PI3K Pathway Mutations Research Articles

A multicenter, single‐arm, open‐label, phase 2 study of apitolisib (GDC‐0980) for the treatment of recurrent or persistent endometrial carcinoma (MAGGIE study)

The current single-arm, open-label trial was designed to evaluate the activity of apitolisib (GDC-0980), a dual phosphoinositide 3-kinase/mammalian target of rapamycin (PI3K/mTOR) inhibitor, in patients with advanced endometrial cancer (EC). Patients with recurrent or persistent EC who were treated with 1 to 2 prior lines of chemotherapy but no prior PI3K/mTOR inhibitor received oral apitolisib at a dose of 40 mg daily during 28-day cycles until disease progression or intolerable toxicity occurred. Patients with type I/II diabetes who required insulin were excluded. The primary endpoints were progression-free survival (PFS) at 6 months and objective response rate. A total of 56 women were enrolled, including 13 (23%) with well-controlled diabetes. Reasons for discontinuation were disease progression (24 patients; 43%), adverse events (13 patients; 23%), and withdrawal by subject (12 patients; 21%). Grade 3/4 apitolisib-related adverse events were hyperglycemia (46%), rash (30%), colitis (5%), and pneumonitis (4%) (toxicities were graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events [version 4.0]). The PFS rate at 6 months was 20% (Kaplan-Meier estimate; 95% confidence interval [95% CI], 7%-33%). The objective response rate was 6% (confirmed). The median PFS was 3.5 months (95% CI, 2.7-3.7 months) and the median overall survival was 15.7 months (95% CI, 9.2-17.0 months). Nineteen patients discontinued the study before the first tumor assessment. Dose reductions were required for 4 diabetic (31%) and 18 nondiabetic (42%) patients. Comprehensive molecular profiling of 46 evaluable archival tumor samples demonstrated that 57% of patients had at least 1 alteration in phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), phosphatase and tensin homolog (PTEN), or AKT1. All 3 patients with a confirmed response had at least 1 alteration in a PI3K pathway gene. The antitumor activity noted with the use of a dose of 40 mg of apitolisib daily was limited by tolerability, especially in diabetic patients. Patients with PI3K pathway mutations may have derived enhanced benefit from apitolisib. Cancer 2016;122:3519-28. © 2016 American Cancer Society.

Mapping cancer signaling networks by an integrated multiplexed tissue imaging platform

Aberrant cellular signaling networks are implicated in major diseases including cancer, but are difficult to reliably quantitate, as many signaling proteins are expressed at low abundance and further reduced following specimen collection. MTIP is an integrated tissue-imaging platform that leverages bright, fluorescent reporters and sensitive spectral instrumentation, along with automated staining, image acquisition and non-parametric image analysis, to attain reproducible, multiplexed quantitation of signaling proteins in tissue. MTIP captured the phosphoactivity of six key PI3K/MAPK proteins (pAKTS473, pAKT308, pPRAS40, pS6, peIF4G and pERK1/2) at high precision (coefficient of variation, CV <10%), four-log dynamic range and subcellular resolution. We demonstrated the MTIP platform's capability to capture a diversity of PI3K/MAPK networks present in breast tumors. These protein networks are heterogeneously distributed across the tumor tissue and associated with subgroups of cells and underscore the importance of accessing information about signaling networks in spatially intact tissue. Analysis of PI3K/MAPK networks by hierarchical clustering showed that PI3K/MAPK networks do not strictly correlate with PI3K pathway mutations, also pointing to the value of functional signaling network profiling together with genomic information. MTIP's reliable quantitative capability can be applied to guide therapeutic development and selection in precision medicine.

Abstract 138: Solid papillary carcinoma with reverse polarization are driven by IDH2 and PI3K pathway mutations

Abstract Background: Solid papillary carcinoma with reverse polarization (SPCRP) is a rare breast cancer subtype, whose most striking morphologic feature is the presence of back-to-back columnar epithelial cells with nuclei situated in an apical rather than in the normal basal location. We sought to characterize the morphologic and genetic landscape of SPCRP and determine whether it represents a distinct subtype of breast cancer underpinned by highly recurrent disease-specific genetic alterations. Material and Methods: Archival sections of 13 SPCRPs were subjected to microdissection. DNA samples extracted from microdissected tumor and matched normal samples were subjected to whole exome sequencing (n = 2) on an Illumina HiSeq2000, and DNA samples from tumors were further subjected to targeted massively parallel sequencing (n = 1), SNaPshot profiling (n = 7) and/or Sanger sequencing. State-of-the-art bioinformatics methods were used to define somatic mutations. Non-malignant breast epithelial MCF10A cells with and without somatic PIK3CA H1047R knock-in were used to assess the functional impact of the mutations identified by sequencing of SPCRPs in two- and three-dimensional cell culture systems. Results: Ten of 13 (77%) SPCRPs were found to harbor IDH2 R172 hotspot mutations, and eight of these cases had concurrent pathogenic mutations affecting PIK3CA or PIK3R1, in particular PIK3CA H1047R hotspot mutations. Functional monolayer and three-dimensional cell culture studies demonstrated that IDH2 R172 and PIK3CA mutations constitute likely drivers of this tumor and contribute to its unusual nuclear polarization phenotype. Conclusions: Our results suggest that SPCRP is a distinct subtype of breast cancer underpinned by IDH2 hotspot mutations in conjunction with mutations affecting canonical genes of the PI3K pathway. Given that IDH2 hotspot mutations have not been described in breast cancer to date, these may be used as a diagnostic ancillary marker for SPCRPs. Furthermore, IDH2 mutations may serve as a potential therapeutic target in SPCRP patients with disseminated disease. Citation Format: Sarah Chiang, Britta Weigelt, Huei-Chi Wen, Salvatore Piscuoglio, Luciano G. Martelotto, Charlotte K Y Ng, Jörg Balss, Gabrielle Baker, Kimberly S. Cole, Andreas von Deimling, Julie M. Batten, Jonathan D. Marotti, Hwei-Choo Soh, Benjamin L. McCalip, Raymond S. Lim, Kalliopi P. Siziopikou, Randi Burke, Song Lu, Xiaolong Liu, Tarek Hammour, Edi Brogi, A John Iafrate, Jorge S. Reis-Filho, Stuart J. Schnitt. Solid papillary carcinoma with reverse polarization are driven by IDH2 and PI3K pathway mutations. [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 138.

Abstract 434: Rational molecular assessment and innovative drug selection (RAIDs): exome data from cervical cancer

Abstract Background Recent retrospective data1,2 identified major molecular alterations in cervical cancer (CC), but so far there has been no prospective assessment on patient outcome using a complete molecular profiling with quality control evaluation of treatment. The Cetuxicol (phase 2) clinical trial showed that the addition of Cetuximab over a 6 week period, did not improve DFS. PI3K pathway mutations in the tumor in the Cetuximab treatment arm led to a worse DFS3. We are lacking prognostic and predictive biomarkers for CC treatment and there is a growing need for the development of biomarkers to follow up the course of the disease. Methodology RAIDs (http://www.raids-fp7.eu) is a multidisciplinary co-operation between academic clinical centers, SMEs and translational research platforms in seven European countries. It includes: 1) a cognitive cohort study (BioRAIDs)4, one of the first prospective trials intended to define patient stratification for targeted therapies, 2) a targeted clinical trial using an HPV directed vaccine and 3) preclinical studies aiming at assessing new treatment strategies. Molecular analysis on quality controlled tumor and sera samples from 500 patients enrolled in BioRAIDs combine Next Generation Sequencing at SeqOmics (Hungary), PIK3CA mutations detection in circulating tumor (ct) DNA at ERASMUS (The Netherlands), Reverse Phase Protein array and HPV insertion sites analyses at Institut Curie (France). Bioinformatics pipelines to detect somatic mutations and clustering methods were developed in order to stratify the patients into different subtypes. Following quality control, the pharmacological profiling of a panel of 20 CC cell lines using a panel of drugs which may potentially synergize with “standard treatment” has been completed. Drugs were chosen so as to interfere with different signaling pathways. Results Molecular profiles including exome sequencing and ctDNA analyses on 48 quality controlled samples from the BioRAIDs patients and 20 CC cell lines will be presented. Preliminary results on HPV insertion sites in tumor and serum will be reported. Somatic and DNA copy number alterations from exome sequencing profiles confirm PI3K pathway mutations to be a dominant feature in CC. Stratifications of patients’ tumors based on their mutation profiles show that some cell lines cluster similarly to patients’ tumors, suggesting that they will be helpful for the prediction of response to drugs for patients within the same cluster. Conclusions The identification of predictive tumor/blood based biomarkers will permit the definition of new strategies for precision medicine in CC. A bioinformatics analysis which will assess drug responsiveness in CC cell lines in relation to specific genomic alterations is ongoing. Its results should permit to select treatments according to genetic “constellations” of the tumors. Citation Format: Suzy Scholl, Maud Kamal, Els Berns, Balzs Balint, Attila Kereszt, Leanne de Koning, Emmanuelle Jeannot, Windy Luscap-rondof, Vonick Sibut, Philippe Hupe, Gemma Kenter, Sanne Samuels, Katja Jordanova, Sandrine Blanchet, Laurence Lafanachere, Marc Billaud, RAIDs consortium. Rational molecular assessment and innovative drug selection (RAIDs): exome data from cervical 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 434.

Abstract 1852: Multiple PI3K pathway mutations predominate in low stage endometrial carcinomas

Abstract Objectives: Endometrial cancers (ECs) frequently harbor mutations in PI3K pathway genes. Our objective was to determine the mutation frequency of the PIK3 pathway in the tumors of EC patients prospectively enrolled on an IRB-approved institutional tumor sequencing initiative (UNCseq) and correlate these with clinical factors. Methods: Snap-frozen and FFPE tissue samples were collected from patients enrolled on UNCseq (NCT01457196). DNA was isolated using the Puregene DNA purification system, and Illumina libraries were prepared separately from tumor and a matched normal sample from each patient. Relevant targets, were enriched by a custom designed Agilent SureSelect hybrid capture enrichment library using standard protocols. Samples were sequenced on Illumina HiSeq machines in a variety of formats. Statistical analysis of clinical correlates was performed in R (v 3.1.1). Results: Data was collected from 109 EC patients. Thirty-six tumors (33.0%), 34 of which were endometrioid, had >2 mutations between PIK3CA and PIK3R1. 20 patients had multiple PIK3CA mutations (18.3%) and 11 had multiple PIK3R1 mutations (10.1%). In all but 5 cases, multiple mutations of PIK3R1 or PIK3CA were coincident with PTEN mutation (p = 0.018). There were strong correlations between PI3K pathway mutations and tumor grade and stage. High stage tumors (stage >2) were less likely (5/36) than stage 1 tumors (31/73) to have multiple PI3K mutations (p = 0.0056). Though not reaching significance, grade 3 tumors (8/37) were less likely than low-grade tumors (28/72) to demonstrate multiple PI3K mutations (p = 0.11). Of the 10 serous ECs, none exhibited multiple PI3K mutations. Conclusions: There is a strong relationship between mutated PIK3 pathway genes and clinically relevant tumor biology in ECs. Multiple mutations in the PIK3 pathway are common, suggesting that PI3K pathway inhibition in EC may need to be directed against PIK3CA and PIK3R1 simultaneously. These multiply mutated tumors are strongly linked to lower stage and grade tumors with concurrent PTEN mutations. We posit that initially PTEN mutant clones may give rise to subclonal populations with various PI3K mutations which may be relatively indolent until additional driver mutations are acquired such that one subpopulation predominates, resulting in higher grade and stage ECs. Citation Format: Dario R. Roque, Will R. Jeck, David N. Hayes, Arthur M. Dizon, Leslie Clark, Stuart Pierce, Weiya Z. Wysham, Tara Castellano, Paola A. Gehrig, Victoria Bae-Jump. Multiple PI3K pathway mutations predominate in low stage endometrial carcinomas. [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 1852.

HER2+ Cancer Cell Dependence on PI3K vs. MAPK Signaling Axes Is Determined by Expression of EGFR, ERBB3 and CDKN1B.

Understanding the molecular pathways by which oncogenes drive cancerous cell growth, and how dependence on such pathways varies between tumors could be highly valuable for the design of anti-cancer treatment strategies. In this work we study how dependence upon the canonical PI3K and MAPK cascades varies across HER2+ cancers, and define biomarkers predictive of pathway dependencies. A panel of 18 HER2+ (ERBB2-amplified) cell lines representing a variety of indications was used to characterize the functional and molecular diversity within this oncogene-defined cancer. PI3K and MAPK-pathway dependencies were quantified by measuring in vitro cell growth responses to combinations of AKT (MK2206) and MEK (GSK1120212; trametinib) inhibitors, in the presence and absence of the ERBB3 ligand heregulin (NRG1). A combination of three protein measurements comprising the receptors EGFR, ERBB3 (HER3), and the cyclin-dependent kinase inhibitor p27 (CDKN1B) was found to accurately predict dependence on PI3K/AKT vs. MAPK/ERK signaling axes. Notably, this multivariate classifier outperformed the more intuitive and clinically employed metrics, such as expression of phospho-AKT and phospho-ERK, and PI3K pathway mutations (PIK3CA, PTEN, and PIK3R1). In both cell lines and primary patient samples, we observed consistent expression patterns of these biomarkers varies by cancer indication, such that ERBB3 and CDKN1B expression are relatively high in breast tumors while EGFR expression is relatively high in other indications. The predictability of the three protein biomarkers for differentiating PI3K/AKT vs. MAPK dependence in HER2+ cancers was confirmed using external datasets (Project Achilles and GDSC), again out-performing clinically used genetic markers. Measurement of this minimal set of three protein biomarkers could thus inform treatment, and predict mechanisms of drug resistance in HER2+ cancers. More generally, our results show a single oncogenic transformation can have differing effects on cell signaling and growth, contingent upon the molecular and cellular context.

Influence of PI3K and MAPK Pathway Mutations on Response to Mono and Dual Treatment with Targeted Kinase Inhibitors

Genetic heterogeneity and drug resistance has hindered the clinical development of targeted kinase inhibitors for treatment of glioblastoma, the most common and aggressive primary brain tumor. We addressed these issues using immortalized astrocytes harvested from genetically engineered mice with and without phosphoinositide 3‐kinase (PI3K) and/or RAS/mitogen activated protein kinase (MAPK) pathway mutations, specifically Pten deletion and/or oncogenic Kras. We found that the potency of PI3K pathway inhibitors (PI3Ki potency: BEZ235 > BKM120 > LY294002) varied and sensitivity was increased 2 – 19 fold by mutations in Pten, Kras, or both. Sensitivity to MEK inhibitors (MEKi potency: GSK1120212 > PD0325901 > AZD6244) was increased 5 – 53 fold by Pten, but not Kras mutations. To determine how BKM120 alters the kinome, we used multiplexed inhibitor bead‐mass spectrometry (MIB‐MS). In immortalized astrocytes with both Pten and Kras mutations, the PI3Ki BKM120 caused dynamic kinome changes, including sustained inhibition of proximal PI3K, inhibition followed by re‐activation of distal PI3K signaling, and alternative activation of MAPK signaling. These results were confirmed by immunoblot. The more potent dual PI3K/mTOR inhibitor BEZ235 inhibited PI3K and induced alternative activation of MAPK at lower concentrations. Conversely, multiple MEKi caused MAPK inhibition and alternative activation of PI3K signaling. Next we determined whether Pten or Kras mutations alone influenced alternative pathway activation by PI3Ki or MEKi. BKM120 inhibited PI3K signaling and induced alternative activation of MAPK independent of Pten and Kras mutation status. In contrast, treatment with the MEKi AZD6244 induced alternative activation of PI3K in immortalized astrocytes with activated Kras, independent of Pten. These findings suggested that alternative activation of PI3K or MAPK signaling promotes drug resistance in response to monotherapy inhibition of the other arm of receptor tyrosine kinase signaling. Combined treatment of immortalized, Pten‐deleted, Kras‐activated astrocytes with both the PI3Ki BKM120 and the MEKi AZD6244, PD0325901, or GSK1120212 ablated alternative pathway activation and functioned synergistically at physiologically achievable concentrations. Moreover, synergism was achieved at lower PI3Ki doses when a more potent MEKi was used. We then examined how Pten and Kras mutations and MEKi potency interact to influence drug synergism. BKM120 and AZD6244 became synergistic at ≥ 10× lower drug concentrations in immortalized astrocytes with Pten and/or Kras mutations. Combining BKM120 with the more potent MEKi GSK212 was more broadly synergistic and independent of mutation status. Taken together, we conclude that PI3K and MAPK mutation status, as well as drug potency, influences targeted kinase inhibitor response and that dual inhibition may be required to achieve clinical benefits.Support or Funding InformationRSM is a Robert H. Wagner Scholar in the Pathobiology and Translational Sciences Program and a trainee in the HHMI Graduate Training Program in Translational Medicine. CRM was a Damon Runyon‐Genentech Clinical Investigator supported by the Damon Runyon Cancer Research Foundation (CI‐45‐09). This work was supported by grants to CRM from the UNC University Cancer Research Fund (UCRF) and the NIH National Center for Advancing Translational Sciences (NCATS, 550KR21202) and a NIH grant to GLJ (GM101141).

Abstract B100: ASN003, a unique B-RAF inhibitor with additional selective activity against PI3K and mTOR kinases, shows strong antitumor activity in multiple xenograft models

Abstract Various genes in RAS-RAF and PI3K pathways are frequently mutated in a wide variety of solid tumors. Concurrent double mutations are also observed quite often in a broad range of tumor types. Combined inhibition of both pathways has been shown to impart greater efficacy in multiple tumor models in animals. In order to simultaneously block these pathways, we have discovered and characterized several molecules showing dual inhibition of the RAS-RAF and PI3K pathways. ASN003 has been identified as a lead compound with potent and highly selective inhibitory activity against B-RAF, PI3K and mTOR kinases (low nM IC50). Within the PI3K family, ASN003 has high selectivity for inhibition of PI3Kα and PI3Kδ over PI3Kβ. In cell-based mechanistic studies, ASN003 did not increase phospho-ERK levels in the KRAS mutant cell line HCT116 and thus may not cause paradoxical activation of RAS/MAPK pathway in tumors. Consistent with its dual inhibition profile, ASN003 showed strong antiproliferative activity in cell lines with B-RAF and PI3K pathway mutations as well as vemurafenib (B-RAF inhibitor)-resistant cell lines. ASN003 suppressed the phosphorylation of downstream targets of B-RAF, PI3K and mTOR in pharmacodynamic studies in multiple tumor models, indicating appropriate target engagement. In in vivo efficacy studies, ASN003 showed regression in a B-RAFV600E mutant A375 xenograft model and also caused significant tumor growth inhibition in RKO and A2058 tumor models, which harbor mutations in PIK3CA or PTEN genes, respectively, in addition to the B-RAF V600E mutation. Dual targeting of the B-RAF and PI3K pathways with ASN003 has the potential to treat and/or prevent the acquired resistance to selective B-RAF inhibitors, and may also treat a broader patient population and provide greater efficacy and survival benefit than selective B-RAF inhibitors or selective PI3K pathway inhibitors alone. Due to the lack of paradoxical activation, treatment with ASN003 may not cause skin toxicities seen with pure B-RAF inhibitors. ASN003 is currently in preclinical development and is expected to enter Phase I/II clinical trials by early 2016. Citation Format: Scott K. Thompson, Mahaboobi Jaleel, Vijay Kumar Nyavanandi, Murali Ramachandra, Hosahalli Subramanya, Aravind Basavaraju, Vaibhav Sihorkar, Roger A. Smith, Niranjan Rao, Sandeep Gupta, Sanjeeva P. Reddy. ASN003, a unique B-RAF inhibitor with additional selective activity against PI3K and mTOR kinases, shows strong antitumor activity in multiple xenograft models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B100.

Genome Analysis of Latin American Cervical Cancer: Frequent Activation of the PIK3CA Pathway.

Cervical cancer is one of the most common causes of cancer mortality for women living in poverty, causing more than 28,000 deaths annually in Latin America and 266,000 worldwide. To better understand the molecular basis of the disease, we ascertained blood and tumor samples from Guatemala and Venezuela and performed genomic characterization. We performed human papillomavirus (HPV) typing and identified somatically mutated genes using exome and ultra-deep targeted sequencing with confirmation in samples from Mexico. Copy number changes were also assessed in the exome sequence. Cervical cancer cases in Guatemala and Venezuela have an average age of diagnosis of 50 years and 5.6 children. Analysis of 675 tumors revealed activation of PIK3CA and other PI3K/AKT pathway genes in 31% of squamous carcinomas and 24% of adeno- and adenosquamous tumors, predominantly at two sites (E542K, E545K) in the helical domain of the PIK3CA gene. This distribution of PIK3CA mutations is distinct from most other cancer types and does not result in the in vitro phosphorylation of AKT. Somatic mutations were more frequent in squamous carcinomas diagnosed after the age of 50 years. Frequent gain of chromosome 3q was found, and low PIK3CA mutation fractions in many tumors suggest that PI3K mutation can be a late event in tumor progression. PI3K pathway mutation is important to cervical carcinogenesis in Latin America. Therapeutic agents that directly target PI3K could play a role in the therapy of this common malignancy.

Inhibiting the PI3K signaling pathway: buparlisib as a new targeted option in breast carcinoma.

Aberrations in the PI3K signaling pathway are frequently observed in patients with breast cancer. Because of that, PI3K inhibitors are attractive options for the treatment of breast cancer because PI3K is the most proximal component of the pathway other than receptor tyrosine kinases. Buparlisib is a potent and highly specific oral pan-class I PI3K inhibitor, which is currently under investigation in patients with breast cancer. In this article, we describe the PI3K signaling pathway, the prognostic value of PI3K pathway mutations, as well as the mechanism of action of buparlisib. Lastly, we discuss preliminary results of preclinical and clinical studies showing the efficacy and safety profile of this agent in breast cancer patients.

Abstract 4697: The PI3K/mTOR pathway is a potential therapeutic target in cancers with ARID1A mutations

Abstract The chromatin remodeling SWI/SNF complex is mutated in 20% of all cancers and ARID1A is the most frequently mutated subunit. However, the tumor suppressive functions of ARID1A are not fully understood and no feasible therapeutic strategies are available for ARID1A-mutant cancers. Recent studies found that loss of ARID1A is associated with increased phosphorylation of AKT. We found that from a study that analyzed data from Project Achilles, a broad shRNA screening project, PIK3CA is the number 2 gene essential for survival of ARID1A-mutant cell lines compared to ARID1A-wildtype cell lines (P = 7.37 × 10-6, FDR < 0.001). Based on these findings, we hypothesized that the PI3K pathway is a potential therapeutic target in ARID1A-mutant cancers. We analyzed the Cancer Genome Atlas (TCGA) datasets and found that mutations in the PI3K pathway co-occur with ARID1A mutations. In addition, the number of co-existing PI3K pathway mutations in the same sample is higher when ARID1A is mutated. We knocked down PIK3CA in ARID1A-wildtype cells (RMG1 and OVCAR3) and ARID1A-mutant cells (OVAS and HCH-1). We found that proliferation was impaired more profoundly in ARID1A-mutant cells. Interestingly, HCH-1 cells are wildtype in PIK3CA, PTEN, PIK3R1 and KRAS, but are still sensitive to PIK3CA depletion. For an unbiased approach, we analyzed the Genomics of Drug Sensitivity in Cancer datasets, which contain drug responses of a large cancer cell line panel to 138 anti-cancer drugs. We compared the drug responses of 49 cell lines harboring inactivating ARID1A-mutations with 266 ARID1A-wildtype cell lines. We found that the presence of inactivating ARID1A mutations is highly associated with sensitivity to mTOR inhibitor AZD8055 (ranked 2nd, P = 2.00 × 10-3) and AKT inhibitor MK2206 (ranked 4th, P = 7.98 × 10-3). This association is still significant for MK2206 when we removed cell lines with PIK3CA, KRAS, PTEN, PIK3R1 and TSC1 alterations (P = 1.32 × 10-2). Finally, we investigated how ARID1A loss can directly increase PI3K/mTOR activity. Using microarray analysis, we found that knockdown of ARID1A up-regulated MYC and MYC target genes, including SLC7A5, an amino-acid transporter required for mTOR activation. Analysis of TCGA datasets showed that MYC amplification and ARID1A mutations are mutual exclusive events, suggesting that overexpression of MYC and loss of ARID1A may converge on the same pathway. In conclusion, we found that ARID1A-mutant cells are highly sensitive to PI3K/mTOR inhibition. Although ARID1A mutations frequently co-occur with PI3K pathway mutations, it is not the sole explanation of this specific sensitivity. ARID1A loss may increase mTOR signaling through MYC target gene SLC7A5. However, increase in PI3K/mTOR activity maybe a long term effect of ARID1A loss. Together, our data identified PI3K/mTOR signaling is essential for survival of ARID1A-mutant cancers and PI3K/mTOR inhibitors can be used as therapeutic strategies. Citation Format: Suet-Yan Kwan, Daisy I. Izaguirre, Xuanjin Cheng, Suet-Ying Kwan, Yvonne TM Tsang, Hoi-Shan Kwan, Kwong-Kwok Wong. The PI3K/mTOR pathway is a potential therapeutic target in cancers with ARID1A mutations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4697. doi:10.1158/1538-7445.AM2015-4697

Abstract 4668: Targeted, massively parallel sequencing identifies novel genetic subsets of cutaneous melanoma

Abstract Despite the prevalence of recurrent, high activating BRAF V600 mutations in 45% of tumors, cutaneous melanoma (CM) is a heterogeneous malignancy resulting from aberrant signaling in multiple pathways. It has been traditionally characterized by activation of the MAPK and PI3K signaling pathways, as well as cell cycle disruption. In recent years, whole-genome and exome sequencing studies have identified several new genes associated with melanomagenesis. However, a comprehensive understanding of concurrent, and mutually exclusive, mutations in tumors is currently lacking. Using a custom targeted capture of 108 genes previously implicated in melanoma pathogenesis, massively parallel sequencing was performed on 94 human melanoma cell lines, 67 patient-derived xenografts (PDX), and 5 cell lines made from PDX, all untreated. Samples were then clustered into groups based on deleterious mutations. 83% of samples had deleterious mutations in the MAPK signaling pathway, including 92 high activity BRAF (55%), 35 RAS codon 61 (21%), 7 with multiple mutations (e.g. low activity BRAF/RAS codons 12/13) (4%) and 10 NF1 (6%) mutated samples. Likely deleterious NF1 mutations were found in several BRAF or NRAS-mutated samples. PI3K pathway mutations were found in 10% of samples, predominantly associated with BRAF mutations. TP53 mutations were found in 24% of samples and were associated with all MAPK signaling mutations. Mutations in chromatin remodeling genes (ARID1A/1B, ARID2, TRRAP, and BAP1) were mutually exclusive with each other and primarily found in tumors with high activity BRAF or NRAS mutations. The majority of BRAF or RAS-mutated samples with a mutation in a chromatin remodeling gene lacked mutations in cell cycle, TP53, and PI3K signaling genes; however, 100% of deleterious, or likely deleterious, NF1-mutated samples with a chromatin remodeling gene mutation harbored additional mutations in cell cycle, TP53, and/or PI3K signaling genes. Of particular interest, five of the 10 NF1-mutated samples (50%) lacked BRAF, RAS, and MEK1/2 mutations but harbored likely deleterious mutations in MAP3K5 or MAP3K9, suggesting the potential involvement of the JNK signal transduction pathway in this particular cohort. Only 4% of samples did not have a deleterious mutation in any of the genes on the panel. These data reveal novel insights into the genetics of melanomas lacking a canonical BRAF V600 mutation. Functional assays are needed to confirm the biological relevance of likely deleterious mutations, which will further facilitate a more thorough classification of CM subsets. Citation Format: Bradley Garman, Clemens Krepler, Katrin Sproesser, Patrica Brafford, Melissa Wilson, Bradley Wubbenhorst, Ravi Amaravadi, Joseph Bennett, Marilda Beqiri, Michael Davies, David Elder, Keith Flaherty, Dennie Frederick, Tara C. Gangadhar, Michael Guarino, David Hoon, Giorgos Karakousis, Nandita Mitra, Nicholas J. Petrelli, Lynn Schuchter, Batool Shannan, Jennifer Wargo, Min Xiao, Wei Xu, Xaiowei Xu, Meenhard Herlyn, Katherine Nathanson. Targeted, massively parallel sequencing identifies novel genetic subsets of cutaneous melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4668. doi:10.1158/1538-7445.AM2015-4668

Abstract 4817: Microsatellite instability status in endometrioid endometrial carcinomas is associated with distinct types and patterns of PI3K pathway mutations

Abstract Background: Endometrioid endometrial carcinomas (EECs) frequently harbor mutations in the PI3K pathway. In contrast with other cancer types (e.g. breast cancer) where PIK3CA mutations are generally mutually exclusive with PTEN mutations, in EECs mutations affecting these genes often co-occur. Here we sought to determine whether the type and pattern of mutations targeting different components of the PI3K pathway are distinct between microsatellite stable (MSS) and high-level microsatellite instable (MSI-H) EECs, and to define the mutational signatures in MSI-H and MSS EECs. Methods: Whole exome massively parallel sequencing-based mutation data from EECs of The Cancer Genome Atlas (TCGA) project were used to define the number, type and pattern of mutations affecting PI3K pathway-related genes (i.e., AKT1, INPP4B, MTOR, PIK3CA, PIK3R1 and PTEN). Based on seven MSI markers assessed by TCGA, EECs were classified as MSI-H (n = 70) and MSS (n = 109). POLE ultramutated cases were excluded. Mutational signatures were defined using EMu, a method based upon the expectation-maximization algorithm. Results: Although the mutation rates of MSS and MSI-H EECs were significantly different, the prevalence of mutations affecting PI3K pathway genes was similar between these two groups (all p>0.05), with the exception of PTEN mutations, which were more prevalent in MSI-H (87%) than in MSS EECs (72%; p = 0.017). The PIK3CA hotspot mutations E542K, E545K, and H1047R were found to be significantly more prevalent in PIK3CA-mutant MSS EECs (36%) than in PIK3CA-mutant MSI-H EECs (13.5%; p = 0.019). In both MSI-H and MSS EECs a mutational signature related to age was identified, characterized by C>T transitions at NpCpG trinucleotides; in MSS tumors a C>T and C>G at TpCpN trinucleotides mutational signature, attributed to the APOBEC family of cytidine deaminases, was identified, whereas in MSI-H tumors, a DNA-MMR deficiency-like signature was found. Conclusion: Although the prevalence of mutations targeting different components of the PI3K pathway is similar between MSS and MSI-H EECs, PIK3CA hotspot mutations, which result in constitutive kinase activation, are significantly more prevalent in MSS than in MSI-H EECs. We have observed that the mutational processes operating in MSI-H and MSS EECs are distinct, and that the landscape of mutations affecting PI3K pathway-related genes might be shaped by multiple mutational processes in these cancers. Our findings warrant further investigation of the role of different types of PIK3CA mutations in and their predictive impact on distinct subtypes of EECs. Citation Format: Caterina Marchio, Maria R. De Filippo, Charlotte KY Ng, Robert A. Soslow, Jorge S. Reis-Filho, Britta Weigelt. Microsatellite instability status in endometrioid endometrial carcinomas is associated with distinct types and patterns of PI3K pathway mutations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4817. doi:10.1158/1538-7445.AM2015-4817

Mutations in the RAS and PI3K pathways are associated with metastatic location in colorectal cancers.

Identification of mutations in the downstream epidermal growth factor receptor (EGFR) signaling pathway could provide important insights of EGFR-targeted therapies in colorectal cancers. We analyzed the mutation spectra of the PI3K/PTEN/AKT and RAS/RAF/MAPK pathways in colorectal cancers and the associations of these mutations with sites of metastases or recurrence. The study population comprised 1,492 retrospectively collected stages I-IV colorectal cancer specimens. Tissue was obtained between 2000 and 2010 at a single hospital. We analyzed 61 hot spots using MALDI-TOF mass spectrometry for nucleic acid analysis. Mutations were found in the RAS pathway in 47.3% of patients and in the PI3K pathway in 14.3% of patients, with 9.2% of patients carrying mutations in both pathways. Both the RAS and PI3K pathway mutations were significantly associated with proximal tumors, mucinous tumors, and microsatellite instability. Tumors carrying a RAS pathway mutation exhibited a higher frequency of lung and peritoneal metastasis than did tumors with a wild-type gene (P = 0.025 and 0.009, respectively). NRAS gene mutation was significantly associated with lung metastasis (P = 0.001). Somatic mutations in the RAS pathway of the primary tumor in colorectal cancer can influence patterns of metastasis and recurrence.

In-depth molecular profiling of the biphasic components of uterine carcinosarcomas.

Uterine carcinosarcoma is a clinically aggressive malignancy composed of a mix of carcinomatous and sarcomatous elements. We performed targeted next‐generation sequencing of 27 uterine cancer and sarcoma genes together with immunohistochemical analyses of selected proteins in 30 uterine carcinosarcomas. This included 13 cases in which the distinct carcinoma and sarcoma components were sequenced separately and 10 cases where the metastatic tumours were analysed in addition to the primary tumours. We identified non‐synonymous somatic mutations in 90% of the cases, with 27 of 30 cases (90%) harbouring TP53 alterations. The PI3K pathway was the most commonly mutated signalling pathway with mutations identified in PIK3CA, PTEN, PIK3R1, and/or PIK3R2 in two‐thirds of the cases. Mutations in FBXW7, PPP2R1A, ARID1A and KRAS were demonstrated in a minority of cases. In cases where the carcinomatous and sarcomatous components were separately analysed, most of the mutations identified were present in both components, indicating a common origin for the two components. Furthermore, the same TP53 alterations and/or PI3K pathway mutations seen in the primary tumours were also identified in the metastatic sites. Overall, carcinosarcomas exhibited heterogeneous molecular features that resemble the heterogeneity seen in endometrial carcinomas, with some showing endometrioid carcinoma‐like and others showing serous carcinoma‐like mutation profiles. While patients with serous‐like tumours presented more frequently with advanced‐stage disease compared to patients with endometrioid‐like tumours, there was no statistical difference in outcome between the two groups. Our results provide insights into the oncogenesis of uterine carcinosarcoma and identify targetable mutations that represent early oncogenic events. The findings of the different molecular types of uterine carcinosarcoma that parallel the different molecular types in endometrial carcinoma may have future treatment implications with targeted therapies.

PIKing the type and pattern of PI3K pathway mutations in endometrioid endometrial carcinomas

ObjectiveThe vast majority of endometrioid endometrial carcinomas (EECs) harbor mutations in the PI3K pathway. Here we sought to determine whether the type and pattern of mutations targeting different components of the PI3K pathway are distinct between microsatellite stable (MSS) and high-level microsatellite instable (MSI-H) EECs. MethodsWhole exome massively parallel sequencing-based mutation data from EECs of The Cancer Genome Atlas (TCGA) were used to define the number, type and pattern of mutations affecting PI3K pathway-related genes, including AKT1, INPP4B, MTOR, PIK3CA, PIK3R1 and PTEN. EECs were classified as MSI-H (n=70) and MSS (n=109) based on seven MSI markers assessed by TCGA. Ultramutated cases were excluded. ResultsAlthough the mutation rates and mutational signatures of MSS and MSI-H EECs were distinct, the prevalence of PI3K pathway mutations was similar between these two groups (all p>0.05), with the exception of PTEN mutations, which were more prevalent in MSI-H (61/70; 87%) than in MSS EECs (78/109; 72%; p=0.017). The PIK3CA hotspot mutations E542K, E545K, and H1047R were found to be significantly more prevalent in PIK3CA-mutant MSS (21/58, 36%) compared to PIK3CA-mutant MSI-H EECs (5/37, 13.5%; p=0.019). ConclusionAlthough the prevalence of mutations targeting PI3K pathway genes is similar between MSS and MSI-H EECs, PIK3CA hotspot mutations, which result in constitutive kinase activation, are significantly more prevalent in MSS than in MSI-H EECs. Our findings warrant further investigation of the role of different types of PIK3CA mutations and their predictive impact on distinct subtypes of EECs.

Coexistent ARID1A-PIK3CA mutations promote ovarian clear-cell tumorigenesis through pro-tumorigenic inflammatory cytokine signalling.

Ovarian clear-cell carcinoma (OCCC) is an aggressive form of ovarian cancer with high ARID1A mutation rates. Here we present a mutant mouse model of OCCC. We find that ARID1A inactivation is not sufficient for tumor formation, but requires concurrent activation of the phosphoinositide 3-kinase catalytic subunit, PIK3CA. Remarkably, the mice develop highly penetrant tumors with OCCC-like histopathology, culminating in hemorrhagic ascites and a median survival period of 7.5 weeks. Therapeutic treatment with the pan-PI3K inhibitor, BKM120, prolongs mouse survival by inhibiting tumor cell growth. Cross-species gene expression comparisons support a role for IL-6 inflammatory cytokine signaling in OCCC pathogenesis. We further show that ARID1A and PIK3CA mutations cooperate to promote tumor growth through sustained IL-6 overproduction. Our findings establish an epistatic relationship between SWI/SNF chromatin remodeling and PI3K pathway mutations in OCCC and demonstrate that these pathways converge on pro-tumorigenic cytokine signaling. We propose that ARID1A protects against inflammation-driven tumorigenesis.

Next-generation sequencing of advanced, relapsed colorectal adenocarcinoma (CRC) to reveal mutations affecting Wnt, MAPK and PI3K pathway signaling: Emergence of novel combinatorial strategies.

605 Background: Advanced relapsed colorectal adenocarcinoma (CRC) is emerging as a cancer not with a singular phenotype but rather as a multitude of ‘diseases’ subdivided based on its molecular genotype. Given the heterogeneity in outcomes of CRC patients on targeted therapy clinical trials, we sought to identify a unique molecular subset within this disease. Methods: To that end, we identified advanced CRC patients in our Phase I clinic on whom adequate tissue was available for next generation sequencing (NGS) on a commercially available platform (FoundationOne, Boston, MA). Over 2000 exons of 186 cancer-related genes plus over 30 introns from 14 genes often rearranged in cancer were fully sequenced for point mutations, insertions/deletions, copy number alterations (CNAs) and select gene fusions. Results: We identified 51 patients with advanced relapsed CRC (26 male, 25 female; median age 54 years; 49 colonic primary lesion, 4 rectum primary). Overall, 44 (86%) harbored a mutation linked to aberrant Wnt pathway signaling (most commonly, APC 40 pts, 78%). Of these 44 patients, half (n=22, 50%) had ≥1 concurrent mutation affecting the PI3K/AKT/MTOR pathway, most commonly PIK3CA in 11 patients (4 E545K, 3 Q546K, 2 E542K, 1 H1047Y, 1 E453K). Furthermore, 20 of these 22 pts with both Wnt and PI3K pathway mutations also demonstrated a concomitant MAPK pathway abnormality, most commonly KRAS (n=16) and 3 BRAF (V600E, G466R, D594G). Overall 20 of 51 (39%) patients had coexisting mutations implicated in all three signaling pathways (Wnt, PI3K, and MAPK). Conclusions: Three prominent pathways linked to the continuum of development and progression of cancer (Wnt, MAPK, PI3K) occur frequently in concurrence in patients with advanced CRC, thereby highlighting the role for an innovative therapeutic combination targeting all three pathways.

Abstract CT229: A phase II and biomarker study of an irreversible pan-human EGF receptor (HER) tyrosine kinase inhibitor, dacomitinib, in patients with recurrent and/or metastatic squamous cell carcinoma of head and neck (R/M-SCCHN)

Abstract Background: Dacomitinib is an oral irreversible tyrosine kinase inhibitor of EGFR, HER2 and HER4 signaling. The goals of this study were to investigate the clinical activity, safety and predictive biomarkers of dacomitinib in R/M-SCCHN. Methods: Patients with R/M-SCCHN were eligible if they had progressed on platinum-based chemotherapy or considered platinum-intolerant, and were treated with dacomitinib 45mg/day. The primary endpoint was objective response rate (ORR) by RECIST v1.1; Secondary endpoints included progression-free survival rate (PFS), overall survival (OS) and safety. We charaterized somatic mutations using the Ion AmpliSeq™ platform (2,800 mutations from 50 genes), gene copy number (86 genes) and gene expression (230 genes) using NanoString nCounter® and CDKN2A (p16INK4A) expression by immunohistochemistry, and investigated their relationship with clinical outcomes. Results: A total of 48 patients were enrolled and evaluable for efficacy and toxicity. Patient and disease characteristics included median age (61 years; range, 30-82); male (85%); ECOG performance status 0/1/2 (4%/75%/21%); locoregional/distant metastatic/both (31/23/46%); oral cavity/larynx/oropharynx primary (38%/19%/23%); prior chemotherapy regimens 0/1/≥2 (8%/52%/40%). Ten patients (21%) had partial responses and 31 patients (65%) had stable diseases. With a median 8.4 months of follow up, the median PFS and OS were 3.9 months (95% CI, 2.9-5.0) and 8.2 months (95% CI, 5.8-10.3). Adverse events (AEs) were mostly grade 1-2 and manageable. The most common AEs were skin toxicities (paronychia, 65%; acneiform dermatitis, 44%) and diarrhea (52%). Results from the biomarker analyses were available in 28 patients for AmpliSeq™, 31 patients for gene copy number, and 12 patients for gene expression. Mutations in PIK3CA and PTEN was significantly associated with shorter PFS (2.5 vs 5.4 months, P=0.013). There were trends toward higher ORR (23% vs 0) for patients with wild PIK3CA and PTEN, with unusually long durations of response (5.1, 5.4, 5.8, 13.1, and 18.9 months, respectively). Increased gene expression of proinflammatory chemokines and inflammatory response modulators (IL6, IL8, PTGS2, PLA2G2A) was significantly associated with shorter PFS (1.9 vs 6.8 months, P=0.001). Patients with low gene expression of proinflammatory chemokines also tend to have higher ORR (40% vs 0), with unusually long durations of response (18.9 and 5.4 months, respectively). Conclusions: Dacomitinib demonstrated promising efficacy with manageable toxicity in platinum-failed R/M-SCCHN patients. Screening of PI3K pathway mutation and proinflammatory chemokines may help identify which R/M-SCCHN patients are likely to gain a benefit from dacomitinib. Citation Format: Han Sang Kim, Hyung Joo Kwon, Myung-Ju Ahn, Byung Woog Kang, Jong-Mu Sun, Dok-Hyun Yoon, Keon Uk Park, Se-Hoon Lee, Yoon Woo Koh, Se Hun Kim, Eun Chang Choi, Hye Ryun Kim, Hyo Song Kim, Joo Hang Kim, Byoung Chul Cho. A phase II and biomarker study of an irreversible pan-human EGF receptor (HER) tyrosine kinase inhibitor, dacomitinib, in patients with recurrent and/or metastatic squamous cell carcinoma of head and neck (R/M-SCCHN). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT229. doi:10.1158/1538-7445.AM2014-CT229

MDM2 antagonists synergize broadly and robustly with compounds targeting fundamental oncogenic signaling pathways.

While MDM2 inhibitors hold great promise as cancer therapeutics, drug resistance will likely limit their efficacy as single agents. To identify drug combinations that might circumvent resistance, we screened for agents that could synergize with MDM2 inhibition in the suppression of cell viability. We observed broad and robust synergy when combining MDM2 antagonists with either MEK or PI3K inhibitors. Synergy was not limited to cell lines harboring MAPK or PI3K pathway mutations, nor did it depend on which node of the PI3K axis was targeted. MDM2 inhibitors also synergized strongly with BH3 mimetics, BCR-ABL antagonists, and HDAC inhibitors. MDM2 inhibitor-mediated synergy with agents targeting these mechanisms was much more prevalent than previously appreciated, implying that clinical translation of these combinations could have far-reaching implications for public health. These findings highlight the importance of combinatorial drug targeting and provide a framework for the rational design of MDM2 inhibitor clinical trials.