PIK3CA Amplification Research Articles

Integrative genomic analysis implicates gain of PIK3CA at 3q26 and MYC at 8q24 in chronic lymphocytic leukemia.

The disease course of chronic lymphocytic leukemia (CLL) varies significantly within cytogenetic groups. We hypothesized that high-resolution genomic analysis of CLL would identify additional recurrent abnormalities associated with short time-to-first therapy (TTFT). We undertook high-resolution genomic analysis of 161 prospectively enrolled CLLs using Affymetrix 6.0 SNP arrays, and integrated analysis of this data set with gene expression profiles. Copy number analysis (CNA) of nonprogressive CLL reveals a stable genotype, with a median of only 1 somatic CNA per sample. Progressive CLL with 13q deletion was associated with additional somatic CNAs, and a greater number of CNAs was predictive of TTFT. We identified other recurrent CNAs associated with short TTFT: 8q24 amplification focused on the cancer susceptibility locus near MYC in 3.7%; 3q26 amplifications focused on PIK3CA in 5.6%; and 8p deletions in 5% of patients. Sequencing of MYC further identified somatic mutations in two CLLs. We determined which catalytic subunits of phosphoinositide 3-kinase (PI3K) were in active complex with the p85 regulatory subunit and showed enrichment for the α subunit in three CLLs carrying PIK3CA amplification. Our findings implicate amplifications of 3q26 focused on PIK3CA and 8q24 focused on MYC in CLL.

Mutation and genomic amplification of the PIK3CA proto-oncogene in pituitary adenomas

The tumorigenesis of pituitary adenomas is poorly understood. Mutations of the PIK3CA proto-oncogene, which encodes the p110-α catalytic subunit of PI3K, have been reported in various types of human cancers regarding the role of the gene in cell proliferation and survival through activation of the PI3K/Akt signaling pathway. Only one Chinese study described somatic mutations and amplification of the PIK3CA gene in a large series of pituitary adenomas. The aim of the present study was to determine genetic alterations of PIK3CA in a second series that consisted of 33 pituitary adenomas of different subtypes diagnosed by immunohistochemistry: 6 adrenocorticotropic hormone-secreting microadenomas, 5 growth hormone-secreting macroadenomas, 7 prolactin-secreting macroadenomas, and 15 nonfunctioning macroadenomas. Direct sequencing of exons 9 and 20 assessed by qPCR was employed to investigate the presence of mutations and genomic amplification defined as a copy number ≥4. Previously identified PIK3CA mutations (exon 20) were detected in four cases (12.1%). Interestingly, the Chinese study reported mutations only in invasive tumors, while we found a PIK3CA mutation in one noninvasive corticotroph microadenoma. PIK3CA amplification was observed in 21.2% (7/33) of the cases. This study demonstrates the presence of somatic mutations and amplifications of the PIK3CA gene in a second series of pituitary adenomas, corroborating the previously described involvement of the PI3K/Akt signaling pathway in the tumorigenic process of this gland.

The Association of PI3 Kinase Signaling and Chemoresistance in Advanced Ovarian Cancer

Evidence that the phosphoinositide 3-kinase (PI3K) pathway is deregulated in ovarian cancer is largely based on the analysis of surgical specimens sampled at diagnosis and may not reflect the biology of advanced ovarian cancer. We aimed to investigate PI3K signaling in cancer cells isolated from patients with advanced ovarian cancer. Ascites samples were analyzed from 88 patients, of whom 61 received further treatment. Cancer cells were immunomagnetically separated from ascites, and the signaling output of the PI3K pathway was studied by quantifying p-AKT, p-p70S6K, and p-GSK3β by ELISA. Relevant oncogenes, such as PIK3CA and AKT, were sequenced by PCR-amplified mass spectroscopy detection methods. In addition, PIK3CA and AKT2 amplifications and PTEN deletions were analyzed by FISH. p-p70S6K levels were significantly higher in cells from 37 of 61 patients who did not respond to subsequent chemotherapy (0.7184 vs. 0.3496; P = 0.0100), and this difference was greater in patients who had not received previous chemotherapy. PIK3CA and AKT mutations were present in 5% and 0% of samples, respectively. Amplification of PIK3CA and AKT2 and deletion of PTEN was seen in 10%, 10%, and 27% of samples, respectively. Mutations of PIK3CA and amplification of PIK3CA/AKT2 or deletion of PTEN did not correlate with levels of p-AKT, p-p70S6K, and p-GSK3β. In patients with advanced ovarian cancer, there is an association between levels of p-p70S6K and response to subsequent chemotherapy. There is no clear evidence that this is driven specifically by PIK3CA or AKT mutations or by amplifications or deletion of PTEN.

Cancer gene profile of metastatic breast cancer.

1015 Background: There is great interest in using genomic information to guide therapy selection in cancer patients. The aim of this study was to determine the spectrum of genomic alterations identified in MBC patients, and evaluate the concordance of alterations between primary and recurrent tumors. Methods: We performed comprehensive profiling on formalin-fixed paraffin embedded samples from 42 patients with MBC using a targeted next generation sequencing (NGS) assay in a CLIA laboratory (Foundation Medicine). Genomic libraries were captured for 3,230 exons in 182 cancer related genes plus 37 introns from 14 genes often rearranged in cancer and sequenced to an average depth of 390X with 99% of bases covered >100X. In total 30 primary and 37 recurrent tumors were profiled, including 3 separate recurrences in 1 patient and matched primary-recurrences in 22 patients. Point mutations, indels, copy number alterations and rearrangements were assessed. Alterations that are targetable with established or investigational therapeutics were considered “actionable”. Results: At least 1 genomic alteration was identified in all but 2 breast samples (both primary tumors). Point mutations were identified in several cancer-related genes including PIK3CA, TP53, PTEN, CDH1, ARID1A, AKT1, NF1, FBXW7 and FGFR3. Amplification was observed in HER2; 11 of 12 HER2 IHC positive samples were found to have HER2 gains by NGS; in addition, a HER2 gain was identified by NGS in a HER2- (1+ IHC) sample. Amplification of PIK3CA, IGF1R, FGFR2, AKT2, MDM2, and MCL1 plus a CDKN2A homozygous deletion were also identified. While the majority of known driver alterations (85%) were concordant in the matched pairs of primary and recurrent tumors, in 11 of 22 sets there was at least 1 discordant driver alteration, and these included both gains and losses of potential therapeutic targets. Overall 32 of 42 patients (76%) had an actionable genomic alteration. Conclusions: Genomic profiling of breast cancer samples reveals genomic alterations in most metastatic breast cancer patients. Over three quarters of patients have actionable findings, suggesting that genomic profiling may assist in individualized pathway-directed therapy.

Frequency and clinical characterization of NSCLC patients harboring PIK3CA mutations identified within a regional screening network.

10526 Background: PIK3CA mutations are a rare oncogenic event of potential therapeutic relevance in NSCLC. Here we report frequency and characteristics of patients with PIK3CA mutated lung tumors. Methods: Patients with NSCLC and PIK3CA mutations were identified within our regional Network for Molecular Screening in Lung Cancer. We further analyzed the presence of BRAF, KRAS, EGFR mutations as well as ALK translocation, ERBB2 and FGFR1 amplifications in PIK3CA mutated samples. Clinical data on age, sex, TNM classification and tumor stage, histological type, grading, overall survival, smoking status, comorbidity, BMI and secondary malignancies were retrieved from clinical charts in accordance with the local ethics committee. Results: PIK3CA mutations were detected with a frequency of 3.7% (24% exon 20,76% exon 9) in 1000 patients. Histologically 32% were defined as squamous cell carcinoma, 48% as adenocarcinoma and 18% other histological subtypes or NSCLC-NOS. Exon 9 mutations were present in the acinar and lepidic subtype, whereas exon 20 mutations were seen in the papillary and solid subtype. Cooccuring genetic lesions were observed in 16% (mutations in KRAS=2, EGFR=1, BRAF=1; FGFR1 amplification=2). 14 were female, 23 male with a mean age of 69 years. 21 of these patients were further clinically annotated. 11 patients presented with stage IIIb/IV eligible for palliative treatment and 10 stage I – IIIa eligible for surgical therapy +/- adjuvant therapy. All but 1 patient were smokers with an average BMI of 26,2kg/m2 with a typical high load of comorbidity mainly of cardiovascular diseases, 8 of 21 patients showed prior malignancies in their medical history. The median overall survival within this population has not been reached yet. Conclusions: Screening for PIK3CA mutations is feasible. A high proportion (38%) of patients with PIK3CA mutated lung cancer have prior malignancies and show a high load of comorbidity. Furthermore PIK3CA mutations are not exclusive to KRAS, EGFR or BRAF mutations or FGFR1 amplifications. Successful identification of patients with oncogenic lesions in lung cancer in a screening network might allow future personalized treatment of these patients.

Pilot trial of molecular profiling and targeted therapies in advanced thoracic malignancies: Non-small cell lung cancer, small cell lung cancer and thymic malignancies (CUSTOM).

TPS7609 Background: For decades, clinical trial design strategies have relied upon the broad classification of tumors into tumor site and histopathology. Several lines of evidence have shown that a molecular approach to patient selection may be better in its capacity to improve patient outcomes. CUSTOM is an innovative clinical trial that allows for the parallel evaluation of multiple targeted therapies in molecularly selected patients with multiple cancer histological subtypes and the prospective evaluation of multiple molecular biomarkers. Methods: All patients with advanced lung cancer and thymic malignancies and a good performance status are eligible independently of previous lines of treatment. The trial begins with tumor biopsy and molecular profiling using a multi-platform approach to identify oncogenic alterations in more than 34 genes for treatment allocation (12 genes) and exploratory purposes. Depending upon the specific biomarker identified, patients are then triaged into 5 experimental arms. Erlotinib for sensitizing EGFR mutations; AZD6244 for KRAS, NRAS, HRAS and BRAF mutations; MK2206 for PIK3CA, AKT and PTEN mutations and PIK3CA amplification; Lapatinib for ERBB2 mutations or amplification; Sunitnib for KIT mutations and PDGFRA mutations and amplification. Patients not eligible for the experimental treatment arms are enrolled into a standard treatment arm. Upon disease progression patients are eligible for repeat biopsy and molecular profiling in order to identify molecular changes and mechanisms of resistance. All patients are followed until death. The primary endpoint is response rate and secondary endpoints include progression-free survival, duration of response and overall survival. With three disease types and five experimental drugs, there are 15 possible treatment arms which will be under active consideration. For each of these, the study will be conducted as an optimal two-stage phase II trial in order to rule out an unacceptably low 10% clinical response rate in favor of a modestly high response rate of 40%. As of January 11, 2012, two hundred and sixteen patients have been enrolled.

Towards individualisation of treatment in endometrial cancer

Endometrial cancer is the most common gynaecological cancer in the Western world, and its incidence is rising in most European countries, largely owing to increasing obesity. The majority are early stage, low-grade tumours associated with a favourable prognosis, and current classifications describe type 1 and 2 categories based primarily on morphological and molecular criteria. Hormone therapy has been shown to be effective predominantly in type 1 tumours and subgroups identified by classical receptor evaluation (Decruze and Green, 2007). Recent clinical interest has focused on identifying poor prognosis tumours and evaluating the benefit of adjuvant cytotoxic chemotherapy. This follows the demonstration of survival gain in advanced disease (Humber et al, 2007), but these therapies are toxic and not suitable for many elderly patients with concurrent medical problems. Activation of the P13K pathway has been observed in ~30% of type 1 endometrial cancers and 20% of type 2 endometrial cancers, although mTOR-directed targeted therapy has shown only modest activity to date. Other PI3K pathway alterations leading to deregulated signalling include PIK3CA amplification or mutation and mutation in the AKT gene (Dedes et al, 2011). These observations provide interesting challenges for a personalised approach to the treatment of these tumours.

Abstract 4602: Preclinical evaluation of the AKT inhibitor MK2206 in nasopharyngeal carcinoma cell lines

Abstract MK2206 is an allosteric inhibitor of AKT with potent activity in cancer cell lines that exhibit genetic aberrations in the PI3K-AKT-PTEN pathway. AKT is frequently activated in nasopharyngeal carcinoma (NPC), while PIK3CA amplification is found in 40-70% of NPC tissues. The preclinical activity of MK2206 was evaluated in NPC cell lines: HONE-1-EBV, HONE-1, CNE-2, HNE-1, and 2 cell lines with PIK3CA amplifications, HK-1 and C666-1. All 6 cell lines showed increased basal level of activated (p-)AKT(ser473), p-GSK3β, p-mTOR, pi-p70S6K and p-4EBP1, except in C666-1 cells where p-70S6K and p-4EBP1 were weakly expressed. Basal expression of p-MAPK was most pronounced in HK-1, CNE-2 and HONE-1-EBV, and weakly expressed in C666-1 and HONE-1. The effect on cell growth was evaluated by exposing cells to increasing concentrations (conc) of MK2206 (100pM, 2nM, 20nM, 0.2µM, 2µM, 30µM) for up to 72 hrs followed by MTT assay. Over 95% of growth inhibition was achieved in all cell lines with respective IC50 values of: CNE-2=0.39µM, HK-1 = 0.48µM, HONE-1= 1.51µM, C666-1= 1.64µM, HNE-1= 1.92µM, HONE1-EBV= 2.36µM. CNE-2, HONE-1-EBV and C666-1 were selected for assessing the effect on AKT signaling, apoptosis, cell cycle and synergism with chemotherapy. Treatment of these cells with MK2206 at IC50 conc for 15 and 24 hours, resulted in G1 arrest in CNE-2 and HONE-1-EBV, and G2 arrest in C666-1 cells. Apoptosis (increased cleaved PARP) was detected in CNE-2 cells only. MK2206 reduced the expression of p-AKT, p-mTOR, p-BAD, p-FKHD and p-GSK3β in CNE-2, HONE-1-EBV and C666-1 cells, while the level of p-4EBP1 level was significantly reduced in C666-1 and CNE-2 cells only. The effect on p-70S6K expression in all 3 cell lines was minimal. MK2206 increased expression of p-MAPK in HONE1-EBV-1 and C666-1 cells, but not CNE-2 cells. A supra-additive effect on cell growth was observed when cisplatin was added to MK2206, while the effect of adding paclitaxel to MK2206 was minimal in all 3 cell lines. Our result suggests that MK2206 has activity in NPC in vitro. The increased expression of p-MAPK observed in some cell lines suggests the presence of compensatory MAPK activation as described with mTOR inhibitors in other cancers. MK-2206 is currently under clinical investigation in recurrent NPC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4602. doi:1538-7445.AM2012-4602

Abstract 2799: BAY 1082439, a highly selective and balanced PI3Kα/β inhibitor demonstrated potent activity in tumors with activated PI3Kα and loss-of-function of PTEN

Abstract The PI3K pathway plays critical roles in cancer cell growth and survival, as well as in intrinsic and acquired resistance to both chemotherapy and targeted agents. These essential roles have led to the clinical development of PI3K pathway inhibitors. Due to the complexity derived from the existence of various PI3K isoforms (≥,α,α,α), and their differential roles in signal transduction, as well as cancer pathology, development of PI3K inhibitors with differential pharmacological profiles would allow exploration in different indications, combinations and dosing regimens. Having identified BAY 80-6946, an intravenously dosed, highly potent and selective PI3K inhibitor which is particularly effective in PIK3CAmut and/or Her2+ tumors, we sought to develop a novel oral PI3K inhibitor particularly effective in PTEN-loss tumors with coexisting mutation or amplification of PIK3CA and/or activation of PI3Kα (e.g., through RTKs). Herein we report the pharmacological profile of a highly selective PI3Kα/α-balanced inhibitor, BAY 1082439. BAY 1082439 has an IC50 ratio of 1:3 in biochemical assays of PI3Kα (4.9 nM) vs. PI3Kα (15.0 nM), and >1000-fold selectivity against mTOR kinase. The balanced PI3Kα and PI3Kα activity of BAY 1082439 is also reflected in cellular mechanistic (p-AKT473) and proliferation assays in PI3Kα- (KPL4, BT474) vs. PI3Kα-driven (PC3, LNCaP) tumor cells. In vivo, BAY 1082439 showed clear advantages over the strong PI3Kα inhibitor BAY 80-6946 in PTEN/PI3Kα-driven tumor models (e.g., PC3 and HEC-1B), when the two compounds were compared at their MTDs. Furthermore, BAY 1082439 has unique pharmacokinetic (PK) properties with very high plasma free fractions across all species tested (33-50%), large Vss, high clearance and intermediate T1/2. The relationship of PK vs. PD and the efficacy vs. dosing regimens were investigated. BAY 1082439 showed strong p-AKT inhibition at 2 and 5 hours post-treatment while p-AKT returned to levels comparable to the vehicle group at 24 hours in all 4 tumor models tested. Interestingly, with once daily dosing, BAY 1082439 could induce tumor regression in KPL4 (PIK3CAmut and HER2+), and tumor stasis in HEC-1B (PTENdel) and in HEC-1A (PIK3CAmut) tumor models, suggesting that continuous inhibition of p-AKT may not be required for anti-tumor efficacy. In addition, comparison of different dosing regimens (QD, Q2D, D1-2/W, D1-3/W, D1-4/W to QW) at MTD indicated that QD and QW dosing produced optimal anti-tumor efficacy. These results support the hypothesis that strong pathway inhibition for a certain time period, rather than maintaining constant inhibition might lead to optimal anti-tumor efficacy along with a maximal therapeutic window. In conclusion, BAY 1082439 represents a new type of PI3K inhibitor with unique pharmacological and pharmacodynamic properties to be further explored in clinical development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2799. doi:1538-7445.AM2012-2799

Abstract 726: Copy number amplification of the PIK3CA gene is associated with poor prognosis in early-stage head and neck squamous cell carcinoma

Abstract Background: Deregulation of the EGFR signaling pathway is one of the most frequently observed genetic abnormalities that drive cancer development. Mutations in downstream components of the EGFR signaling pathway, including KRAS, BRAF and PIK3CA, have been reported in numerous cancers. However, extensive mutation analysis of these genes in clinical samples has not been performed for head and neck squamous cell carcinoma (HNSCC). Patient and methods: We examined mutations and copy number alterations of KRAS, BRAF and PIK3CA in 115 clinical specimens of HNSCC obtained from surgically treated patients. Results: We identified three mutations in K-RAS and two mutations in PIK3CA. Copy number amplification was found in 42 cases (36.4%) for PIK3CA, 12 cases (10.4%) for K-RAS and nine cases (7.8%) for BRAF. Tumor recurrence and death occurred in 49 (42.6%) and in 22 (19.3 %) patients during the median follow-up of 723 days, respectively. Kaplan-Meier survival analysis revealed that in patients with early-stage carcinoma (Stages I-III), copy-number amplification of PIK3CA was drastically associated with cancer relapse. Conclusion: Copy number amplification of the PIK3CA is associated with poor prognosis in early-stage HNSCC. The PIK3CA copy number status will serve as a marker of poor prognosis in patients with HNSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 726. doi:1538-7445.AM2012-726

Abstract 2817: Preclinical studies of the PI3K/mTOR dual inhibitors in endometrial cancer cell lines

Abstract The PI3K pathway plays a pivotal role in many cellular functions that include regulation of cell proliferation, survival, growth, angiogenesis, and motility. Dysregulation of the PI3K/AKT pathway is a common occurrence in cancer and could be a result of mutations in PIK3CA and PTEN, as well as loss of heterozygosity of PTEN. In endometrial cancer patients, the most frequent aberration is the activation of the PI3K/mAKT pathway with; (1) 80% loss of PTEN function and 30% PIK3CA mutations in type I endometrial cancer and (2) 20% PIK3CA mutation and 46% PIK3CA amplification in type II endometrial cancer. In this study, we investigated the functional consequences of the dual PI3K/mTOR inhibitors PF-04691502 and PF-05212384 in endometrial cancer cell lines for antiproliferative effects, pathway signaling inhibition and tumor growth inhibition. Both compounds had antiproliferative activity in vitro that translated to tumor growth inhibition in vivo. Moreover, in the MFE-280 xenograft model that harbors a PIK3CA mutation (P1047R), the dual PI3K/mTOR inhibitors showed tumor regression. These studies support the use of the dual PI3K/mTOR inhibitors for endometrial cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2817. doi:1538-7445.AM2012-2817

Abstract 4821: Biomarker evaluation In phase I clinical trials of selective PI3K and PI3K/mTOR inhibitors

Abstract Background: The PI3K/Akt pathway is frequently activated in cancer by multiple mechanisms including PI3K activating mutations, PTEN loss, RTK activation, and other means. GDC-0941 and GDC-0980 are selective PI3K and dual PI3K/mTOR inhibitors, respectively, which are currently in phase II clinical development. Assays for candidate predictive and pharmacodynamic biomarkers were conducted on patient samples collected from phase I studies of GDC-0941 and GDC-0980. The purpose was to confirm pathway inhibition at tolerable doses, as well as look for association between anti-tumor activity and candidate predictive biomarkers. Methods: Archival tumor tissue samples were analyzed using a 6 gene mutation assay (PIK3CA, EGFR, KRAS, BRAF, NRAS, AKT1), an immunohistochemistry assay for PTEN, and a fluorescence in situ hybridization (FISH) assay for PIK3CA. Select samples were analyzed for an expanded mutation panel and genome-wide copy number alterations using the Oncoscan platform (Affymetrix). For pharmacodynamic biomarker assays, analyses were initially limited to pre and post treatment phospho-AKT levels in platelet rich plasma. Once substantial downmodulation of pAKT in surrogate tissue was observed, pre- and post treatment biopsies were collected from a subset of patients and analyzed for phospho-S6 levels by immunohistochemistry. Results: Predictive biomarker assays were conducted on over 150 samples from the phase I study. Overall we found a prevalence of 7% PIK3CA mutations and 15% loss of PTEN in these samples. PIK3CA amplification was observed in several samples from ovarian cancer patients. Overall there were five confirmed partial responses in the studies, and two of these occurred in patients with activating mutations in PIK3CA. Robust PD knockdown of pAKT in surrogate tissue was observed in the early stage of dose-escalation. Moreover, substantial knockdown of pS6 was seen in tumor tissues in patients treated at or below the recommended phase II dose. Conclusions: Pharmacodynamic assays confirmed effective pathway knockdown at safe and tolerated clinical doses of GDC-0941 and GDC-0980. Anti-tumor activity was observed in patients with PIK3CA mutations, as well as some patients whose tumors did not harbor pathway alterations. These data support patient stratification in phase II clinical studies to determine whether a predictive biomarker will be useful in identifying responsive patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4821. doi:1538-7445.AM2012-4821

High frequency of allelic loss at the BRCA1 locus in ovarian cancers: clinicopathologic and molecular associations

BRCA1 dysfunction may occur by different mechanisms that are rarely evaluated concomitantly. We aimed to analyze BRCA1 germline mutations, loss of heterozygosity (LOH) and promoter methylation in unselected ovarian carcinomas in the context of their clinicopathologic characteristics and other molecular changes. BRCA1 mutations were analyzed in 257 carcinomas using single-strand conformation polymorphism (SSCP), heteroduplex, and sequencing methods. LOH at the BRCA1 locus was screened for in 180 cancers. Methylation analysis was performed for 241 tumors using quantitative methylation specific PCR(qMSP). BRCA1 alterations, comprising germline mutations, allelic loss, and/or aberrant promoter methylation, were found in 77.6% (125/161) of ovarian carcinomas. Patients with germline mutations were younger than non-carriers (P < 0.0001). Germline mutations and LOH were associated with advanced stages (P=0.009, P < 0.0001), high tumor grade (P=0.005, P<0.0001), and TP53 mutations (P=0.003, P < 0.0001, for mutations and LOH, respectively). LOH was also associated with the serous histological type (P=0.004) and PIK3CA amplification (P=0.003). Aberrant promoter methylation was associated with LOH (P=0.017) and absence of germline mutations (P=0.037). The high frequency of LOH at the BRCA1 locus suggests that LOH may be an important mechanism of BRCA1 deficiency in ovarian carcinomas. Tumors with various BRCA1 alterations have a similar phenotype of high-grade, high-stage carcinomas with frequent TP53 mutations.

Highly frequent PIK3CA amplification is associated with poor prognosis in gastric cancer

BackgroundThe phosphoinositide 3-kinase (PI3K)/Akt pathway plays a fundamental role in cell proliferation and survival in human tumorigenesis, including gastric cancer. PIK3CA mutations and amplification are two major causes of overactivation of this pathway in human cancers. However, until this work, there was no sound investigation on the association of PIK3CA mutations and amplification with clinical outcome in gastric cancer, particularly the latter.MethodsUsing direct sequencing and real-time quantitative PCR, we examined PIK3CA mutations and amplification, and their association with clinicopathological characteristics and clinical outcome of gastric cancer patients.ResultsPIK3CA mutations and amplification were found in 8/113 (7.1%) and 88/131 (67%) gastric cancer patients, respectively. PIK3CA amplification was closely associated with increased phosphorylated Akt (p-Akt) level. No relationship was found between PIK3CA mutations and clinicopathological characteristics and clinical outcome in gastric cancer. PIK3CA amplification was significantly positively associated with cancer-related death. Importantly, Kaplan-Meier survival curves revealed that the patients with PIK3CA amplification had significantly shorter survival times than the patients without PIK3CA amplification.ConclusionsOur data showed that PIK3CA mutations were not common, but its amplification was very common in gastric cancer and may be a major mechanism in activating the PI3K/Akt pathway in gastric cancer. Importantly, Kaplan-Meier survival curves revealed that PIK3CA amplification was significantly positively associated with poor survival of gastric cancer patients. Collectively, the PI3K/Akt signaling pathway may be an effective therapeutic target in gastric cancer.

Abstract A167: First-in-patient study of PF-05212384, a small molecule intravenous dual inhibitor of PI3K and mTOR in patients with advanced cancer: Update on safety, efficacy, and pharmacology.

Abstract Background: Activation of the PI3K pathway, by PIK3CA mutation or amplification or PTEN deletion, has been implicated in a wide variety of human cancers. PF-05212384 is a dual-specificity inhibitor of PI3K and mTOR that is formulated for intravenous (IV) administration. PF-05213384 is the first IV PI3K/mTOR inhibitor to be tested in a clinical trial. Methods: In this ongoing phase 1 trial, PF-05212384 was administered once weekly, using a modified continual reassessment method (CRM) for estimation of the maximum tolerated dose (MTD). Assessments included safety (NCI CTC AE v4.0), pharmacokinetics (PK), pharmacodynamics (PD), and antitumor activity (RECIST v1.1). PD assessments included blood glucose and immunohistochemistry analysis of phosphoprotein biomarkers in tumor and surrogate tissue (hair follicles). The population was actively enriched for patients with dysregulation in the PI3K pathway. Results: As of 15 March 2011, 47 patients have been enrolled at doses ranging from 10 mg to 319 mg. Median age was 58. Represented tumor types included CRC (8), NSCLC (4), ovarian/endometrial/cervical (8), salivary (3) and gastric/esophageal (3). Analysis of archived tumor biopsies revealed PI3K pathway alterations in 12/26 evaluable patients; alterations included PTEN loss, PIK3CA mutation or amplification. PF-05212384 was well tolerated with the most common AEs being mucositis (44%), nausea (42%), fatigue (28%), rash (28%), and hyperglycemia (26%). DLTs included mucositis, rash, transaminase elevation, and hyperglycemia. An MTD of 154 mg weekly has been estimated. PF-05212384 is eliminated with a half-life of approximately 16 hours, with low clearance (5–9 L/h) and relatively high volume of distribution (54–144 L). Changes in blood glucose have been observed in 13 patients, including 2 patients with Grade 3 hyperglycemia, a known effect of PI3K/Akt pathway suppression. No objective tumor response has been observed; 12 patients have achieved stable disease (SD) in excess of 16 weeks, with 9 patients on study for ≥24 weeks and one patient (salivary) on study for &amp;gt;46 weeks. Enrollment to MTD expansion cohorts is ongoing. Conclusions: Weekly administration of PF-05212384 is safe and tolerable across dose levels with multiple patients deriving clinical benefit with prolonged SD. The occurrence of hyperglycemia is suggestive of target modulation. Updated data for safety, PK, PD and antitumor activity will be presented Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A167.

High sensitivity detection and robust enrichment for PIK3CA somatic mutations using ice COLD-PCR technology.

e13546 Background: High sensitivity detection of cancer biomarkers facilitates early diagnosis and provides insight for intervention including stratification for drug treatment and monitoring changes in tumor mutation profiles by analyzing DNA isolated from serum/plasma or circulating tumor cells. PIK3CA, encoding the PI3K p110α catalytic subunit, is mutated frequently in common cancers, including breast and colon carcinomas. 80% of these mutations are represented by three amino acid substitutions in the enzyme’s helical (E542K and E545K) or kinase (H1047R) domains. Methods: In this study, we have applied improved and complete enrichment CO-amplification at lower denaturation temperature PCR (Ice COLD-PCR) technology for high sensitivity detection and robust enrichment of any mutations present in a DNA sample. Ice-COLD-PCR preferentially amplifies low levels of any mutant DNA in a sample containing a vast excess of wild-type DNA. The use of a reference sequence oligonucleotide complimentary to one of the wild-type DNA strands results in linear amplification of one of the wild type strands while both mutant strands amplify exponentially during ice COLD- PCR. After Ice COLD-PCR, there is sufficient mutant allele enrichment to allow Sanger sequencing confirmation of the mutation. Results: The use of Ice COLD-PCR allows for the enrichment of mutations any of the key mutations in the PIK3CA gene that cannot be detected by standard PCR followed by Sanger sequencing methods. Initial limit of detection experiments indicate that the detection and sequence confirmation of mutations in PIK3CA more sensitive than standard PCR amplification or allele specific probe methodologies. Conclusions: After Ice COLD-PCR, low level PIK3CA mutant alleles are enriched over the wild-type allele such that simple sequence confirmation on multiple downstream systems such as Sanger Sequencing or allele-specific probe platforms is possible. Ice COLD-PCR amplification of PIK3CA and other genes requires only a standard thermocycler coupled to any downstream sequence analysis platform and therefore can be developed as a powerful, highly sensitive diagnostic assay.

PIK3CA mutations and amplification in endometrioid endometrial carcinomas: relation to other genetic defects and clinicopathologic status of the tumors

Alterations of the PIK3CA gene occur in endometrial carcinomas, but their role in the carcinogenesis of those malignancies is still poorly understood. In this study, PIK3CA mutations and amplification in 196 endometrioid endometrial carcinomas and 20 endometrial hyperplasias were assessed by single-strand conformation polymorphism (PCR-SSCP), sequencing, and quantitative polymerase chain reaction. Results were correlated with mutations in the PTEN, KRAS, and CTNNB1 genes and with the clinicopathologic parameters of the tumors. PIK3CA mutations were found in 39 (20%) carcinomas. Six new mutations were identified. No PIK3CA mutations were found in endometrial hyperplasias. PIK3CA amplification was observed in 24 (12.2%) carcinomas and in 2 (10%) hyperplasias. The PIK3CA mutations and amplifications (with the exception of 6 cases) occurred independently. PIK3CA mutations were significantly associated with PTEN mutations (P = .0414) and tended to be associated with CTNNB1 (P = .0833), but not with KRAS mutations. Conversely, the PIK3CA amplifications significantly negatively correlated with PTEN mutations (P = .0038) and did not coexist with CTNNB1 and KRAS mutations. The PIK3CA mutations were significantly associated with poorly differentiated tumors (P = .0423). Interestingly, PIK3CA amplifications, but not mutations, were strongly associated with older age (≥63 years, P = .0018). Our data show that mutations and amplification of PIK3CA are significant genetic alterations in endometrioid endometrial carcinomas associated with adverse clinicopathologic parameters (grade and stage). These data also demonstrate that PIK3CA mutations cooperate with PTEN mutations, suggesting an additive effect to PTEN, whereas PIK3CA amplification can, as an isolated event, enable the development of those tumors. Moreover, for the first time, a possible role of PIK3CA amplification in initiation and progression of endometrial carcinomas in older women is suggested, but this preliminary suggestion requires further research.

Highly frequent promoter methylation and PIK3CA amplification in non-small cell lung cancer (NSCLC)

BackgroundLung cancer is the leading cause of cancer-related death worldwide. Genetic and epigenetic alterations have been identified frequently in lung cancer, such as promoter methylation, gene mutations and genomic amplification. However, the interaction between genetic and epigenetic events and their significance in lung tumorigenesis remains poorly understood.MethodsWe determined the promoter methylation of 6 genes and PIK3CA amplification using quantitative methylation-specific PCR (Q-MSP) and real-time quantitative PCR, respectively, and explore the association of promoter methylation with PIK3CA amplification in a large cohort of clinically well-characterized non-small cell lung cancer (NSCLC).ResultsHighly frequent promoter methylation was observed in NSCLC. With 100% diagnostic specificity, excellent sensitivity, ranging from 45.8 to 84.1%, was found for each of the 6 genes. The promoter methylation was associated with histologic type. Methylation of CALCA, CDH1, DAPK1, and EVX2 was more common in squamous cell carcinomas (SCC) compared to adenocarcinomas (ADC). Conversely, there was a trend toward a higher frequency of RASSF1A methylation in ADC than SCC. In addition, PIK3CA amplification was frequently found in NSCLC, and was associated with certain clinicopathologic features, such as smoking history, histologic type and pleural indentation. Importantly, aberrant promoter methylation of certain genes was significantly associated with PIK3CA amplification.ConclusionsOur data showed highly frequent promoter methylation and PIK3CA amplification in Chinese NSCLC population, and first demonstrated the associations of gene methylation with PIK3CA amplification, suggesting that these epigenetic events may be a consequence of overactivation of PI3K/Akt pathway.

Abstract 4116: Predictive biomarkers for the PI3 kinase inhibitor GDC-0941 in NSCLC

Abstract The class I phosphatidylinositol 3’ kinase (PI3K) plays a major role in proliferation and survival in a wide variety of human cancers. A key factor in successful development of drugs targeting the PI3K pathway is likely to be the identification of responsive patient populations with predictive diagnostic biomarkers. GDC-0941 is a pan-inhibitor of all 4 isoforms of class 1 PI3K that is currently undergoing testing in the clinic. We were interested in the predictive value of candidate biomarkers in NSCLC to help inform clinical trial biomarker strategies in this indication. We assembled a focused panel of cell lines with key alterations in the PI3K pathway isolated from other genetic events to the extent possible. The approach involved pharmacogenomic and proteomic profiling. We found that models harboring key pathway alterations were exquisitely sensitive to GDC-0941. In particular, cell lines with EGFR mutations, PIK3CA mutations or amplification were the most sensitive. Inhibition of PI3K in mechanism of action studies showed effects consistent with induction of apoptosis, cell cycle arrest, and impairment of translation in models with these key alterations. In addition, gene expression changes of genes downstream of PI3K were seen upon treatment with GDC-0941 in sensitive cell lines but not those that were resistant. We extended these studies to NSCLC tumor samples and evaluated the prevalence of important pathway alterations, including PTEN loss, PIK3CA amplification, LKB1 loss in tumors representing distinct histopathological subtypes. We find that certain candidate biomarkers show subtype specific associations, and will discuss the implications for the development of PI3K-targeting agents in NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4116. doi:10.1158/1538-7445.AM2011-4116

The frequent co-expression of the oncogenes PIK3CA and PAK1 in oral carcinomas

PIK3CA and PAK1 are critical genes in the PI3K/AKT/PAK pathway. Amplification and strong expression of PIK3CA and PAK1 were identified in around 50% and 88% oral carcinomas, respectively, while co-expression of PIK3CA and PAK1 together was found to be present in 80% of oral carcinomas. PIK3CA and PAK1 amplification was more obvious in recurrent tumors than their primary counterparts. Low grade amplification of PAK1 was prevalent in tumor risk individuals. Knockdown of expression of PIK3CA and PAK1 reduced the oncogenic potential of tumor cells. This study is the first to demonstrate the concordant PIK3CA and PAK1 alterations in oral carcinoma.