PIK3CA H1047R Research Articles

Abstract CT328: Exploratory genetic analysis of tumors from a phase I/II dose escalation study of GSK2636771 in patients (pts) with PTEN deficient advanced tumors

Abstract Background: GSK2636771 is a potent, orally bioavailable and selective inhibitor of PI3Kβ (apparent Ki = 0.89 nM), with >900-fold selectivity over PI3Kα/PI3Kγ and 10-fold over PI3Kδ. It inhibits AKT phosphorylation and downstream signaling measured as decrease of PRAS40-, GSK3β-, and RPS6-phosphorylation in PTEN deficient cell lines. Methods: An ongoing phase I/IIa FTIH, open label dose escalation study of GSK2636771, once daily (QD), is being conducted to evaluate safety, pharmacokinetics (PK), pharmacodynamics (PD) and efficacy in pts with advanced tumors deficient in PTEN. The study comprised 3 parts: starting-dose selection, 3+3 dose escalation, and phase II expansion. PD was assessed in tumor and platelet rich plasma samples. Archival tumor samples were analyzed for alterations in a custom panel of select oncogenes and tumor suppressors by next-generation sequencing and copy number analyses. Results: As of Nov 2014, 62 pts (38 m: 24 f, mean 60 yrs) were enrolled and dosed at 7 dose levels (25 - 500 mg). In total 5 DLTs (3x hypophosphatemia/ 2x hypocalcemia G3) were observed in 3 pts at 500 mg, defining MTD. AEs ≥20% (all grades) included diarrhoea, nausea, vomiting, fatigue, abdominal pain, anemia, decreased appetite and headache. Cmax was reached 4-6 h after single and repeat dosing, with a mean t1/2 of 17.1-38.6 h across cohorts. Increases in mean Cmax and AUC(0-24 h) were dose-proportional up to 350 mg, but less than proportional at 400 and 500 mg. Phospho/total AKT ratio was decreased by >50% in platelet-rich plasma in 21 of 23 pts across various doses. Similarly, phospho/total AKT ratio was decreased (median 35%) in post-treatment tumor biopsies compared to pre-treatment tumors in 4 of 5 pts treated at 400 mg dose. One pt (prostate) had a RECIST PR and 17 pts had SD, 8 of whom were on study for ≥6 months. Exploratory genomic analyses of tumor samples from 10 prostate cancer pts revealed a PIK3CB L1049R mutation in the tumor of one of the pts who remained on study for 33 weeks and an increased PIK3CB gene copy number in the tumor of a pt who achieved PR. Preclinical characterization of PIK3CB L1049R mutation is ongoing, as it is homologous to the oncogenic PIK3CA H1047R mutation. These data suggest potential clinical benefit with GSK2636771 in pts with tumors harboring genetic alterations in PIK3CB. Tumors from other histologies are being analyzed for alterations in key cancer-relevant genes, including PIK3CB. Conclusions: MTD and recommended phase II dose of GSK2636771 is 400 mg QD. Pathway inhibition was observed with limited anti-tumor activity in pts with PTEN deficient tumors. Pts with tumors harboring concomitant genetic alterations in PIK3CB may benefit from GSK2636771 treatment. Recruitment into phase II expansion is ongoing. Citation Format: Johann de Bono, Hendrik-Tobias Arkenau, Joaquin Mateo, Jeffrey R. Infante, Howard A. Burris, Yung-Jue Bang, Joseph Eder, Sunil Sharma, Hyun C. Chung, Shaun Decordova, Karen E. Swales, Michelle D. Garrett, Desamaparados Roda-Perez, Meichun Ding, Mark Russo, Li Yan, Ben Suttle, Jerry M. Tolson, Wendy S. Halsey, Ganji Gopi, Harjeet K. Van Der Keyl, Shanker Kalyana-Sundaram, Ganesh M. Sathe, Monica Motwani, Rakesh Kumar. Exploratory genetic analysis of tumors from a phase I/II dose escalation study of GSK2636771 in patients (pts) with PTEN deficient advanced tumors. [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 CT328. doi:10.1158/1538-7445.AM2015-CT328

Inhibition of PI3K Pathway Reduces Invasiveness and Epithelial-to-Mesenchymal Transition in Squamous Lung Cancer Cell Lines Harboring PIK3CA Gene Alterations

A prominent role in the pathogenesis of squamous cell carcinoma of the lung (SQCLC) has been attributed to the aberrant activation of the PI3K signaling pathway, due to amplification or mutations of the p110α subunit of class I phosphatidylinositol 3-kinase (PIK3CA) gene. The aim of our study was to determine whether different genetic alterations of PIK3CA affect the biologic properties of SQCLC and to evaluate the response to specific targeting agents in vitro and in vivo. The effects of NVP-BEZ235, NVP-BKM120, and NVP-BYL719 on two-dimensional/three-dimensional (2D/3D) cellular growth, epithelial-to-mesenchymal transition, and invasiveness were evaluated in E545K or H1047R PIK3CA-mutated SQCLC cells and in newly generated clones carrying PIK3CA alterations, as well as in a xenograft model. PIK3CA mutated/amplified cells showed increased growth rate and enhanced migration and invasiveness, associated with an increased activity of RhoA family proteins and the acquisition of a mesenchymal phenotype. PI3K inhibitors reverted this aggressive phenotype by reducing metalloproteinase production, RhoA activity, and the expression of mesenchymal markers, with the specific PI3K inhibitors NVP-BKM120 and NVP-BYL719 being more effective than the dual PI3K/mTOR inhibitor NVP-BEZ235. A xenograft model of SQCLC confirmed that PIK3CA mutation promotes the acquisition of a mesenchymal phenotype in vivo and proved the efficacy of its specific targeting drug NVP-BYL719 in reducing the growth and the expression of mesenchymal markers in xenotransplanted tumors. These data indicate that PIK3CA mutation/amplification may represent a good predictive feature for the clinical application of specific PI3K inhibitors in SQCLC patients.

Comparison of cell stabilizing blood collection tubes for circulating plasma tumor DNA

ObjectivesCirculating plasma DNA is being increasingly used for biomedical and clinical research as a substrate for genetic testing. However, cell lysis can occur hours after venipuncture when using standard tubes for blood collection, leading to an increase in contaminating cellular DNA that may hinder analysis of circulating plasma DNA. Cell stabilization agents can prevent cellular lysis for several days, reducing the need for immediate plasma preparation after venipuncture, thereby facilitating the ease of blood collection and sample preparation for clinical research. However, the majority of cell stabilizing reagents have not been formally tested for their ability to preserve circulating plasma tumor DNA. Design & methodsIn this study, we compared the properties of two cell stabilizing reagents, the cell-free DNA BCT tube and the PAXgene tube, by collecting blood samples from metastatic breast cancer patients and measuring genome equivalents of plasma DNA by droplet digital PCR. We compared wild type PIK3CA genome equivalents and also assayed for two PIK3CA hotspot mutations, E545K and H1047R. ResultsOur results demonstrate that blood stored for 7days in BCT tubes did not show evidence of cell lysis, whereas PAXgene tubes showed an order of magnitude increase in genome equivalents, indicative of considerable cellular lysis. ConclusionsWe conclude that BCT tubes can prevent lysis and cellular release of genomic DNA of blood samples from cancer patients when stored at room temperature, and could therefore be of benefit for blood specimen collections in clinical trials.

Abstract A37: PI3-kinase signaling is rate limiting for KRASG12D-driven lung tumorigenesis but is not required for tumor maintenance

Abstract In genetically engineered mice (GEM), KRASG12D-driven lung tumorigenesis is dependent on signaling through both the RAF>MEK>ERK and the PI3'K>AKT signaling pathways. However, there is an extended period of latency prior to the onset of KRASG12D-initiated lung tumorigenesis. Moreover, the level of KRASG12D-activated signaling (pERK & pAKT) is only weakly detectable in early-stage initiated tumor cells. To test the hypothesis that PI3'K signaling is rate-limiting for KRASG12D-driven tumorigenesis we generated GEM models that express both KRASG12D and a constitutively active form of PIK3CA (PIK3CAH1047R) in the lung epithelium. As shown previously, expression of PIK3CAH1047R alone has no effect on the adult mouse lung. However, when PIK3CAH1047R was co-expressed with KRASG12D we noted a dramatic increase in overall lung tumor burden and a dramatic decrease in tumor latency. In addition, a larger fraction of KRASG12D/PIK3CAH1047R expressing lung tumors progressed to non-small cell lung cancer than in the KRASG12D alone controls. Hence, unlike oncogenic KRASG12D or BRAFV600E that serve as initiators of lung tumorigenesis, PIK3CAH1047R does not appear to be an initiator but rather a promoter of lung cancer progression. Next we tested the importance of PI3'-kinase signaling for lung tumor maintenance. Perhaps surprisingly, pharmacological inhibition of class 1 PI3'-kinases or the protein kinases AKT1-3 did not lead to substantial regression of established KRASG12D/PIK3CAH1047R expressing lung cancers. At best, these agents elicited cytostatic effects that led to slower tumor growth but with no statistically significant effect on overall mouse survival. These results stand in contrast to the effects of a MEK inhibitor that elicited frank regression of KRASG12D-induced lung tumors. Using mouse or human lung tumor derived cell lines expressing mutationally activated KRAS and PIK3CA we observed that pharmacological inhibition of either the ERK1/2 MAP kinase or the PI3'-kinase pathways resulted in a G1 cell cycle arrest that was enhanced when both pathways were inhibited simultaneously. However, only limited apoptosis was observed in cells treated with combined pathway inhibitors. Immunoblot analysis of downstream effectors indicated the ERK1/2 MAP kinase and the PI3'-kinase pathways coordinately regulate expression of c-MYC, p27KIP1, Cyclins D1 and A and the phosphorylation of the Retinoblastoma protein pRB thereby explaining the strong cooperative effects we see in cell cycle progression when both pathways are activated. Ongoing work aims to further analyze nodes of dual regulation and the factors that render KRASG12D/PIK3CAH1047R expressing lung cancers resistant to dual pathway inhibition. Identifying the signals generated by KRAS and PIK3CA to promote lung tumorigenesis will provide a better understanding of how best to target such cancers in the clinic and perhaps how best to combine pathway-targeted inhibitors to enhance apoptosis and thereby elicit tumor regression. Citation Format: Shon Green, Martin McMahon. PI3-kinase signaling is rate limiting for KRASG12D-driven lung tumorigenesis but is not required for tumor maintenance. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A37.

Activating PIK3CA Mutations Induce an Epidermal Growth Factor Receptor (EGFR)/Extracellular Signal-regulated Kinase (ERK) Paracrine Signaling Axis in Basal-like Breast Cancer*

Mutations in PIK3CA, the gene encoding the p110α catalytic subunit of phosphoinositide 3-kinase (PI3K) have been shown to transform human mammary epithelial cells (MECs). These mutations are present in all breast cancer subtypes, including basal-like breast cancer (BLBC). Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified 72 protein expression changes in human basal-like MECs with knock-in E545K or H1047R PIK3CA mutations versus isogenic MECs with wild-type PIK3CA. Several of these were secreted proteins, cell surface receptors or ECM interacting molecules and were required for growth of PIK3CA mutant cells as well as adjacent cells with wild-type PIK3CA. The proteins identified by MS were enriched among human BLBC cell lines and pointed to a PI3K-dependent amphiregulin/EGFR/ERK signaling axis that is activated in BLBC. Proteins induced by PIK3CA mutations correlated with EGFR signaling and reduced relapse-free survival in BLBC. Treatment with EGFR inhibitors reduced growth of PIK3CA mutant BLBC cell lines and murine mammary tumors driven by a PIK3CA mutant transgene, all together suggesting that PIK3CA mutations promote tumor growth in part by inducing protein changes that activate EGFR.

Using the MCF10A/MCF10CA1a Breast Cancer Progression Cell Line Model to Investigate the Effect of Active, Mutant Forms of EGFR in Breast Cancer Development and Treatment Using Gefitinib.

BackgroundBasal-like and triple negative breast cancer (TNBC) share common molecular features, poor prognosis and a propensity for metastasis to the brain. Amplification of epidermal growth factor receptor (EGFR) occurs in ~50% of basal-like breast cancer, and mutations in the epidermal growth factor receptor (EGFR) have been reported in up to ~ 10% of Asian TNBC patients. In non-small cell lung cancer several different mutations in the EGFR tyrosine kinase domain confer sensitivity to receptor tyrosine kinase inhibitors, but the tumourigenic potential of EGFR mutations in breast cells and their potential for targeted therapy is unknown.Materials and MethodsConstructs containing wild type, G719S or E746-A750 deletion mutant forms of EGFR were transfected into the MCF10A breast cells and their tumorigenic derivative, MCF10CA1a. The effects of EGFR over-expression and mutation on proliferation, migration, invasion, response to gefitinib, and tumour formation in vivo was investigated. Copy number analysis and whole exome sequencing of the MCF10A and MCF10CA1a cell lines were also performed.ResultsMutant EGFR increased MCF10A and MCF10CA1a proliferation and MCF10A gefitinib sensitivity. The EGFR-E746-A750 deletion increased MCF10CA1a cell migration and invasion, and greatly increased MCF10CA1a xenograft tumour formation and growth. Compared to MCF10A cells, MCF10CA1a cells exhibited large regions of gain on chromosomes 3 and 9, deletion on chromosome 7, and mutations in many genes implicated in cancer.ConclusionsMutant EGFR enhances the oncogenic properties of MCF10A cell line, and increases sensitivity to gefitinib. Although the addition of EGFR E746-A750 renders the MCF10CA1a cells more tumourigenic in vivo it is not accompanied by increased gefitinib sensitivity, perhaps due to additional mutations, including the PIK3CA H1047R mutation, that the MCF10CA1a cell line has acquired. Screening TNBC/basal-like breast cancer for EGFR mutations may prove useful for directing therapy but, as in non-small cell lung cancer, accompanying mutations in PIK3CA may confer gefitinib resistance.

Abstract P4-01-14: Whole exome sequencing of circulating and disseminated tumour cells in patients with metastatic breast cancer

Abstract Introduction: Circulating tumour cells (CTC) found in the blood of patients with cancer offer the potential to provide a repeatedly accessible source of tumour cells for the real-time assessment of tumour characteristics in patients with metastatic breast cancer (MBC). Questions remain to what extent CTC are truly representative of the actually present tumour mass in a patient at a specific moment in time and the molecular heterogeneity within the CTC population is only now being explored. Here, we report on the first results of an ongoing comparative study of mutation profiles of CTC and synchronously isolated disseminated tumour cells (DTC) from metastatic effusions or biopsies of solid metastases of patients with clinically progressive MBC. Materials and methods: For this project CTC are isolated from 7.5 ml blood samples of patients with MBC using the CellSearch system. CellSearch enriched CTC samples are subsequently further purified and sorted into several batches of 1-125 CTC per patient using the DEPArray system. DNA is isolated and amplified using the Ampli1 whole genome amplification (WGA) kit and subjected to whole exome paired-end sequencing (WES). DTC from metastatic effusions, fresh frozen tissue from solid metastases or the primary tumour, or - in patients with extremely high CTC counts (>10.000/7.5 ml) - pooled CTC from the CellSearch Profile sample, are sequenced as a comparator for mutation profiles. DNA from the buffy coat of white blood cells are sequenced to enable somatic mutation analysis. Results: Eight samples of 1-125 CTC and a CellSearch Profile sample of one patient with MBC who had ca. 30.000 CTC/7.5 ml of blood (patient 1) and 4 CTC samples of 5-10 CTC, 2 temporally matched DTC samples of 10 and 20 DTC from a pleural effusion and a fresh frozen tissue sample of the primary tumour of a second patient (patient 2) have been sequenced so far. Average base coverages were 13.6x (patient 1) and 11.8x (patient 2) for CTC/DTC samples and 175x and 120x for the CellSearch profile sample (patient 1) and the primary tumour sample (patient 2) respectively. Between 29.64% and 53.57% of the exomes of amplification products of CTC/DTC DNA were uncovered, probably due to technical limitations of the WGA procedure. Overall, if adequately covered, good concordances were observed for variants identified with MuTect in 28 frequently mutated genes in breast cancer between samples of amplification products of 1-125 CTC and the CellSearch Profile sample of patient 1. In patient 2, the same H1047R PIK3CA mutation was identified in the primary tumour and all CTC and DTC samples. In-depth analyses of the full exome data are being conducted. Discussion: Our data provide insight into clinically relevant questions to what extent CTC reflect mutational profiles in temporally matched metastatic tumour cells, and – by analysing multiple CTC samples of the same patient – genetic heterogeneity between CTC in patients with MBC. Sample accrual and analysis is being expanded and updated results will be presented at the conference. Citation Format: Dieter JE Peeters, Ken Op De Beeck, Anja Brouwer, Geert Vandeweyer, Patrick Pauwels, Marc Peeters, Peter B Vermeulen, Peter A van Dam, Steven J Van Laere, Guy Van Camp, Luc Y Dirix. Whole exome sequencing of circulating and disseminated tumour cells in patients with metastatic breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-01-14.

Co-activation of PIK3CA and Yap promotes development of hepatocellular and cholangiocellular tumors in mouse and human liver.

Activation of the PI3K and Yes-associated protein (Yap) signaling pathways has been independently reported in human hepatocellular carcinoma (HCC). However, the oncogenic interactions between these two cascades in hepatocarcinogenesis remain undetermined. To assess the consequences of the crosstalk between the PI3K and Yap pathways along liver carcinogenesis, we generated a mouse model characterized by combined overexpression of activated mutant forms of PIK3CA (PIK3CAH1047R) and Yap (YapS127A) in the mouse liver using hydrodynamic transfection (PIK3CA/Yap). In addition, suppression of PI3K and Yap pathways was conducted in human HCC and cholangiocarcinoma (CCA) cell lines. We found that concomitant activation of PI3K and Yap pathways triggered rapid liver tumor development in mice. Histologically, tumors were pure HCC, CCA, or mixed HCC/CCA. At the molecular level, PIK3CA/Yap tumors were characterized by activation of the mTORC1/2, ERK/MAPK, and Notch pathways. Simultaneous activation of PI3K and Yap pathways frequently occurred in human liver tumor specimens and their combined suppression was highly detrimental for the growth of HCC and CCA cell lines. In conclusion, our study demonstrates the oncogenic cooperation between PI3K and Yap pathways along liver carcinogenesis. The PIK3CA/Yap mouse represents an important preclinical liver tumor model for the development of novel therapeutics against this malignancy.

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.

Investigating the Structure and Dynamics of the PIK3CA Wild-Type and H1047R Oncogenic Mutant

The PIK3CA gene is one of the most frequently mutated oncogenes in human cancers. It encodes p110α, the catalytic subunit of phosphatidylinositol 3-kinase alpha (PI3Kα), which activates signaling cascades leading to cell proliferation, survival, and cell growth. The most frequent mutation in PIK3CA is H1047R, which results in enzymatic overactivation. Understanding how the H1047R mutation causes the enhanced activity of the protein in atomic detail is central to developing mutant-specific therapeutics for cancer. To this end, Surface Plasmon Resonance (SPR) experiments and Molecular Dynamics (MD) simulations were carried out for both wild-type (WT) and H1047R mutant proteins. An expanded positive charge distribution on the membrane binding regions of the mutant with respect to the WT protein is observed through MD simulations, which justifies the increased ability of the mutated protein variant to bind to membranes rich in anionic lipids in our SPR experiments. Our results further support an auto-inhibitory role of the C-terminal tail in the WT protein, which is abolished in the mutant protein due to loss of crucial intermolecular interactions. Moreover, Functional Mode Analysis reveals that the H1047R mutation alters the twisting motion of the N-lobe of the kinase domain with respect to the C-lobe and shifts the position of the conserved P-loop residues in the vicinity of the active site. These findings demonstrate the dynamical and structural differences of the two proteins in atomic detail and propose a mechanism of overactivation for the mutant protein. The results may be further utilized for the design of mutant-specific PI3Kα inhibitors that exploit the altered mutant conformation.

Abstract A35: A recurrent neomorphic mutation in MYOD1 defines a clinically aggressive subset of embryonal rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma diagnosed in children. To identify novel and recurrent oncogenic mutations in RMS, we performed whole exome sequencing on 20 RMS samples, including 9 alveolar RMS, and 11 embryonal RMS (ERMS). Two ERMS samples showed the same c.365T>G point mutation in MYOD1, leading to an L122R substitution. Both were primary tumor samples and, in each case, normal tissue did not harbor the mutation, confirming its somatic nature. The L122R mutation occurs in the conserved basic region of MYOD1, at a highly specific residue that is a leucine in all myogenic bHLH transcription factors (MYOD1, MYF5, myogenin, MRF4) but is an arginine in MYC. Interestingly, previous studies of artificial mutants of the MYOD1 basic region found that MYOD1 L122R competes wild-type MYOD1 but without leading to target gene transactivation and also binds to MYC consensus sequences, regulating target genes similarly to MYC. We genotyped an additional 97 ERMS samples for the MYOD1 L122R point mutation using a mass spectrometry-based platform. The mutation was detected in 8 additional cases, resulting in an overall prevalence of 10% (10/103) in our ERMS samples. Corresponding normal tissue was available in 6 of the 8 additional cases, and, again, no mutations were identified in germline DNA. The 10 ERMS tumors harboring the MYOD1 L122R mutation displayed highly cellularity and frequent spindle cell morphology with uniform strong positivity for MYOD1 by IHC, features associated with the adult spindle cell variant of ERMS. Notably, these ERMS with MYOD1 mutations were from patients diagnosed in later childhood or adulthood (mean age = 25; median age = 28), and were more likely to arise in the head/neck (8/10 vs 16/80; p=0.0003). Furthermore, the overall survival of this subgroup of patients was significantly poorer than for ERMS lacking this mutation (0% vs 48% at 10 yrs; p=0.02). Transfection of MYOD1 L122R or wild type MYOD1 into C2C12 mouse myoblasts showed that MYOD1 L122R favors proliferation and transformation over myogenic differentiation. In a previous report, we had identified activating PIK3CA mutations in 3 of 60 ERMS samples. Remarkably, all 3 of these PIK3CA-mutated tumor samples are among the 10 MYOD1 L122R cases identified here. Therefore, we examined the combined effect of PIK3CA H1047R and MYOD1 L122R in soft-agar colony formation assays in C2C12 cells; this resulted in an increased number of colonies compared to either PIK3CA H1047R alone or PIK3CA H1047R + wild type MYOD1. Finally, expression profiling experiments in C2C12 cells and in BJ human fibroblasts comparing MYOD1 L122R, wild type MYOD1, and MYC showed that MYOD1 L122R shifts gene expression from a myogenic program to a MYC-like program. In summary, we have identified a distinctive subset of ERMS with recurrent somatic mutations in MYOD1, a transcription factor that functions as a critical regulator of skeletal muscle differentiation, altering its function resulting in impaired myogenic differentiation and enhanced proliferation. The tumors tend to present in older patients, usually arise in the head/neck, overlap pathologically with the recently described spindle cell variant of ERMS, and have especially poor outcomes. This represents a novel, molecularly defined subset of RMS that should be considered for high risk protocols and targeted therapeutic development. Citation Format: Neerav Shukla, Shinji Kohsaka, Nabahet Ameur, Angela Yannes, Agnes Viale, Li-Xuan Qin, Snjezana Dogan, Raf Sciot, Julia Bridge, Paul Meyers, Leonard Wexler, Jonathan Fletcher, Marc Ladanyi. A recurrent neomorphic mutation in MYOD1 defines a clinically aggressive subset of embryonal rhabdomyosarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A35.

Abstract 3925: The PIK3CA H1047R mutation can initiate tumorigenesis in the colon

Abstract Background: Our understanding of how mutations within the intestinal epithelium result in tumor development and progression is continuously evolving. We have previously described the FC PIK3ca* murine colon cancer model which expresses a constitutively activated phosphoinositide-3 kinase (PI3K) in the intestinal epithelium. The expression of this dominantly active form of PI3K resulted in invasive mucinous adenocarcinomas in the proximal colon through a non-canonical mechanism. We sought to determine if the hotspot PIK3CA H1047R mutation, commonly found in human colorectal cancers, can also induce intestinal tumorigenesis. Methods: Female FVB mice carrying a conditional PIK3CA H1047R mutation (PIK3CAH1047R) were crossed to B6 males carrying atransgene in which the fatty acid binding protein promoter is fused to Cre recombinase (FC). (FVB x B6)F1 FC PIK3CAH1047R progeny express this constitutively active form of PI3K in epithelial cells of the distal small bowel and colon. 22 F1 FC PIK3CAH1047R mice were examined. Necropsy was performed at approximately 200 days of age or once moribund. Results: 50% of FC PIK3CAH1047R mice develop invasive adenocarcinomas within the colon. These cancers are highly mucinous and often penetrate through the serosal layer of the colon. These lesions can be found throughout the colon, but are more common proximally. No tumors were identified within the small intestine. These cancers can have associated invasion into the regional lymphatics and metastases to the mesentery. Interestingly these cancers have a minimal luminal component compared to the amount of submucosal extension. Precursor lesions are able to be identified and are not polypoid. These cancers demonstrate activation of the AKT signaling pathway, but do not demonstrate nuclear localization of β-catenin. Conclusion: This is the first report of the hotspot PIK3CA H1047R mutation leading to intestinal tumorigenesis through a non-canonical mechanism, resulting in flat mucinous invasive adenocarcinomas. Citation Format: Cheri Pasch, Molly Maher, Dustin Deming. The PIK3CAH1047R mutation can initiate tumorigenesis in the colon. [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 3925. doi:10.1158/1538-7445.AM2014-3925

Abstract 1876: Combination of PI3KCA targeting with irradiation: A preclinical study on head and neck cancer cell lines

Abstract Purpose In complement to anti-EGFR therapy, the targeting of PI3KCA/AKT/mTOR signalling pathway is of particular interest in the management of HNSCC. Its activation is recognized as an important mechanism in tumor progression and resistance to EGFR inhibitors. Of importance, PI3KCA inhibitors have shown their ability to selectively inhibit the PI3KCA/AKT signalling and to enhance the effects of conventional cytotoxic agents. Therefore, it would be particularly interesting to assess the effects of PI3KCA inhibition combined with anti-EGFR monoclonal antibody (cetuximab) and/or irradiation (RT). Experimental procedures We identified two HNSCC cell lines (Cal 33, PIK3CA H1047R mutated and Cal 27, wild type) and examined the effects of the combination of BKM120 (specific inhibitor of PI3KCA), cetuximab and RT. Experimental conditions were: BKM120, cetuximab, RT alone; sequence 1 (BKM120 followed by cetuximab), sequence 2 (cetuximab followed by BKM120) and combination (BKM120 + cetuximab together) with/without RT given ahead of all treatments. Drugs were administered at their IC50. Cell survival was assessed by MTT test. Combination effects were analyzed by the Combination Index (CI) and Compusyn software. Biochemical factors were examined for the impact on proliferation, apoptosis (caspases), DNA repair (ERCC1, XRCC1, DNA-PK, ATM) and PI3KCA/AKT/mTOR pathway p-P70/S6k, p-Akt/Akt, PTEN). Gene expression was determined by real time-PCR and protein expression by western blot. Results We evidenced sequence 2 as the best combined treatment in inhibiting cell proliferation in both cell lines (ΔIC50 = - 7.91*10-6 M in Cal 7 and ΔIC50 = - 5.7*10-6 M in Cal 33; being ΔIC50 the difference of IC50 between sequence 2 and BKM120 alone). CI values confirmed a synergistic interaction between the two drugs given sequentially cetuximab + BKM120 in both cell lines. Addition of RT ahead of all treatments did not add any anti-proliferative effect to the treatments on Cal 33. Of note, on Cal 27 the addition of RT to BKM120 increased the antiproliferative effect of this drug (p<0.05). Overall, preliminary data on protein expression indicated an upregulation of caspase 3 and 9 only in Cal 27 suggesting an apoptotic pathway activation after sequence 1 and 2, not seen in Cal 33. Moreover Akt was upregulated in Cal 27 after sequence 1 and 2. Analysis of pAkt is on going to verify if blocking of PI3KCA by BKM120 in our models could lead to a control of the feedback loop induced by mTORC1 inhibition. This may be supported by the observed upregulation of p4EBP1 (downstream) following sequence 1 and 2. Akt upregulation due to the treatments may also explain the lack of a significative effect of RT on cell proliferation, mainly in Cal 27 wild-type cells. Conclusions The results of this in vitro could serve as a basis for early clinical trials associating BKM120 and RT in locally advanced HNSCC, providing interesting biochemical markers to conduct personalised therapy. Citation Format: Laura Lattanzio, Federica Tonissi, Martino Monteverde, Luca Gianello, Elvio Russi, Marco C. Merlano, Cristiana Lo Nigro, Gerard Milano. Combination of PI3KCA targeting with irradiation: A preclinical study on head and neck cancer cell lines. [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 1876. doi:10.1158/1538-7445.AM2014-1876

Hotspot mutations in common oncogenes are infrequent in nasopharyngeal carcinoma.

Oncogene mutations contribute to carcinogenesis and can provide potential therapeutic targets for clinical anticancer management. However, oncogene mutation patterns in nasopharyngeal carcinoma(NPC) have yet to be fully elucidated. To gain insight into mutation patterns in NPC, a high-throughput OncoCarta panel assay was used to determine 238 hotspot mutations across 19 common oncogenes in 8 NPC cell lines and 160 NPC patient samples from southern China. Statistical analyses were further conducted to identify associations between oncogene mutations and selected clinicopathological characteristics. In total, we identified 24 mutations across 11 oncogenes in 17 (10.6%) NPC patients. Four patients exhibited mutations in at least one oncogene. We also identified a PIK3CA H1047R mutant in 7 NPC cell lines. In addition, oncogene mutations showed no correlation with either risk habits (smoking and drinking) or other clinical characteristics except for TNM stage. KIT mutations were associated with poorer overall and relapse-free survival. Furthermore, KIT mutations together with age and N stage were independent prognostic factors in NPC. Taken together, the present study is the first report on mutations in multiple oncogenes in NPC. We found that hotspot oncogene mutations are infrequent in NPC patients from southern China. The lack of hotspot mutations requires a comprehensive characterization of gene mutations in NPC for developing new therapeutic targets in the future.

Assessing PIK3CA and PTEN in Early-Phase Trials with PI3K/AKT/mTOR Inhibitors

Despite a wealth of preclinical studies, it is unclear whether PIK3CA or phosphatase and tensin homolog (PTEN) gene aberrations are actionable in the clinical setting. Of 1,656 patients with advanced, refractory cancers tested for PIK3CA or PTEN abnormalities, PIK3CA mutations were found in 9% (146/1,589), and PTEN loss and/or mutation was found in 13% (149/1,157). In multicovariable analysis, treatment with a phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) inhibitor was the only independent factor predicting response to therapy in individuals harboring a PIK3CA or PTEN aberration. The rate of stable disease ≥6 months/partial response reached 45% in a subgroup of individuals with H1047R PIK3CA mutations. Aberrations in the PI3K/AKT/mTOR pathway are common and potentially actionable in patients with diverse advanced cancers. This work provides further important clinical validation for continued and accelerated use of biomarker-driven trials incorporating rational drug combinations.

Abstract B38: Evaluation of the OnTarget™ system for highly sensitive mutation detection.

Abstract Background: Effective non-invasive methods for patient characterization and monitoring during treatment are desired. Cell-free circulating tumor DNA in plasma is elevated in cancer patients, and can be a valuable tool for evaluating mutation profile over time. Low mutant allele fraction in a limited amount of low quality DNA makes it very challenging to accurately characterize plasma samples. Therefore, a highly sensitive and robust testing method is required for genetic characterization of circulating tumor DNA. The goals of this study were to: • Evaluate assay performance and limit of detection of the Boreal Genomics OnTarget™ system 18-mutation panel using human germline DNA • Assess the relative sensitivity of the OnTarget™ system for KRAS G13D and PIK3CA H1047R mutation detection using cell line derived DNA • Compare assay performance and sensitivity with multiple DNA input amounts Materials and methods: DNA was extracted from normal healthy human blood and HCT116 (KRAS G13D and PIK3CA H1047R heterozygous mutant) cell line pellets. The cell line DNA was diluted with the human blood DNA to produce samples with known ratios of mutant and wild-type alleles. Samples were run with the OnTarget™ mutation detection technology platform 18-mutation panel. The platform utilizes oligo probes and a gel based system to enrich targeted mutant fragments from a DNA sample, followed by PCR amplification and next generation sequencing with the Illumina MiSeq™ platform. The 18-plex panel tests mutant allele percentage of 18 common cancer mutations in the BRAF, EGFR, KRAS, and PIK3CA genes. Results: Repeat testing of germline DNA from multiple human donors was conducted to determine the background variability (noise) of the 18-plex assay. The mean mutant allele percentage for 10 of the 18 mutations assessed was under 0.001%. 7 of the mutations were under 0.01%, with one other mutation was just over 0.01%. Comparing mutant allele percentages of the titrated cell line DNA samples assessed limit of detection of two of the assays at the 300ng input level. The PIK3CA H104R assay detected mutant DNA over the background levels at the 0.01 and 0.001% input amount of mutant DNA. The KRAS G13D assay signal was over background at the 0.01% input, but within the assay noise at the 0.001% input. When testing the same samples at 30ng of DNA input, all mutation detection was similar, except the PIK3CA mutation was not detected at the 0.001% input amount. Summary: The OnTarget™ mutation enrichment system followed by NGS provides high sensitivity mutation profiling of DNA with high multiplexing capabilities. The low DNA input requirement and limit of detection make it a useful platform for testing of cell-free circulating plasma DNA. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B38. Citation Format: Russell M. Walker, Sara Dunaj, Dalin Chan, Sunita Badola. Evaluation of the OnTarget™ system for highly sensitive mutation detection. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B38.

Abstract B26: Actionable mutations in cell-free DNA in plasma of patients with advanced cancers referred for experimental targeted therapies.

Abstract Background: Actionable mutations confer a survival advantage to cancer cells and can provide therapeutic targets. Cell-free (cf) DNA in the plasma of cancer patients offers an easily obtainable, low-risk, and inexpensive source of biologic material for mutation analysis. Methods: cfDNA in plasma samples from patients with advanced cancers who progressed on systemic therapy was tested for 21 mutations in BRAF (codon 600), EGFR (exons 19-21), KRAS (codons 12, 13), and PIK3CA (exons 9, 20) using the BEAMing method. Results were compared to mutation analysis results from archival primary or metastatic tumor tissue from a CLIA-certified laboratory. Results: cfDNA was extracted from 155 patients with advanced cancers. BRAF mutations were detected in 27% (33/122) plasma samples and in 30% (37/122) of archival tumor samples resulting in agreement in 88.5% cases (kappa=0.72, 95% confidence interval [CI] 0.58- 0.86). EGFR mutations were detected in 8% (6/77) plasma samples and in 6% (5/77) tumor samples resulting in agreement in 98.7% cases (kappa=0.90, 95% CI 0.71- 1.09). KRAS mutations were detected in 50% (55/110) of plasma samples and in 53% (58/110) of tumor samples, resulting in agreement in 82.7% cases (kappa=0.66, 95% CI 0.51- 0.80). PIK3CA mutations were detected in 20% (19/94) of plasma samples and in 14% (13/94) of tumor samples, resulting in concordance in 91.5% cases (kappa=0.70, 95% CI 0.51- 0.89). Of interest, 2 of 9 patients with BRAF mutations (both melanoma) in the tumor, but not in cfDNA had plasma collection at the time of progression on prior BRAF targeting therapy. Also, a patient with V600E BRAF-mutated melanoma, who previously progressed on a MEK inhibitor, displayed a G12R KRAS mutation in cfDNA and a patient with V600K BRAF-mutated melanoma with additional E542K and H1047R PIK3CA mutations in cfDNA was ultimately refractory to BRAF inhibitor treatment. Also, a V600K BRAF mutation was found in the cfDNA of a patient with colorectal cancer and a G12V KRAS mutation. In addition, 2 patients with EGFR-mutated lung adenocarcinoma (exon 19 and L858R), who progressed on erlotinib, had T790M EGFR (n=2) and E545K PIK3CA (n=1) mutations in cfDNA, plausibly explaining secondary resistance to erlotinib. A patient with Erdheim-Chester disease and a V600E BRAF mutation also had a T790M EGFR mutation in cfDNA. Finally, a patient with H1047R PIK3CA mutant ovarian cancer showed an additional G12C KRAS mutation in cfDNA and experienced a partial response followed by early progression on a PI3K inhibitor. Patient characteristics, mutation types and detailed discrepancy analysis will be presented. Conclusions: Detecting actionable mutations in cfDNA is a noninvasive alternative to mutation testing in tumor tissue with an acceptable level of concordance, and should be investigated further for selecting appropriate targeted therapies and monitoring mutation profiles. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B26. Citation Format: Filip Janku, Philipp Angenendt, Aung Naing, Gerald S. Falchook, Apostolia M. Tsimberidou, Veronica R. Holley, Siqing Fu, David S. Hong, Jennifer J. Wheler, Sarina A. Piha-Paul, Ralph G. Zinner, Daniel D. Karp, Vivek Subbiah, Kevin B. Kim, Funda Meric-Bernstam, Frank Diehl, Razelle Kurzrock. Actionable mutations in cell-free DNA in plasma of patients with advanced cancers referred for experimental targeted therapies. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B26.

Abstract 2069: Combination of mTOR targeting with cetuximab and conventional chemotherapy: a preclinical study on an orthotopic xenograft model of head and neck cancer.

Abstract Background: Preclinical and clinical studies on head and neck squamous cell carcinoma (HNSCC) demonstrated synergistic anti-tumoral effects when combining anti-EGFR agents with conventional chemotherapeutic drugs such as cisplatin (C) and 5-fluorouracil (F). In a previous experimental study, our group pointed out an enhancement of PIK3CA signaling (pAKT) in tumors re-growing after cetuximab-based treatment (Bozec et al. Br J Cancer 2007). This context justifies investigations on mTOR inhibitors combined with cetuximab (Cetux) in HNSCC. Methods: The effects on tumor growth of treatments combining an mTOR inhibitor (Temsirolimus (Tem)), an anti-EGFR (Cetux) and a clinically validated-chemotherapeutic regimen (C+F) were examined in an orthotopic (floor of the mouth) model of human HNSCC (CAL33 cell line, over-expressing EGFR, secreting VEGF-A, H1047R PIK3CA mutated). In parallel, in-vitro experiments were conducted in order to strengthen the data arising from the in-vivo study. On day 0, cells were injected in nude NMRI mice. Treatment was started on day 3 and consisted of 8 groups (10 mice/group) treated as follows: Control, Tem (5 mg/kg i.p. five times a week), Cetux (2.5 mg/kg i.p. once a week), cisplatin (6 mg/kg) + 5-fluorouracil (17 mg/kg) (i.p. once a week), concomitant treatment combining Tem with either Cetux, C+F, or Cetux+C+F. The tumor growth was recorded by IVIS imaging and on day 13 after animal euthanasia with a caliper ruler. Results: All tested treatments produced significant growth inhibition as compared to controls (p<0.01). The Tem-Cetux and Tem-Cetux+C+F regimens produced the strongest growth-inhibiting effects, however the addition of C+F to the Tem-Cetux regimen did not result in a significantly greater effect as compared to Tem-Cetux (p = 0.18). The growth-inhibiting effects of Tem-Cetux were significantly stronger than those of Tem or Cetux alone (p<0.004). Moreover, a significant synergism was demonstrated between Temsirolimus and Cetuximab effects (significant interaction: p = 0.001, ANOVA). Furthermore, in contrast to mice receiving the Tem-Cetux+C+F regimen, mice treated with Tem-Cetux had no clinical signs of secondary effects. Accordingly, the mice weight loss measured on day 12 was significantly lower in mice receiving Tem-Cetux regimens as compared to mice receiving the Tem-Cetux+C+F regimen (p=0.005). IHC analysis on tumor vasculature (VEGFR2), proliferation status (Ki67) and downstream signaling phospho-proteins (pAKT, p-P70/S6k, pERK) strengthen our data on tumor growth and proliferation. Conclusion: These data on the association of temsilorimus with cetuximab could serve as a strong basis for innovative treatments aiming at an optimal management of patients with recurrent/metastatic HNSCC. Citation Format: Alexandre Bozec, Nathalie Ebran, Martino Monteverde, Nicolas Toussan, Anne Cayre, Marie-Cristine Etienne-Grimaldi, Cristiana Lo Nigro, Anne Sudaka, Marco Merlano, Frederique Penault-Llorca, Gerard Milano. Combination of mTOR targeting with cetuximab and conventional chemotherapy: a preclinical study on an orthotopic xenograft model of head and neck cancer. [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 2069. doi:10.1158/1538-7445.AM2013-2069

R132C IDH1 Mutations Are Found in Spindle Cell Hemangiomas and Not in Other Vascular Tumors or Malformations

Spindle cell hemangioma (SCH) is a rare, benign vascular tumor of the dermis and subcutis. The lesions can be multifocal and are overrepresented in Maffucci syndrome, in which patients also have multiple enchondromas. Somatic mosaic R132C IDH1 hotspot mutations were recently identified in Maffucci syndrome. We evaluated the presence of mutations in solitary and multiple SCHs in patients without multiple enchondromas and tested a range of other vascular lesions that enter into the differential diagnosis. The R132C IDH1 mutation was identified by hydrolysis probes assay and confirmed by Sanger sequencing in 18 of 28 (64%) SCHs; of the 10 negative cases, 2 harbored a mutation in IDH2 (R172T and R172M) by Sanger sequencing. None of 154 other vascular malformations and tumors harbored an IDH1 R132C mutation, and R132H IDH1 mutations were absent in all 182 cases. All 16 SCHs examined by immunohistochemistry were negative for expression of HIF-1α. In conclusion, 20 of 28 (71%) SCHs harbored mutations in exon 4 of IDH1 or IDH2. Given that mutations were absent in 154 other vascular lesions, the mutation seems to be highly specific for SCH. The mutation does not induce expression of HIF-1α in SCH, and therefore the exact mechanism by which mutations in IDH1 or IDH2 lead to vascular tumorigenesis remains to be established.

Network quantification of EGFR signaling unveils potential for targeted combination therapy

The epidermal growth factor receptor (EGFR) signaling network is activated in most solid tumors, and small-molecule drugs targeting this network are increasingly available. However, often only specific combinations of inhibitors are effective. Therefore, the prediction of potent combinatorial treatments is a major challenge in targeted cancer therapy. In this study, we demonstrate how a model-based evaluation of signaling data can assist in finding the most suitable treatment combination. We generated a perturbation data set by monitoring the response of RAS/PI3K signaling to combined stimulations and inhibitions in a panel of colorectal cancer cell lines, which we analyzed using mathematical models. We detected that a negative feedback involving EGFR mediates strong cross talk from ERK to AKT. Consequently, when inhibiting MAPK, AKT activity is increased in an EGFR-dependent manner. Using the model, we predict that in contrast to single inhibition, combined inactivation of MEK and EGFR could inactivate both endpoints of RAS, ERK and AKT. We further could demonstrate that this combination blocked cell growth in BRAF- as well as KRAS-mutated tumor cells, which we confirmed using a xenograft model.