Activating Mutations In PIK3CA Research Articles

Abstract 3610: Synergy between androgen receptor (AR)-targeting agents seviteronel (SEVI) or enzalutamide (ENZA) and mTOR or HER2 pathway-targeting agents in breast cancer

Abstract Background: AR is expressed in ~60% of HER2+ and up to 50% of triple negative breast cancer (TNBC) with growing evidence that AR plays a role in BC growth and survival. Next-generation AR-targeting agents including SEVI (selective CYP17-lyase and AR inhibitor) and ENZA (AR inhibitor) have shown promise in preclinical and clinical models of prostate cancer and are currently being evaluated in BC. Approved therapeutics targeting mTOR such as everolimus (EVEROL) and HER2-targeting agents provide significant clinical benefit in some BC patients; however, de novo and acquired resistance remain critical issues that may be partially attributed to compensatory AR action. Hypothesis: Due to cross-regulation between AR and the HER2/PI3K/mTOR pathway in BC, targeting AR in combination with approved agents for these pathways may improve responses. Methods: Human BC cell lines HER2+ (BT474, BT474-HR20, SKBR3) and TNBC (MDAMB453, BT549) and an AR+ TNBC mouse mammary carcinoma model (Met-1) were used. Cells were treated with multiple doses of AR-targeting agents SEVI or ENZA and EVEROL, alone or in combination and assayed over time using IncuCyte instrumentation and combination indices calculated using Calcusyn. Anchorage independence was tested by growth on soft agar. Pathway component genes and proteins were measured. In vivo, NOD/SCID mice with HER2+ trastuzumab-resistant BT474-HR20 xenografts were treated with ENZA, EVEROL, or the combination. Similar in vivo experiments with SEVI are ongoing. Results: Both SEVI and ENZA significantly inhibited proliferation of both HER2+ and TNBC cell lines. Both significantly inhibited growth on soft agar in l Met-1 cells (p=0.0005 for SEVI, p=0.032 for ENZA). Using 9 dose combinations of EVEROL with SEVI or ENZA, synergy was observed in multiple cell lines in vitro, but only in lines containing activating PIK3CA mutations. DHT induced a cell line-specific increase in pHER2 and/or pHER3 that was attenuated by AR inhibition. EVEROL alone caused an increase in total AR, pHER2, and pHER3, and these effects were also abrogated by AR inhibition. In vivo, ENZA inhibited BT474-HR20 xenografts, and combining ENZA with EVEROL decreased tumor viability more than either single agent (p<0.001, repeated measures ANOVA). Similar in vivo studies with SEVI are planned. Conclusion: Agents targeting AR synergize with EVEROL. Combination therapies targeting AR and the mTOR and HER2/HER3 pathways may provide benefit for HER2+ and TNBC patients. SEVI was a stronger inhibitor of anchorage-independent growth than ENZA and will thus be further investigated in combination with approved HER2/PI3K/mTOR pathway-directed therapeutics in vivo. This work was funded by DOD BCRP - Clinical Translational Award BC120183 W81XWH-13-1-0090 to JKR and 0091 to AE and R01 CA187733 to JKR Note: This abstract was not presented at the meeting. Citation Format: Michael A. Gordon, Nicholas D'Amato, Jessica Christenson, Beatrice Babbs, Haihua Gu, Julia Wulfkuhle, Emmanuel Petricoin, Anthoy Elias, Jennifer K. Richer. Synergy between androgen receptor (AR)-targeting agents seviteronel (SEVI) or enzalutamide (ENZA) and mTOR or HER2 pathway-targeting agents in breast cancer [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 3610. doi:10.1158/1538-7445.AM2017-3610

Safety and tolerability of the dual PI3K/mTOR inhibitor LY3023414 in combination with fulvestrant in treatment refractory advanced breast cancer patients.

1064 Background: Thephosphatidylinositol 3-kinase (PI3K) /mammalian target of rapamycin (mTOR) pathway is frequently activated in breast cancer. LY3023414 (LY) is an oral ATP- competitive inhibitor that selectively and potently inhibits class I PI3K isoforms, mTOR, and DNA-PK. The recommended phase 2 dose (RP2D) of LY monotherapy was previously established to be 200 mg twice daily (BID). Here we present the safety and preliminary activity data of LY in combination with fulvestrant (F) for breast cancer patients (pts) as part of a multi-cohort Phase 1 study. Methods: Pts with advanced HR+, HER2- breast cancer refractory to standard treatment received 200 mg LY BID + 500 mg F (day 1 and 15, then once monthly). Eligible pts had measurable disease and baseline tumor tissue available. Primary objective was to determine a RP2D. Other objectives included assessment of pharmacokinetics (PK), antitumor activity, and biomarker analysis. Results: 9 pts received LY + F in the breast cancer expansion cohort. All pts had multiple lines of prior systemic therapy (range 3-12), including chemotherapy. Dose limiting toxicity was observed in one pt in the form of grade (Gr) 3 oral mucositis. Common possibly related adverse events included nausea (5 pts), vomiting (4 pts), oral mucositis (4 pts), decreased appetite (3 pts), fatigue (3 pts), mucosal inflammation (2 pts), and paresthesia (2 pts). No obvious impact of LY on F PK or of F on LY PK was observed. Median duration of treatment was 15 weeks (range 3-63). In the 6 pts evaluable for tumor response, there was 1 durable partial response according to RECIST (still on treatment for ≥11 months) and 4 further pts had a decrease in their target lesions for a disease control rate of 56%. The median progression-free survival for this cohort is 4.2 months (90% CI 1.8, NA). Of note, the partial response was observed in a pt harboring an activating PIK3CA mutation (H1047R). Further biomarker analysis is ongoing. Conclusions: The RP2D of LY in combination with F is 200mg BID and may cause tumor regression or stabilization in breast cancer pts. Clinical trial information: NCT01655225.

Phase Ib study of cetuximab + BYL719 + IMRT in stage III-IVB head and neck squamous cell carcinoma (HNSCC).

6081 Background: Activation of the PI3K/mTOR signaling pathway is common in HNSCC. PI3K inhibitors have been shown to enhance radiosensitivity. BYL719 is an α-specific PI3K inhibitor that is synergistic and efficacious when combined with cetuximab, an FDA-approved radiosensitizing agent in HNSCC. This study evaluates the addition of BYL719 to cetuximab and radiation in the treatment of locally advanced HNSCC. Methods: This is a single-institution, phase I study. Patients with Stage III-IVB HNSCC received cetuximab 400 mg/m2 IV loading dose prior to intensity modulated radiation therapy (IMRT), followed by 250 mg/m2 weekly infusions during IMRT. BYL719 was given orally during IMRT in 3 dose levels (DLs): 1) 200 mg, 2) 250 mg, and 3) 300 mg per day in a standard 3 + 3 dose escalation design. Results: 11 patients are evaluable; median age-60 years (36-73); Stage III-1, IVA-9, IVB-1; oropharynx primary-9, unknown primary-2; HPV-positive -10. 8 patients completed treatment. 3 patients were treated on DL 1 and 2 without a dose limiting toxicity (DLT). Both patients on DL 3 experienced a DLT of grade 3 mucositis. Related adverse events (AEs) of any grade experienced by all 8 patients include: mucositis, weight loss, and hyperglycemia. Related grade 3 and 4 AEs in at least 2 patients include: mucositis (4), dysphagia (4), and decreased lymphocyte count (2). An additional 3 patients have been enrolled onto DL 2 and are undergoing treatment. All other patients had a complete response (CR) on post-treatment PET (from 6/2015 - 9/2016) and remain free of disease. Of 6 patients with mutational analysis, 1 had an activating PIK3CA mutation associated with a rapid response on serial intra-treatment MRIs. Conclusions: BYL719 administered at DLs 200 mg or 250 mg oral daily with cetuximab and IMRT resulted in expected AEs of radiation-based treatment with cetuximab and tolerable class-related AEs of PI3K inhibitors. BYL719 300 mg oral daily exceeds the maximum tolerated dose; the phase II recommended dose is pending completion of expansion of DL 2. All patients who completed treatment had a CR. Further evaluation of the addition of BYL719 to the platinum-sparing regimen of cetuximab + IMRT in the treatment of locally advanced HNSCC is warranted. Clinical trial information: NCT02282371.

Feasibility of multiplexed gene mutation detection in plasma samples of colorectal cancer patients by mass spectrometric genotyping.

Mutation analysis of circulating tumor DNA (ctDNA) has recently been introduced as a noninvasive tumor monitoring method. In this study, we tested the mass spectrometric-based MassARRAY platform for multiplexed gene mutation analysis of plasma samples from colorectal cancer (CRC) patients. A total of 160 patients, who underwent curative resection of either primary or metastatic CRC harboring KRAS mutations between 2005 and 2012, were included. Circulating DNA was isolated from plasma was analyzed on the MassARRAY platform with or without selective amplification of mutant DNA fragments. Tumor-specific KRAS mutations were detected in 39.6% (42/106) of patients with distant metastasis, and in 5.6% (3/54) of patients without distant metastasis. Selective amplification of the mutant allele increased sensitivity to 58.5% (62/106) for patients with distant metastasis, and 16.7% (9/54) for patients without distant metastasis. These mutation detection rates were no less than those of droplet digital polymerase chain reaction. Among patients with distant metastasis, detectable plasma KRAS mutations correlated with larger primary tumors and shorter overall survival rate (P = 0.014 and P = 0.003, respectively). In addition, activating PIK3CA mutations were detected together with KRAS mutations in two plasma samples. Taken together, massARRAY platform is a cost-effective, multigene mutation profiling technique for ctDNA with reasonable sensitivity and specificity.

Cell-Free DNA from Ascites and Pleural Effusions: Molecular Insights into Genomic Aberrations and Disease Biology

Collection of cell-free DNA (cfDNA) from the blood of individuals with cancer has permitted noninvasive tumor genome analysis. Detection and characterization of cfDNA in ascites and pleural effusions have not yet been reported. Herein, we analyzed cfDNA in the ascites and pleural effusions from six individuals with metastatic cancer. In all cases, cfDNA copy number variations (CNV) were discovered within the effusate. One individual had a relevant alteration with a high copy amplification in EGFR in a never smoker with lung cancer, who showed only MDM2 and CDK4 amplification in a prior tissue biopsy. Another subject with metastatic breast cancer had cytology-positive ascites and an activating PIK3CA mutation identified in the tissue, blood, and ascites collectively. This individual had tumor regression after the administration of the mTOR inhibitor everolimus and had evidence of chromotripsis from chromosomal rearrangements noted in the cell-free ascitic fluid. These results indicate that cfDNA from ascites and pleural effusions may provide additional information not detected with tumor and plasma cell-free DNA molecular characterization, and a context for important insights into tumor biology and clonal dynamic change within primary tumor and metastatic deposits. Mol Cancer Ther; 16(5); 948-55. ©2017 AACR.

PIK3CA-mutated melanoma cells rely on cooperative signaling through mTORC1/2 for sustained proliferation.

Malignant conversion of BRAF- or NRAS-mutated melanocytes into melanoma cells can be promoted by PI3'-lipid signaling. However, the mechanism by which PI3'-lipid signaling cooperates with mutationally activated BRAF or NRAS has not been adequately explored. Using human NRAS- or BRAF-mutated melanoma cells that co-express mutationally activated PIK3CA, we explored the contribution of PI3'-lipid signaling to cell proliferation. Despite mutational activation of PIK3CA, melanoma cells were more sensitive to the biochemical and antiproliferative effects of broader spectrum PI3K inhibitors than to an α-selective PI3K inhibitor. Combined pharmacological inhibition of MEK1/2 and PI3K signaling elicited more potent antiproliferative effects and greater inhibition of the cell division cycle compared to single-agent inhibition of either pathway alone. Analysis of signaling downstream of MEK1/2 or PI3K revealed that these pathways cooperate to regulate cell proliferation through mTORC1-mediated effects on ribosomal protein S6 and 4E-BP1 phosphorylation in an AKT-dependent manner. Although PI3K inhibition resulted in cytostatic effects on xenografted NRASQ61H /PIK3CAH1047R melanoma, combined inhibition of MEK1/2 plus PI3K elicited significant melanoma regression. This study provides insights as to how mutationally activated PIK3CA acts in concert with MEK1/2 signaling to cooperatively regulate mTORC1/2 to sustain PIK3CA-mutated melanoma proliferation.

Syndromes hypertrophiques secondaires aux mutations de PIK3CA

This review presents an overview of a recently characterized spectrum of overgrowth syndrome: phosphoinositide-3 kinase (PI3K)-related overgrowth spectrum (PROS). This spectrum encompasses overgrowth syndromes associated with somatic mosaic activating PIK3CA mutations such as megalencephaly-capillary malformation (MCAP) syndrome, dysplatic megalencephaly (DMEG), congenital lipomatous asymmetric overgrowth of the trunk, lymphatic, capillary, venous, and combined-type vascular malformations, epidermal nevi, skeletal and spinal anomalies (CLOVES) syndrome, hemihyperplasia-multiple lipomatosis (HHML), fibroadipose overgrowth and Klippel-Trenaunay syndrome. Mosaic gain of function mutation in PIK3CA gene leads to abnormal AKT-mTOR pathway activation and is responsible of the clinical manifestations. Here, we summarize the current knowledge on this disorder.

MYOD1 (L122R) mutations are associated with spindle cell and sclerosing rhabdomyosarcomas with aggressive clinical outcomes

Recurrent mutations in the myogenic transcription factor MYOD1 and PIK3CA were initially described in a subset of embryonal rhabdomyosarcomas. Recently, two independent studies demonstrated presence of MYODI (L122R) mutations as the basis to re-classify a spindle cell rhabdomyosarcoma, along with a sclerosing rhabdomyosarcoma, distinct from an embryonal rhabdomyosarcoma. We analyzed a much larger cohort of 49 primary rhabdomyosarcoma tumor samples of various subtypes, collected over a period of 9 years, for the presence of MYOD1 (L122R), PIK3CA (H1047), and PIK3CA (E542/E545) mutations, along with immunohistochemical analysis of desmin, myogenin, and MYOD1. Although activating PIK3CA mutations were absent across the sample set analyzed, we report 20% MYOD1 (L122R) mutation in rhabdomyosarcomas, found exclusively in 10 of 21 spindle cell and sclerosing rhabdomyosarcomas, occurring mostly in the head and neck region along with extremity sites (64%), than the paratesticular and intra-abdominal sites. Furthermore, while all 10 MYOD1 mutant spindle cell and sclerosing rhabdomyosarcoma samples showed diffuse and strong MYOD1 immunoexpression, 7 of 31 samples of rhabdomyosarcoma with wild-type MYOD1 were negative for MYOD1 expression. Clinically, a striking correlation was found between MYOD1 mutation and the clinical outcomes available for 15 of 21 cases: 5 of 7 patients with spindle cell and sclerosing rhabdomyosarcomas, harboring MYOD1 mutation, were alive-with-disease and 2 of 8 patients with spindle cell and sclerosing rhabdomyosarcomas, with mutant MYOD1, were free-of-disease. Taken together, we present the first report of MYOD1 (L122R) mutation in the largest cohort of 49 rhabdomyosarcomas reported so far, that are associated with a relatively aggressive clinical course. Moreover, consistent with the earlier two studies, this study further reinforces a relationship between spindle cell and the sclerosing rhabdomyosarcoma—now recognized as a single subtype, distinct from an embryonal rhabdomyosarcoma.

PIK3CA mutation in a mixed dysembryoplastic neuroepithelial tumor and rosette forming glioneuronal tumor, a case report and literature review

BackgroundRosette forming glioneuronal tumors are rare, World Health Organization (WHO) grade I novel tumors frequently affecting the fourth ventricle or posterior fossa with typical neuronal pseudorosettes. RGNTs have been described as possessing additional histologic features of DNETs or pilocytic astrocytomas. Activating PIK3CA mutations have been identified as recurring genetic event in RGNTs. MethodsWe report a 35year old man who presented with binocular diplopia, headache, and was found to have a third ventricle tumor. Tumor pathology and oncogene evaluation were conducted. ResultsThe tumor demonstrated histologic features consistent with mixed RGNT/DNET. Genetic studies revealed a PIK3CA mutation in exon 9 (E545K, C. 1633G>A) without IDH1, p53, 1p19q chromosomal co-deletion, or BRAF mutations. A literature search revealed six cases of PIK3CA mutations in RGNTs and seven cases of mixed RGNT/DNET. No cases of mixed RGNT/DNET with a PIK3CA mutation have been described. ConclusionThis is the first documented case of an RGNT/DNET with an activating PIK3CA mutation. The presence of a PIK3CA mutation aids histologic classification in the setting of mixed histology, and may have implications for targeting the PI3K/AKT/mTOR pathway in this tumor type.

Genomic profiling is predictive of response to cisplatin treatment but not to PI3K inhibition in bladder cancer patient-derived xenografts.

PurposeEffective systemic therapeutic options are limited for bladder cancer. In this preclinical study we tested whether bladder cancer gene alterations may be predictive of treatment response.Experimental designWe performed genomic profiling of two bladder cancer patient derived tumor xenografts (PDX). We optimized the exome sequence analysis method to overcome the mouse genome interference.ResultsWe identified a number of somatic mutations, mostly shared by the primary tumors and PDX. In particular, BLCAb001, which is less responsive to cisplatin than BLCAb002, carried non-sense mutations in several genes associated with cisplatin resistance, including MLH1, BRCA2, and CASP8. Furthermore, RNA-Seq analysis revealed the overexpression of cisplatin resistance associated genes such as SLC7A11, TLE4, and IL1A in BLCAb001. Two different PIK3CA mutations, E542K and E545K, were identified in BLCAb001 and BLCAb002, respectively. Thus, we tested whether the genomic profiling was predictive of response to a dual PI3K/mTOR targeting agent, LY3023414. Despite harboring similar PIK3CA mutations, BLCAb001 and BLCAb002 exhibited differential response, both in vitro and in vivo. Sustained target modulation was observed in the sensitive model BLCAb002 but not in BLCAb001, as well as decreased autophagy. Interestingly, computational modelling of mutant structures and affinity binding to PI3K revealed that E542K mutation was associated with weaker drug binding than E545K.ConclusionsOur results suggest that the presence of activating PIK3CA mutations may not necessarily predict in vivo treatment response to PI3K targeted therapies, while specific gene alterations may be predictive for cisplatin response in bladder cancer models and, potentially, in patients as well.

Frequent PIK3CA activating mutations in nipple adenomas.

Nipple adenoma (NA) is a rare benign epithelial tumour occurring in the nipple. Histologically, it exhibits variable and often mixed adenosis-like and usual ductal hyperplasia-like growth patterns. Morphologically, it is similar to other benign proliferative breast lesions occurring in the breast parenchyma, which have been shown to harbour activating mutations in PIK3CA, AKT1 or, less frequently, in RAS in more than 50% of cases. In this study, we aimed to analyse the mutation status of PIK3CA, AKT1, RAS and BRAF in NAs and correlated the mutation status with the histological features. Mutation analysis of PIK3CA, AKT1, RAS and BRAF was performed in 24 NAs by Sanger sequencing. Our results showed that activating PIK3CA mutations were identified in eight of the 15 NAs (53%) with a predominantly adenosis-like pattern and four of the nine NAs (44%) with a predominantly usual ductal hyperplasia-like pattern. One tumour with a PIK3CA H1047R mutation also had a KRAS Q61H mutation. Two tumours with an adenosis-like pattern had BRAF V600E mutations. Overall, half of the NAs (12 of 24, 50%) in our series had PIK3CA mutations and 58% (14 of 24) had PIK3CA, RAS or BRAF mutations. Our data indicate that, similar to other benign proliferative lesions occurring in the breast parenchyma, activating PIK3CA mutations are very common in NAs, and KRAS mutation may occur concurrently with PIK3CA mutation. In addition, as BRAF mutation has not been identified in benign proliferative lesions in previous studies, BRAF-mutated NAs appear to have distinct pathogenesis.

PIK3CA dependence and sensitivity to therapeutic targeting in urothelial carcinoma.

BackgroundMany urothelial carcinomas (UC) contain activating PIK3CA mutations. In telomerase-immortalized normal urothelial cells (TERT-NHUC), ectopic expression of mutant PIK3CA induces PI3K pathway activation, cell proliferation and cell migration. However, it is not clear whether advanced UC tumors are PIK3CA-dependent and whether PI3K pathway inhibition is a good therapeutic option in such cases.MethodsWe used retrovirus-mediated delivery of shRNA to knock down mutant PIK3CA in UC cell lines and assessed effects on pathway activation, cell proliferation, migration and tumorigenicity. The effect of the class I PI3K inhibitor GDC-0941 was assessed in a panel of UC cell lines with a range of known molecular alterations in the PI3K pathway.ResultsSpecific knockdown of PIK3CA inhibited proliferation, migration, anchorage-independent growth and in vivo tumor growth of cells with PIK3CA mutations. Sensitivity to GDC-0941 was dependent on hotspot PIK3CA mutation status. Cells with rare PIK3CA mutations and co-occurring TSC1 or PTEN mutations were less sensitive. Furthermore, downstream PI3K pathway alterations in TSC1 or PTEN or co-occurring AKT1 and RAS gene mutations were associated with GDC-0941 resistance.ConclusionsMutant PIK3CA is a potent oncogenic driver in many UC cell lines and may represent a valuable therapeutic target in advanced bladder cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2570-0) contains supplementary material, which is available to authorized users.

Abstract 4666: Phosphatidylinositol 3-kinase (PI3K) and mTOR inhibitors demonstrate broad efficacy and synergy in head and neck cancer cell lines and patient-derived xenografts

Abstract Background: There is an urgent need for improved therapeutics in head and neck squamous cell cancer (HNSCC) to improve survival and decrease treatment morbidity. The Phosphatidylinositol 3-kinase (PI3K) pathway is frequently altered in HNSCC, particularly in human papillomavirus (HPV) positive disease. PI3K pathway inhibitors are under active investigation in preclinical models and clinical trials, however it is not clear which patient population will benefit most from these agents. Methods: Thirty unique HNSCC cell lines including five HPV positive lines were characterized with whole exome sequencing and copy number arrays. All lines were treated over a 10-point dose range with a PIK3CA specific inhibitor (BYL719), a pan PI3K inhibitor (GDC-0941), a combined pan-PI3k and mTOR inhibitor (BEZ-235), mTOR inhibitor (everolimus) and a combination of BYL719 and everolimus. Five unique patient derived xenografts (PDX) were treated with BYL719 and everolimus alone and in combination. Results: The selective PIK3CA inhibitor BYL719 was preferentially active in cell lines with PIK3CA activating mutations and amplifications (>100% relative PIK3CA amplification), however HRAS mutant lines were resistant. These genomic markers of sensitivity and resistance did not apply to the pan-PI3K inhibitor GDC-0941 or combined PI3K-mTOR inhibitor BEZ-235, however these agents were more active in HPV-positive cell lines (P<0.05). BYL719 and everolimus were individually effective at decreasing tumor growth in four PDX models including a model with an activating PIK3CA mutation, two PIK3CA amplified models and a PIK3CA wild-type model. Combined PIK3CA and mTOR inhibition appeared to be synergistic and evolved resistance never developed to this treatment, however the combination caused significant weight loss in the PDX models. Interpretation: PI3K inhibitors were broadly active in HNSCC cell lines and PDXs and their application should not be solely restricted to tumors with activating PIK3CA mutations or amplifications. Agents with broader isoform activity and combined PI3K and mTOR treatment were more potent, however combined inhibitor treatment appeared toxic in the PDX models. This trade off of toxicity and efficacy needs to balanced in the clinical setting. Citation Format: Anthony C. Nichols, Laurie Ailkes, Ren Sun, Morgan Black, Alessandro Datti, Frederick Vizeacoumar, Nicole Pinto, Kara Ruicci, John Yoo, Kevin Fung, Danielle MacNeil, Joe Mymryk, Paul C. Boutros, John W. Barrett. Phosphatidylinositol 3-kinase (PI3K) and mTOR inhibitors demonstrate broad efficacy and synergy in head and neck cancer cell lines and patient-derived xenografts. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4666.

Abstract 4509: Clinical genomic profiling of 1000 metastatic breast cancer patients: actionable targets, novel alterations, and clinical correlations

Abstract Most large genomic profiling efforts in breast cancer have focused on primary breast tumors. Profiling of metastatic breast cancer (MBC) could more accurately define actionable genomic alterations and reveal novel alterations that arise under the selective pressure and clonal evolution of prior therapy. Utilizing a 410-gene targeted capture-based sequencing platform (MSK-IMPACT), we analyzed 920 tumors (62% metastatic, 38% primary) from 874 MBC patients for somatic mutations, DNA copy number alterations, and structural rearrangements (planned final analysis 1000 patients). Detailed clinical data including treatment outcomes was collected for all patients. The cohort was representative of the well characterized clinical subtypes of breast cancer with 71% ER+ or PR+ and HER2-, 17% HER2+, and 12% triple negative breast cancer (TNBC). Our analysis revealed recurrent alterations in multiple pathways including PI3K/AKT/mTOR (56%), cell cycle regulation (42%), RTK signaling (40%), epigenetic regulation (33%) and MAPK/ERK (19%) pathways. The most frequent actionable alterations include: PIK3CA mutation (36%), ERBB2 amplification (15%), FGFR1 amplification (12%), ESR1 mutation (11%), PTEN mutation/deletion (10%), AKT1 mutation (6%), and ERBB2 mutation (5%). The genomic landscape was significantly different across breast cancer subtypes. For example, the PI3K/AKT/mTOR pathway was altered in ER+ MBC mainly through activating PIK3CA mutations whereas PTEN deletion/mutations were most common in TNBC. We also identified significant differences in the genomic profiles of metastatic and primary tumor samples. Gene enrichment analyses revealed a subset of genes more frequently altered in metastatic tumors (STK11: 13 vs 2; ROS1: 14 vs 2; FGFR4: 15 vs 1) suggesting that mutations in these genes may play a role in breast tumor metastasis and/or therapy resistance. ESR1 mutations were predominantly present in metastatic tumors (88%) and were significantly associated with duration of prior hormonal therapy (P<0.0001). ESR1 mutations were also associated with a poor response to the ER degrader fulvestrant (median PFS: 4.8 vs 13.7 months, mutated vs wild type; P = 0.01). Analysis for potentially novel hotspot mutations revealed recurrent RHOA G17 mutations in 6 MBC patients, nominating RHOA, a GTPase transducer of membrane receptors, as a candidate driver in a subset of breast cancers. While identified previously in other tumors, RHOA G17 has never been implicated in breast cancer. In summary, our genomic analyses of this large cohort of MBC patients revealed actionable alterations in over 60% of the patients. 29% of patients with these alterations were enrolled in clinical trials of matched targeted therapies to date (PIK3CA 27%, AKT1 30%, ESR1 23%, ERBB2 39%) suggesting that prospective genomic characterization can accelerate enrollment of patients with MBC onto therapeutic clinical trials. Citation Format: Pedram Razavi, Matthew T. Chang, Sumit Middha, Dara S. Ross, Ahmet Zehir, Tracy A. Proverbs-Singh, Cyriac Kandoth, Sarat Chandarlapaty, Maura N. Dickler, Jorge S. Reis-Filho, Sujata Patil, Venkatraman Seshan, Lillian Smyth, Neil M. Iyengar, Komal Jhaveri, Shanu Modi, Chau T. Dang, Mark E. Robson, Larry Norton, Clifford A. Hudis, Marc Ladanyi, Maurizio Scaltriti, Nikolaus Schultz, David Hyman, Michael F. Berger, Barry S. Taylor, David B. Solit, José Baselga. Clinical genomic profiling of 1000 metastatic breast cancer patients: actionable targets, novel alterations, and clinical correlations. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4509.

Abstract 29: MEK and PI3K signaling cooperate through mTORC1/2 to promote PIK3CA mutant melanoma cell proliferation

Abstract Oncogenic transformation of mutationally activated BRAF or NRAS melanocytes often requires the cooperation of genetic aberrations in the phosphoinositide 3-kinase (PI3K) pathway to promote melanomagenesis. Activating point mutations in the p110α catalytic subunit of PI3K are detected at a low frequency in both BRAF and NRAS mutant melanomas, yet the principle mechanism of this cooperation remains elusive. Thus, using BRAFV600E/PIK3CAH1047R, NRASQ61H/PIK3CAH1047R, and PIK3CAE545K mutant melanoma cells derived from metastatic melanoma patients treated with pathway-targeted inhibitors, we examined the contribution of mutational activation of PIK3CA to melanoma maintenance and signaling and the consequential response of these PIK3CA mutant cells to α-specific PI3K inhibition. Combined MEK and isoform-selective PI3K inhibition elicited more potent anti-proliferative effects and greater suppression of S-phase progression of the cell cycle compared to single-agent inhibition of either pathway. Analysis of signaling downstream of MEK or PI3K revealed that these pathways cooperated to regulate PIK3CA cell proliferation through mTORC1-mediated effects on ribosomal protein S6 and 4E-BP1 phosphorylation in an AKT-dependent manner. Despite the profound anti-proliferative and biochemical effects of α-specific or PI3Kβ-sparing class I PI3K inhibition in vitro, these agents elicited largely cytostatic effects on PIK3CA xenograft tumors. However, combined inhibition of PI3Kβ-sparing class I PI3K and MEK did significantly cooperate to reduce tumor growth compared to the corresponding monotherapies. Furthermore, this study provides a biochemical mechanism to explain how mTORC1/2 moderates the cooperation of MEK and PI3K signaling for the maintenance of PIK3CA mutant melanoma cell proliferation. Citation Format: Jillian M. Silva, Marian M. Deuker, Bruce C. Baguley, Martin McMahon. MEK and PI3K signaling cooperate through mTORC1/2 to promote PIK3CA mutant melanoma cell proliferation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 29.

Abstract 242: Deciphering the role of PTEN as a predictive biomarker to next generation isoform-selective PI3K inhibitors in PIK3CA mutant breast cancer cells

Abstract The phosphoinositide-3 kinase (PI3K) pathway is one of the most frequently activated signaling pathways in human cancer and represents a promising therapeutic target. Taselisib (GDC-0032), a potent and selective inhibitor of the PI3K alpha, delta and gamma isoforms, but less potent in the beta isoform, displays increased sensitivity in models that are driven by mutant PIK3CA (Ndubaku CO et al, J Med Chem, 2013). Selective pressure caused by, small molecules such as PI3K inhibitors will ultimately lead to clinical resistance and disease progression. Recently, a published mechanism of acquired resistance to the PI3K inhibitor alpelisib (BYL-719, a PI3K alpha-selective inhibitor) reported that genomic loss of the tumor suppressor PTEN led to clinical progression in a metastatic breast cancer patient harboring an activating PIK3CA mutation (Juric D et al, Nature, 2014). As taselisib and alpelisib are next generation PI3K inhibitors, we sought to determine if there are any differences to drug sensitivity in PIK3CA mutant cells that have concomitant loss of PTEN. To investigate this hypothesis, we first measured viability in breast cancer cells, each with differing genetic backgrounds treated with two PI3K inhibitors, taselisib and alpelisib. We found that PIK3CA mutant and PIK3CA mutant/PTEN null cells were sensitive to treatment when compared to wild-type and PTEN null cells. However, PIK3CA mutant/PTEN null cells displayed higher IC50 values for both inhibitors when compared to PIK3CA mutant cells. To control for differences in genetic backgrounds, we next tested whether knockdown (KD) of PTEN expression in PIK3CA mutant breast cancer cells would promote resistance and reactivate the PI3K signaling cascade even in the presence of a PI3K inhibitor. Transient, siRNA-targeted KD of PTEN in the PIK3CA mutant breast cancer cell lines EFM19 and T47D conferred resistance to alpelisib after 6 days of drug treatment, an observation that was less pronounced in taselisib treated cells. Additionally, PTEN KD in DMSO-treated cells led to an increase in cell proliferation and enhanced downstream PI3K signaling as measured by phosphorylated AKT and S6 proteins when compared to non-targeting siRNA controls. The reactivated, pro-survival signaling with PTEN KD was maintained upon treatment with both inhibitors when compared to control cells, an observation that was more pronounced in alpelisib treated cells. These data suggest that PIK3CB can compensate for PIK3CA inhibition and that PTEN loss could lead to clinical resistance to alpelisib, but may not constitute a resistance mechanism to taselisib. As both molecules have entered phase 3 studies in estrogen receptor positive breast cancer, understanding how biomarker-defined genotypes respond to next generation PI3K inhibitors is critical for identifying patients most likely to gain therapeutic benefit. Citation Format: Heather M. Moore, Heidi M. Savage, Mark R. Lackner, Timothy R. Wilson. Deciphering the role of PTEN as a predictive biomarker to next generation isoform-selective PI3K inhibitors in PIK3CA mutant breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 242.

Abstract 2278: Next-generation sequencing (NGS) analysis from a phase II single-agent gedatolisib study in patients with endometrial cancer

Abstract Genetic alterations in the PI3K pathway are abundant in endometrial cancer. Hence it is hypothesized that PI3K/mTOR inhibitors will have utility for treatment of endometrial cancer. Gedatolisib, also known as PF-384, is a potent and selective dual PI3K-mTOR inhibitor with broad anti-tumor activity in preclinical studies. Gedatolisib delivered weekly intravenously was investigated in a Phase II single-agent study of advanced endometrial cancer. The cumulative clinical benefit rate from 38 response evaluable patients was 39.5%, comparable to historical rates with mTOR inhibitors. A retrospective NGS analysis of archival biopsies from patients in the study was conducted using the Foundation Medicine Inc (FMI) Foundation One Test to explore potential predictive biomarkers for clinical benefit. Of the 26 patient samples submitted for analysis, data was obtained from 19 samples of which 17 had evaluable response data. Of these 17 samples, 6 were from patients who exhibited progressive disease (PD), 6 were from patients with stable disease (SD) and 5 were from patients that showed a partial response (PR) to treatment. In general, the best responders had a low mutation load while the worst responders had a higher mutation load. Whether large mutation burden was due to mismatch-repair defects determined by MSI status of the tumors is currently under investigation. PTEN alterations were found in tumors from patients that exhibited PD and SD, but not PR. Multiple genetic alterations in ARID1A were observed in 3 of 6 tumors from patients with PD. While PIK3CA alterations were frequently present, 2 of 5 patients with PR and 1 with SD exhibited an activating PIK3CA mutation at H1047R. Among 175 genes that were observed to be mutated in the analysis of 17 independent tumor samples, the top ranking genes associated with a reduction in tumor size (% change from baseline by RECIST 1.0; Wilcoxon rank sum test) were MAP3K1 (p = 0.027) and CTNNB1 (p = 0.050). Activating mutations in β-catenin were observed in tumor from 3 of 5 patients with PR and were not present in tumor from patients with PD or SD. Tumor from a patient who exhibited an outlier clinical response (stayed on study after 2 years on treatment) had an activating mutation in Akt at E17K and also a novel mutation in mTOR at F2184L. Computational modeling analysis revealed that the mutation did not have a significant impact on kinase structure or ligand binding. KRAS mutations did not appear to correlate with clinical response. This study highlights both the wealth of information provided by NGS analysis, but also the complexity of NGS data analysis and interpretation. Citation Format: Nuzhat Pathan, Patricia English, Keith Ching, Stephen Huang, Mehran Jalaie, Kristen Pierce, Jennifer Vermette. Next-generation sequencing (NGS) analysis from a phase II single-agent gedatolisib study in patients with endometrial cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2278.

380 - Relevance of TERT promoter mutations in poorly differentiated and anaplastic thyroid carcinomas. Association with RAS, BRAF or PIK3CA activating mutations

380 - Relevance of TERT promoter mutations in poorly differentiated and anaplastic thyroid carcinomas. Association with RAS, BRAF or PIK3CA activating mutations

Incorporation of Next-Generation Sequencing into Routine Clinical Care to Direct Treatment of Head and Neck Squamous Cell Carcinoma.

The clinical impact of next-generation sequencing (NGS) in patients with head and neck squamous cell carcinoma (HNSCC) has not been described. We aimed to evaluate the clinical impact of NGS in the routine care of patients with HNSCC and to correlate genomic alterations with clinical outcomes. Single-center study examining targeted NGS platform used to sequence tumor DNA obtained from 213 HNSCC patients evaluated in outpatient head and neck oncology clinic between August 2011 and December 2014. We correlated tumor genomic profiling results with clinical outcomes. PI3K/RTK pathway activation occurred frequently [activating PIK3CA mutation or amplification (13%), PTEN inactivation (3%), RAS activation (6%), EGFR or ERBB2 activation (9%)]. Alterations in pathways affecting cell-cycle regulation [CCND1 amplification (9%), CDKN2A inactivation (17%), BRCA2 inactivation (2%)] and squamous differentiation [NOTCH1 inactivation (8%) andEP300 inactivation (6%)] were identified. PIK3CA amplification (n = 43), not PIK3CA mutation, was associated with significantly poorer progression-free survival (P = 0.0006). Oncogenic RAS mutations (n = 13) were associated with significantly poorer progression-free survival (P = 0.0001) and lower overall survival (P = 0.003). Eight patients with advanced, treatment-refractory HNSCC enrolled on clinical trials matched to tumor profiling results, and 50% achieved a partial response. Incorporation of NGS clinical assays into the routine care of patients with HNSCC is feasible and may readily facilitate enrollment into clinical trials of targeted therapy with a higher likelihood of success. Data can be utilized for discovery of genomic biomarkers of outcome. PIK3CA amplification and RAS mutations were frequently identified and associated with poorer prognosis in this cohort. Clin Cancer Res; 22(12); 2939-49. ©2016 AACR.

Genomic insights into head and neck cancer.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide and is frequently impervious to curative treatment efforts. Similar to other cancers associated with prolonged exposure to carcinogens, HNSCCs often have a high burden of mutations, contributing to substantial inter- and intra-tumor heterogeneity. The heterogeneity of this malignancy is further increased by the rising rate of human papillomavirus (HPV)-associated (HPV+) HNSCC, which defines an etiological subtype significantly different from the more common tobacco and alcohol associated HPV-negative (HPV-) HNSCC. Since 2011, application of large scale genome sequencing projects by The Cancer Genome Atlas (TCGA) network and other groups have established extensive datasets to characterize HPV- and HPV+ HNSCC, providing a foundation for advanced molecular diagnoses, identification of potential biomarkers, and therapeutic insights. Some genomic lesions are now appreciated as widely dispersed. For example, HPV- HNSCC characteristically inactivates the cell cycle suppressors TP53 (p53) and CDKN2A (p16), and often amplifies CCND1 (cyclin D), which phosphorylates RB1 to promote cell cycle progression from G1 to S. By contrast, HPV+ HNSCC expresses viral oncogenes E6 and E7, which inhibit TP53 and RB1, and activates the cell cycle regulator E2F1. Frequent activating mutations in PIK3CA and inactivating mutations in NOTCH1 are seen in both subtypes of HNSCC, emphasizing the importance of these pathways. Studies of large patient cohorts have also begun to identify less common genetic alterations, predominantly found in HPV- tumors, which suggest new mechanisms relevant to disease pathogenesis. Targets of these alterations including AJUBA and FAT1, both involved in the regulation of NOTCH/CTNNB1 signaling. Genes involved in oxidative stress, particularly CUL3, KEAP1 and NFE2L2, strongly associated with smoking, have also been identified, and are less well understood mechanistically. Application of sophisticated data-mining approaches, integrating genomic information with profiles of tumor methylation and gene expression, have helped to further yield insights, and in some cases suggest additional approaches to stratify patients for clinical treatment. We here discuss some recent insights built on TCGA and other genomic foundations.