David B. Solit

PTEN Loss-of-Function Alterations Are Associated With Intrinsic Resistance to BRAF Inhibitors in Metastatic Melanoma

Purpose The clinical use of BRAF inhibitors in patients with melanoma is limited by intrinsic and acquired resistance. We asked whether next-generation sequencing of pretreatment tumors could identify coaltered genes that predict for intrinsic resistance to BRAF inhibitor therapy in patients with melanoma as a prelude to rational combination strategies. Patients and Methods We analyzed 66 tumors from patients with metastatic BRAF-mutant melanoma collected before treatment with BRAF inhibitors. Tumors were analyzed for > 250 cancer-associated genes using a capture-based next-generation sequencing platform. Antitumor responses were correlated with clinical features and genomic profiles with the goal of identifying a molecular signature predictive of intrinsic resistance to RAF pathway inhibition. Results Among the 66 patients analyzed, 11 received a combination of BRAF and MEK inhibitors for the treatment of melanoma. Among the 55 patients treated with BRAF inhibitor monotherapy, objective responses, as assessed by Response Evaluation Criteria in Solid Tumors (RECIST), were observed in 30 patients (55%), with five (9%) achieving a complete response. We identified a significant association between alterations in PTEN that would be predicted to result in loss of function and reduced progression-free survival, overall survival, and response grade, a metric that combines tumor regression and duration of treatment response. Patients with melanoma who achieved an excellent response grade were more likely to have an elevated BRAF-mutant allele fraction. Conclusion These results provide a rationale for cotargeting BRAF and the PI3K/AKT pathway in patients with BRAF-mutant melanoma when tumors have concurrent loss-of-function mutations in PTEN. Future studies should explore whether gain of the mutant BRAF allele and/or loss of the wild-type allele is a predictive marker of BRAFi sensitivity.

Genomic Characterization of Upper-Tract Urothelial Carcinoma in Patients With Lynch Syndrome

Purpose Patients with Lynch syndrome (LS) have a significantly increased risk of developing upper-tract urothelial carcinoma (UTUC). Here, we sought to identify differences in the patterns of mutational changes in LS-associated versus sporadic UTUCs. Patients and Methods We performed targeted sequencing of 17 UTUCs from patients with documented LS-associated germline mutations (LS-UTUCs) using the Memorial Sloan Kettering Integrated Molecular Profiling of Actionable Cancer Targets targeted exon capture assay and compared the results with those from a recently characterized cohort of 82 patients with sporadic UTUC. Results Patients with LS-UTUC were significantly younger, had had less exposure to tobacco, and more often presented with a ureteral primary site compared with patients with sporadic UTUC. The median number of mutations per tumor was significantly greater in LS-UTUC tumors than in tumors from the sporadic cohort (58; interquartile range [IQR], 47-101 v 6; IQR, 4-10; P < .001), as was the MSIsensor score (median, 25.1; IQR, 17.9-31.2 v 0.03; IQR, 0-0.44; P < .001). Differences in the genetic landscape were observed between sporadic and LS-associated tumors. Alterations in KMT2D, CREBBP, or ARID1A or in DNA damage response and repair genes were present at a significantly higher frequency in LS-UTUC. CIC, NOTCH1, NOTCH3, RB1, and CDKN1B alterations were almost exclusive to LS-UTUC. Although FGFR3 mutations were identified in both cohorts, the R248C hotspot mutation was highly enriched in LS-UTUC. Conclusion LS- and sporadic UTUCs have overlapping but distinct genetic signatures. LS-UTUC is associated with hypermutation and a significantly higher prevalence of FGFR3 R248C mutation. Prospective molecular characterization of patients to identify those with LS-UTUC may help guide treatment.

Abstract 4078: Tumors with class 3 BRAF mutants are sensitive to the inhibition of activated RAS

Approximately two hundred mutant BRAF alleles have been identified in human tumors. Physiologic activation of RAF isoforms requires RAS-dependent induction of their dimerization. Activating BRAF mutants cause ERK dependent feedback inhibition of RAS.GTP and are RAS independent. As we have shown previously, they signal either as active monomers or RAS-independent constitutively active dimers. Here, we characterized a third class of BRAF mutants—those that have impaired kinase activity or are kinase dead. These class 3 BRAF mutants are sensitive to ERK-mediated feedback and they function in a RAS-dependent manner. In tumors, they bind more tightly to active RAS, thus enhancing their heterodimerization with CRAF. This is associated with the amplification of RAS-RAF-MEK-ERK signaling. Since these mutants are sensitive to ERK-dependent feedback inhibition of RAS, their enhancement of ERK signaling in tumors requires concurrent dysregulation of RAS activation. Thus, melanomas with Class 3 mutations usually harbor coexistent RAS mutation or NF1 mutants/deletion, whereas receptor tyrosine kinase signaling is activated in lung and colorectal cancers with these mutants. Our model suggests that these tumors will be sensitive to the inhibition of RAS activation. Currently, no direct inhibitors of RAS activation are available. However, in support of this idea, inhibitors of activated RTK signaling in carcinomas with Class 3 BRAF mutants and wild type RAS is sufficient to markedly inhibit ERK signaling and their growth in in vivo murine models and in patients. We have thus defined a third subset of BRAF mutants, which is RAS-dependent. Tumors harboring such mutants are sensitive to tyrosine kinase inhibitors in tumors expressing wild type RAS and NF1. Citation Format: Zhan Yao, Rona Yaeger, Vanessa S. Rodrik-Outmezguine, Anthony Tao, Neilawattie M. Torres, Matthew T. Chang, Matthias Drosten, Huiyong Zhao, Fabiola Cecchi, Todd Hembrough, Judith Michels, Herve Baumert, Linde Miles, Naomi M. Campbell, Elisa de Stanchina, David B. Solit, Mariano Barbacid, Barry S. Taylor, Neal Rosen. Tumors with class 3 BRAF mutants are sensitive to the inhibition of activated RAS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4078. doi:10.1158/1538-7445.AM2017-4078

Abstract 392: Delineating genomic heterogeneity in paired primary and metastatic colorectal cancer by massively parallel sequencing.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Colorectal cancer (CRC) is the third most common cause of cancer and cancer death for men and women in the United States. While the genetic steps necessary for carcinogenesis are well-defined, the subsequent genetic alterations driving tumor evolution to metastasis are not well characterized. We have performed deep sequencing of 230 key cancer-associated genes in 60 patient-matched primary tumor, metastatic tumor, and normal samples from CRC patients. Genes were selected on the basis of recurrent and/or “actionable” mutations reported in human tumors. Our custom captured-based sequencing assay, termed IMPACT (Integrated Mutation Profiling of Actionable Cancer Targets), provided a median sequence coverage of 644-fold, allowing for the identification of low frequency genetic events involving target genes. Somatic mutations, insertions/deletions (indels), and copy number alterations were identified and compared in both primary and metastatic samples. The most frequently mutated genes have all been previously implicated in CRC: APC, TP53, KRAS, PIK3CA, and SMAD4. Most genetic events were identical between primary and metastatic tumors; however, we identified particular alterations specific to the metastatic samples. The majority of these alterations were members of two important proliferative pathways, further lending credence that they might play an important role in metastatic progression. For patients with metastasis-specific mutations, we have sampled multiple microdissected regions from primary tumors to quantify the extent of intratumor heterogeneity and ascertain whether these differences are caused by sampling biases. The identification of these genetic variations between primary and metastatic tumors could have significant impact on biomarker analysis as well as treatment decisions for CRC patients. Citation Format: Angela Rose Brannon, Efsevia Vakiani, Sasinya N. Scott, Brooke Sylvester, Krishan Kania, Agnes Viale, Nancy Kemeny, Martin Weiser, David B. Solit, Michael F. Berger. Delineating genomic heterogeneity in paired primary and metastatic colorectal cancer by massively parallel sequencing. [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 392. doi:10.1158/1538-7445.AM2013-392

Abstract 4846: Genomic analysis of high grade bladder cancer: Defining genetic subtypes and therapeutic targets

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: Genomic instability, as manifested by copy number alteration (CNA) and mutation, is a hallmark of cancer. We sought to characterize the spectrum of CNAs in 97 high-grade bladder tumors (HGBT) using high-resolution comparative genomic hybridization (CGH). We then performed an integrated analysis of CNA and targeted mutation testing to construct a comprehensive profile of genomic alterations in these tumors and to identify any correlation with clinical outcome. Methods: Genomic DNA from 97 frozen HGBTs (tumor content>70% or greater) and commercially available normal human DNA (Roche) were labeled with cyanine-based fluorescent dyes and hybridized onto a one-million oligonucleotide microarray (Agilent) followed by scanning and image analysis. High-throughput Sanger sequencing of select genes was performed with validation of hotspot oncogene mutations using a mass spectrometry based multiplex Sequenom assay. Results: We found that 47% of HGBT samples possessed a high degree of CNA with widespread amplifications and deletions while 53% contained markedly fewer numbers of events. A non-overlapping pattern of mutations and amplifications was observed when events were grouped within the context of specific signal transduction or cell cycle pathways. Preliminary survival analysis suggests that patients in the high CNA group have a worse prognosis than those in the low CNA group: fifteen out of 46 patients (33%) in the high CNA group have died of their disease vs. 9 of 51 (18%) in the low CNA group. Highly focal amplification events were noted within HER2 (5% of samples), E2F3 (11%), and CCND1 (10%). Immunohistochemistry (IHC) confirmed 3+ overexpression of HER2 in 5 of 5 samples exhibiting genetic amplification. Deletion events were most frequent in CDKN2A (13%). A subset of 11 tumors with small-cell histologic features was included within the samples analyzed and 45% of these tumors possessed E2F3 amplification vs. only 8% within the non-small cell tumor set. Targeted sequencing of select genes revealed that the majority of mutation events occurred within the high-CNA HGBTs. Conclusions: Distinct genetic subsets exist within HGBT and can be classified based on CNA and mutational analysis. The finding of focal amplification events within genes known to drive tumorigenesis in other contexts, such as HER2, underscores the need for global genomic approaches in identifying targets for therapy in bladder cancer. Small-cell bladder cancer may possess a distinct genetic signature characterized by E2F3 amplification. The non-overlapping pattern of CNAs and mutations seen within specific mitogenic pathways suggests that focal driver lesions can be identified for targeted therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4846. doi:10.1158/1538-7445.AM2011-4846

Abstract 4504: Genetic determinants of AKT-dependence in epithelial ovarian cancer

Ovarian cancer is a genetically complex disease. Recent data compiled by The Cancer Genome Atlas Project highlights the heterogeneity within this disease, though distinct genetic signatures, such as PI3K pathway aberration, KRas alteration and Rb loss, are emerging that may condition treatment response. The high frequency of AKT activation in ovarian cancer, via PTEN mutation/deletion, PIK3CA/AKT mutation/amplification, RAS mutation, or mutation/amplification of upstream receptor tyrosine kinases (RTK), suggests that inhibitors of this pathway may be particularly effective treatments for this disease. We utilized a selective, allosteric inhibitor of AKT to determine the AKT-dependence of a panel of ovarian cancer cell lines. This AKT1/2 inhibitor (Merck AKTi-1/2) has been extensively tested against panels of kinases and PH-domain containing proteins and based upon these studies appears to be highly selective for AKT1 and AKT2. The drug effectively inhibited AKT activity as measured by a decrease in AKT phosphorylation at serine 473 in all cell lines. Of the 17 cell lines tested to date, 3 were hypersensitive (IC50 = 0.1um) to AKT inhibition, while 1 demonstrated intermediate sensitivity (IC50 = 2uM) and 13 were resistant (IC50 >=5uM). Levels of AKT phosphorylation at serine 473 were inversely correlated with Merck AKTi-1/2 IC50 (p = 0.036) but AKT activation alone was insufficient to confer sensitivity. Activation of the MAPK pathway, via KRas/BRaf/MEK1 mutation or KRas amplification, and Rb loss were predictive of resistance to AKT inhibition. In sensitive cell lines, AKT inhibition resulted in hypophosphorylation of RB and accumulation of cells in G1. Significant induction of apoptosis was not observed even in PTEN null, AKT2 amplified and PIK3CA mutant models. Treatment of a PTEN-mutant xenograft model of ovarian cancer with the AKTi-1/2 was also effective in inhibiting AKT phosphorylation and downstream signaling and resulted in a cytostatic tumor response. Ongoing studies are focused on the identification of rational combination strategies that result in induction of apoptosis in the cell lines in which the AKTi-1/2 alone induces growth arrest. The long-term goal will be to identify genetic predictors of AKT-dependence and AKT-inhibitor resistance to guide patient selection for future clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4504.

Abstract 2521: The pan-RAF inhibitor PLX4032 inhibits ERK signaling and tumor cell proliferation in a V600E BRAF mutant-selective manner

Activation of the RAS/RAF/MEK/ERK pathway occurs often in human cancer. Tumors with mutant BRAF and some with mutant RAS are dependent upon ERK signaling for proliferation and their growth is suppressed by selective inhibitors of MEK. In contrast, tumor cells with HER-kinase activation proliferate in a MEK-independent manner. These findings have led to the development of RAF and MEK inhibitors as anticancer agents. Like MEK inhibitors, the pan-RAF kinase inhibitor PLX4032 inhibits the proliferation of V600E BRAF mutant tumor cells, but not that of HER kinase dependent tumors. However, tumors with RAS mutation that are sensitive to MEK inhibition are insensitive to PLX4032. Whereas MEK inhibitors inhibit ERK phosphorylation in all normal and tumor cells, PLX4032 only inhibits ERK signaling in tumor cells with BRAF mutation. In contrast, the drug activates MEK and ERK phosphorylation in cells with wild-type BRAF. In mutant BRAF cells, the MEK and RAF inhibitors affect the expression of a common set of genes, whereas they have opposite effects on the expression of these genes in tumors with mutant RAS. Furthermore, PLX4032 inhibits ERK signaling output in mutant BRAF cells, but transiently activates it in tumor cells with wild-type RAF. Thus, PLX4032 inhibits ERK signaling output in a BRAF mutant-selective manner. These data suggest that PLX4032 may have a broader therapeutic index and help to explain the greater antitumor activity observed with this drug compared to MEK inhibitors, which inhibit ERK signaling in all cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2521.