumed precursor to genomic instability. In our samples, ATRX mutation was entirely restricted to IDH-mutant tumors, closely correlated with TP53 mutation and astrocytic differentiation, and mutually exclusive with 1p/19q codeletion, the molecular hallmark of oligodendroglioma. Moreover, ATRX mutation was highly enriched in tumors of so-called early progenitor-like transcriptional subclass (~85%), which our prior work has linked to specific cells of origin in the forebrain subventricular zone. Finally, ATRX mutation correlated with ALT, providing a mechanistic link to genomic instability. In summary, our findings both identify ATRX mutation as a defining molecular determinant for a large subset of IDH-mutant gliomas and have direct implications on pathogenic mechanisms across the wide spectrum of LGGs.

PURPOSE: To compare the mutational and copy number profiles of primary and metastatic colorectal carcinomas (CRCs) using both unpaired and paired samples derived from primary and metastatic disease sites. PATIENTS AND METHODS: We performed a multiplatform genomic analysis of 736 fresh frozen CRC tumors from 613 patients. The cohort included 84 patients in whom tumor tissue from both primary and metastatic sites was available and 31 patients with pairs of metastases. Tumors were analyzed for mutations in the KRAS, NRAS, BRAF, PIK3CA, and TP53 genes, with discordant results between paired samples further investigated by analyzing formalin-fixed, paraffin-embedded tissue and/or by 454 sequencing. Copy number aberrations in primary tumors and matched metastases were analyzed by comparative genomic hybridization (CGH). RESULTS: TP53 mutations were more frequent in metastatic versus primary tumors (53.1% v 30.3%, respectively; P < .001), whereas BRAF mutations were significantly less frequent (1.9% v 7.7%, respectively; P = .01). The mutational status of the matched pairs was highly concordant (> 90% concordance for all five genes). Clonality analysis of array CGH data suggested that multiple CRC primary tumors or treatment-associated effects were likely etiologies for mutational and/or copy number profile differences between primary tumors and metastases. CONCLUSION: For determining RAS, BRAF, and PIK3CA mutational status, genotyping of the primary CRC is sufficient for most patients. Biopsy of a metastatic site should be considered in patients with a history of multiple primary carcinomas and in the case of TP53 for patients who have undergone interval treatment with radiation or cytotoxic chemotherapies.