Faculty Bibliography

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.

Leukemic transformation (LT) of myeloproliferative neoplasms (MPNs) is associated with a poor prognosis and resistance to therapy. Although previous candidate genetic studies have identified mutations in MPN patients who develop acute leukemia, the complement of genetic abnormalities in MPN patients who undergo LT is not known nor have specific molecular abnormalities been shown to have clinical relevance in this setting. We performed high-throughput resequencing of 22 genes in 53 patients with LT after MPN to characterize the frequency of known myeloid mutations in this entity. In addition to JAK2 and TET2 mutations, which occur commonly in LT after MPN, we identified recurrent mutations in the serine/arginine-rich splicing factor 2 (SRSF2) gene (18.9%) in acute myeloid leukemia (AML) transformed from MPNs. SRSF2 mutations are more common in AML derived from MPNs compared with LT after myelodysplasia (4.8%) or de novo AML (5.6%), respectively (P=.05). Importantly, SRSF2 mutations are associated with worsened overall survival in MPN patients who undergo LT in univariate (P=.03; HR, 2.77; 95% CI, 1.10-7.00) and multivariate analysis (P<.05; HR, 2.11; 95% CI, 1.01-4.42). These data suggest that SRSF2 mutations contribute to the pathogenesis of LT and may guide novel therapeutic approaches for MPN patients who undergo LT.

PURPOSE: Diffuse gliomas represent the most prevalent class of primary brain tumor. Despite significant recent advances in the understanding of glioblastoma [World Health Organization (WHO) IV], its most malignant subtype, lower grade (WHO II and III) glioma variants remain comparatively understudied, especially in light of their notable clinical heterogeneity. Accordingly, we sought to identify and characterize clinically relevant molecular subclasses of lower grade diffuse astrocytic gliomas. EXPERIMENTAL DESIGN: We conducted multidimensional molecular profiling, including global transcriptional analysis, on 101 lower grade diffuse astrocytic gliomas collected at our own institution and validated our findings using publically available gene expression and copy number data from large independent patient cohorts. RESULTS: We found that IDH mutational status delineated molecularly and clinically distinct glioma subsets, with IDH mutant (IDH mt) tumors exhibiting TP53 mutations, platelet-derived growth factor receptor (PDGFR)A overexpression, and prolonged survival, and IDH wild-type (IDH wt) tumors exhibiting EGFR amplification, PTEN loss, and unfavorable disease outcome. Furthermore, global expression profiling revealed three robust molecular subclasses within lower grade diffuse astrocytic gliomas, two of which were predominantly IDH mt and one almost entirely IDH wt. IDH mt subclasses were distinguished from each other on the basis of TP53 mutations, DNA copy number abnormalities, and links to distinct stages of neurogenesis in the subventricular zone. This latter finding implicates discrete pools of neuroglial progenitors as cells of origin for the different subclasses of IDH mt tumors. CONCLUSION: We have elucidated molecularly distinct subclasses of lower grade diffuse astrocytic glioma that dictate clinical behavior and show fundamental associations with both IDH mutational status and neuroglial developmental stage.

Activation of the epidermal growth factor receptor (EGFR) in glioblastoma (GBM) occurs through mutations or deletions in the extracellular (EC) domain. Unlike lung cancers with EGFR kinase domain (KD) mutations, GBMs respond poorly to the EGFR inhibitor erlotinib. Using RNAi, we show that GBM cells carrying EGFR EC mutations display EGFR addiction. In contrast to KD mutants found in lung cancer, glioma-specific EGFR EC mutants are poorly inhibited by EGFR inhibitors that target the active kinase conformation (e.g., erlotinib). Inhibitors that bind to the inactive EGFR conformation, however, potently inhibit EGFR EC mutants and induce cell death in EGFR-mutant GBM cells. Our results provide first evidence for single kinase addiction in GBM and suggest that the disappointing clinical activity of first-generation EGFR inhibitors in GBM versus lung cancer may be attributed to the different conformational requirements of mutant EGFR in these 2 cancer types. SIGNIFICANCE: Approximately 40% of human glioblastomas harbor oncogenic EGFR alterations, but attempts to therapeutically target EGFR with first-generation EGFR kinase inhibitors have failed. Here, we demonstrate selective sensitivity of glioma-specific EGFR mutants to ATP-site competitive EGFR kinase inhibitors that target the inactive conformation of the catalytic domain.