E17K Mutation Research Articles

A protein-targeting strategy used to develop a selective inhibitor of the E17K point mutation in the PH domain of Akt1.

Ligands that can selectively bind to proteins with single amino acid point mutations offer the potential to detect or treat an abnormal protein in the presence of the wildtype. However, it is difficult to develop a selective ligand if the point mutation is not associated with an addressable location, such as a binding pocket. Here we report an all-chemical, synthetic epitope-targeting strategy which we used to discover a 5-mer peptide with selectivity for the E17K transforming point mutation in the Pleckstrin Homology Domain of the Akt1 oncoprotein. A fragment of Akt1 containing the E17K mutation and a I19[Propargylglycine] substitution was synthesized to form an addressable synthetic epitope. Azide-presenting peptides that covalently clicked onto this alkyne-presenting epitope were selected from a library using in situ screening. One peptide exhibits a 10:1 in vitro selectivity for the oncoprotein relative to wildtype, with a similar selectivity in cells. This 5-mer peptide was expanded into a larger ligand that selectively blocks the E17K Akt1 interaction with its PIP3 substrate.

Response of head and neck squamous cell carcinoma cells carrying PIK3CA mutations to selected targeted therapies.

The PIK3CA mutation is one of the most common mutations in head and neck squamous cell carcinoma (HNSCC). Through this research we attempt to elicit the role of oncogene dependence and effects of targeted therapy on this PIK3CA mutation. (1) To determine the role of oncogene dependence on PIK3CA-one of the more common and targetable oncogenes in HNSCC, and (2) to evaluate the consequence of this oncogene on the effectiveness of newly developed targeted therapies. This was a cell culture-based, in vitro study performed at an academic research laboratory assessing the viability of PIK3CA-mutated head and neck cell lines when treated with targeted therapy. PIK3CA-mutated head and neck cell lines were treated with 17-AAG, GDC-0941, trametinib, and BEZ-235. Assessment of cell viability of HNSCC cell lines characterized for PIK3CA mutations or SCC25 cells engineered to express the PIK3CA hotspot mutations E545K or H1047R. Surprisingly, in engineered cell lines, the hotspot E545K and H1047R mutations conferred increased, rather than reduced, IC50 assay measurements when treated with the respective HSP90, PI3K, and MEK inhibitors, 17-AAG, GDC-0941, and trametinib, compared with the SCC25 control cell lines. When treated with BEZ-235, H1047R-expressing cell lines showed increased sensitivity to inhibition compared with control, whereas those expressing E545K showed slightly increased sensitivity of unclear significance. (1) The PIK3CA mutations within our engineered cell model did not lead to enhanced oncogene-dependent cell death when treated with direct inhibition of the PI3K enzyme yet did show increased sensitivity compared with control with dual PI3K/mTOR inhibition. (2) Oncogene addiction to PIK3CA hotspot mutations, if it occurs, is likely to evolve in vivo in the context of additional molecular changes that remain to be identified. Additional study is required to develop new model systems and approaches to determine the role of targeted therapy in the treatment of PI3K-overactive HNSCC tumors.

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.

Activated Mutant p110α Causes Endometrial Carcinoma in the Setting of Biallelic Pten Deletion

PTEN and PIK3CA mutations occur with high frequency in uterine endometrioid carcinoma (UEC). Although PTEN mutations are present in complex atypical hyperplasia and carcinoma, PIK3CA mutations are restricted to carcinoma. We generated mouse models harboring Pten loss and/or activated Pik3ca in the endometrial epithelium to investigate their respective roles in the pathogenesis of UEC. Presence of an activated mutant Pik3ca on the background of Pten loss led to aggressive disease, with 100% of mice exhibiting carcinoma. Expression of Pik3ca with E545K mutation alone was unable to cause hyperplasia or cancer in the uterus and did not activate Akt as effectively as Pten deletion in short-term cultures of mouse endometrial epithelium, likely explaining the lack of phenotype in vivo. We also report that nuclear localization of FOXO1 correlated with PTEN mutational status irrespective of the PIK3CA status in endometrial cancer cell lines. Furthermore, gene expression profiles resulting from Pten loss or activation of Pik3ca in primary mouse endometrial epithelial cells exhibit minimal overlap. Thus, Pten and Pik3ca have distinct consequences on the activation of the phosphatidylinositol 3-kinase pathway in endometrial epithelium and are likely to affect other nonoverlapping cellular mechanisms involved in the development and progression of the most common type of uterine cancer.

Novel mutations in neuroendocrine carcinoma of the breast: possible therapeutic targets.

Primary neuroendocrine carcinoma of the breast is a rare variant, accounting for only 2% to 5% of diagnosed breast cancers, and may have relatively aggressive behavior. Mutational profiling of invasive ductal breast cancers has yielded potential targets for directed cancer therapy, yet most studies have not included neuroendocrine carcinomas. In a tissue microarray screen, we found a 2.4% prevalence (9/372) of neuroendocrine breast carcinoma, including several with lobular morphology. We then screened primary or metastatic neuroendocrine breast carcinomas (excluding papillary and mucinous) for mutations in common cancer genes using polymerase chain reaction-mass spectroscopy (643 hotspot mutations across 53 genes), or semiconductor-based next-generation sequencing analysis (37 genes). Mutations were identified in 5 of 15 tumors, including 3 with PIK3CA exon 9 E542K mutations, 2 of which also harbored point mutations in FGFR family members (FGFR1 P126S, FGFR4 V550M). Single mutations were found in each of KDR (A1065T) and HRAS (G12A). PIK3CA mutations are common in other types of breast carcinoma. However, FGFR and RAS family mutations are exceedingly rare in the breast cancer literature. Likewise, activating mutations in the receptor tyrosine kinase KDR (VEGFR2) have been reported in angiosarcomas and non-small cell lung cancers; the KDR A1065T mutation is reported to be sensitive to VEGFR kinase inhibitors, and fibroblast growth factor receptor inhibitors are in trials. Our findings demonstrate the utility of broad-based genotyping in the study of rare tumors such as neuroendocrine breast cancer.

BRAF mutations in patients with well-differentiated pancreatic neuroendocrine tumors (WD pNETs).

321 Background: Whole exome sequencing in WD pNETs demonstrated an increased number of mutations in genes implicated in chromatin remodeling and in the PI3K/Akt/mTOR pathway. Next-generation sequencing (NGS) allows us to bring this technology to the clinic to better characterize WD pNETs and potentially offer tailored therapies. Methods: A NGS platform developed at Memorial Sloan Kettering Cancer Center (MSKCC) termed MSK-IMPACT (Integrated Mutation Profiling of Actionable Cancer Targets) was used. This platform screens for 341 cancer genes commonly altered across tumor types. Thus far, 14 patients with WD pNETs have consented to MSK-IMPACT. Results: To date, mostpatients harbor no clinically validated or actionable mutations. Two patients, however, harbor different and potentially actionable BRAF mutations. The first patient is a 39 year-old woman with a BRAF V600E mutation. She presented with somatostatin receptor-negative, aggressive disease involving the liver, lymph nodes, adnexa, and peritoneum. She was initially diagnosed elsewhere as having a signet ring cell adenocarcinoma, and responded to FOLFOX and bevacizumab. Pathology review at MSKCC, however, identified the tumor as metastatic WD pNET. Upon disease progression, a repeat biopsy confirmed metastatic WD pNET. She is currently on capecitabine and temozolomide with clinical improvement. The second patient is a 22 year-old woman with metastatic WD pNET with a BRAF K601E mutation. Her tumor also has an aggressive behavior; she has progressed on all conventional and nonconventional therapies. Given the finding of a BRAF K601E mutation, she received trametinib and dabrafenib, but developed disease progression after 3 months. Conclusions: Two potentially actionable BRAF mutations were identified from NGS of WD pNETs. BRAF mutations have not been previously reported in pNETs. It is unclear whether BRAF mutations are oncogenic drivers, and whether BRAF-targeted treatments would be beneficial. Both patients show an atypical, aggressive presentation and course. Further testing is warranted to determine the role and clinical significance of BRAF in pNETs. Evaluation using pNET tissue microarrays is ongoing and will be reported at the meeting.

BRAF Mutations in an Italian Regional Population: Implications for the Therapy of Thyroid Cancer.

Background. Molecular diagnostics has offered new techniques for searching for mutations in thyroid indeterminate lesions. The study's aim was to evaluate the BRAF mutations' incidence in an Italian regional population. Subjects and Methods. 70 Caucasian patients born in Liguria with indeterminate or suspicious cytological diagnoses. Results. A BRAF gene mutation was successfully analyzed in 56/70 patients. The mutation was BRAF V600E in 12/56 cases (21%) and BRAF K601E in 2/56 (4%). Of the BRAF mutated samples on cytological diagnosis (14/56 cases), 2/14 cases (14%) were benign on final histology and 12/14 (86%) were malignant. All BRAF-mutated cases on cytology that were found to be benign on histological examination carried the K601E mutation. Of the nonmutated BRAF cases (42/56, 75%) which were later found to be malignant on definitive histology, 5 cases were follicular carcinomas (36%), 3 cases were incidentally found to be papillary microcarcinomas (22%), 2 were cases papillary carcinomas (14%), 1 was case follicular variant of papillary carcinoma (7%), 1 was case medullary carcinoma (7%), 1 case was Hurtle cell tumor (7%), and 1 case was combined cell carcinoma and papillary oncocytic carcinoma (7%). Conclusions. The presence of the BRAF V600E mutation may suggest a more aggressive surgical approach. BRAF K601E mutation did not correlate with malignancy indexes.

Abstract A45: Delineating roles of Pten and Pik3ca in endometrial carcinoma using mouse models

Abstract Uterine endometrioid carcinoma (UEC) are indolent cancers often arising in the setting of unopposed estrogen from complex atypical hyperplasia (CAH) the non-invasive precursor. The light microscopic distinction between UEC and CAH on endometrial biopsy or curettage samples is challenging. Given that approximately 25-40% of cases initially diagnosed with CAH progress to UEC, there is a compelling need to identify molecular diagnostic and/or prognostic markers distinguishing CAH from UEC. Mutational profiling of CAH and UEC has revealed distinct molecular differences between the two types of lesions. While PTEN (the most frequently mutated gene in UEC) mutations were found at an equal frequency in both CAH and UEC, PIK3CA mutations were almost exclusively found in UEC. Furthermore, 48% of UEC with PTEN mutations have PIK3CA mutations. With a Pten+/- mouse model, we demonstrated that CAH and UEC completely lose expression of Pten due to loss of heterozygosity (LOH) or intragenic mutations in the wild type allele with similar frequencies in CAH and UEC, suggesting that biallelic inactivation of Pten, though an early event, may not be sufficient for progression to invasive carcinoma and may require additional events such as mutations in Pik3ca. To test this hypothesis and to unravel the role of Pik3ca in UEC, we have used mice with one Pik3ca allele harboring an activating lox-stop-lox E545K mutation (Pik3caE545K/+ mice) and crossed them with PtenloxP/loxP mice. Driving expression of Cre under two epithelial cell-specific promoters, we show that mutant Pik3ca leads to CAH and/or UEC only in the setting of simultaneous biallelic Pten inactivation. Presence of activated Pik3ca alone does not result in hyperplasia or carcinoma. To dissect out molecular mechanisms underlying these observations, primary uterine epithelial cell cultures were prepared from PtenloxP/loxP;Pik3ca+/+, Pten+/+ ; Pik3caE545K/+ and PtenloxP/loxP;Pik3caE545K/+ mice. Pten deletion and/or activation of Pik3ca was achieved in vitro by treatment with adenovirus expressing Cre (Ad-Cre) for 48 hrs. RNA sequencing was performed on RNA extracted from Ad-Cre treated cells. Cells treated with GFP expressing virus were used as control. PtenloxP/loxP;Pik3ca+/+, Pten+/+ ; Pik3caE545K/+ and PtenloxP/loxP;Pik3caE545K/+ cells exhibited distinct and minimally overlapping gene expression profiles, suggesting that Pten inactivation, Pik3ca activation or both lead to different downstream responses. These results, in conjunction with the human studies, support distinct roles for PTEN and PIK3CA mutations in the development and progression of endometrial hyperplasia/carcinoma. Citation Format: Ayesha Joshi, Christopher Miller, Lora H. Ellenson. Delineating roles of Pten and Pik3ca in endometrial carcinoma using mouse models. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr A45.

Abstract 3685: BAY 1125976, is a selective allosteric AKT1/2 inhibitor with high efficacy in AKT1-mutated cancers

Abstract The PI3K-AKT-mTOR signaling cascade is activated in human cancers by elevated membrane receptor activity, mutation, amplification, and deletion of genes encoding components of the pathway. An oncogenic, single hotspot mutation in the AKT1 gene, G49A:E17K, in the pleckstrin homology domain of AKT1, was described in human cancers with highest relative incidence in breast cancer. The E17K mutation in AKT1 results in PI3K-independent membrane recruitment of AKT1. Recently we reported the development of a highly selective, potent allosteric AKT1/2 inhibitor BAY 1125976 with strong in vitro and in vivo activity in tumor models with activated AKT signaling and strong synergistic activity in combination. The efficacy of BAY 1125976 was evaluated in tumor models carrying activation of the PI3K-AKT pathway by either deletion of PTEN or activating mutation in PI3K. We investigated whether BAY 1125976 can also inhibit AKT signaling in cell lines carrying an activating mutation in AKT. To this extend, KU-19-19 (AKT1E17K; NRASQ61R) bladder cancer as well as LAPC4 (AKT1E17K) prostate cancer cell lines both bearing the AKT1E17K mutation were profiled. BAY 1125976 potently inhibited AKT activation as well as downstream signaling in KU-19-19 and LAPC4 cells. Furthermore, anti-tumor efficacy of BAY 1125976 was tested in the patient-derived anal cancer xenograft AXF 984 (AKT1E17K). BAY 1125976 shows dose-dependent potent pathway activity in AKT1mut patient derived tumor model with inhibition of pAKT473 as well as downstream targets p- PRAS40 and p-FOXO3a compared to selected PI3K, AKT and mTOR inhibitors. Continuous daily treatment of AXF 984 (AKT1E17K) mouse xenografts with 25 mg/kg and 50 mg/kg QD p.o. BAY 1125976 resulted in very potent statistically significant anti-tumor efficacy. All animals of these treatment groups exhibited tumor shrinkage or disease control with response rates of 88% and 83%, respectively. Furthermore, for animals treated with 50 mg/kg BAY 1125976 the delay of reaching a relative tumor volume of 600% was statically significant compared to the vehicle-treated control group. In summary, BAY 1125976 showed superior anti-tumor activity in AKT1E17K mutated models compared to PI3K and mTOR inhibitors. These results indicate that the clinical development of BAY 1125976 in patients with activating mutation in AKT could result in an innovative and more effective alternative to current treatments. Citation Format: Oliver Politz, Arne Scholz, Andrea Haegebarth, Ningshu Liu, Lars Baerfacker, Stuart Ince, Roland Neuhaus, Ulf Boemer, Martin Michels, Dominik Mumberg. BAY 1125976, is a selective allosteric AKT1/2 inhibitor with high efficacy in AKT1-mutated cancers. [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 3685. doi:10.1158/1538-7445.AM2014-3685

Abstract 2371: Heterogeneity of resistance mechanisms in lung adenocarcinoma patients with acquired resistance to EGFR inhibitors

Abstract Drug resistance is a major obstacle in overcoming cancer. Understanding the origin, extent and heterogeneity of resistance mechanisms within a patient is crucial to guide treatment and developing drugs/combinations to circumvent resistance. To this end, we studied autopsy samples collected from four EGFR-mutant lung adenocarcinoma patients who developed resistance to EGFR inhibitors. We used whole-exome sequencing to characterize genomic profiles of multiple metastasis tumors within each patient. To address the challenge of non-uniform variant detection power at sequenced genetic loci across multiple samples, we developed a computational framework that tackles variant detection from pooled tumor followed by a genotyping module. After the identification of somatic alterations, we determined the extent of heterogeneity of resistance mechanisms between multiple tumor samples. Our results revealed that multiple metastasis samples in a single patient can potentially use one common resistance mechanism. For example, one patient harbored a resistant EGFR T790M mutation in all 11 metastasis samples with a correlated allele frequency to the original L858R mutation. However, in some other cases, we found heterogeneous resistance mechanisms coexisting in multiple metastasis samples with various allele frequencies. For instance, one patient harbored both the resistance EGFR T790M mutation and an activating PIK3CA E542K mutation that appear with a range of allelic frequencies in multiple metastatic samples. Furthermore, branched evolutionary patterns of tumors were constructed using a phylogenetic approach, which inform resistance in the context of tumor evolution. Our findings are consistent with the model that cells harboring a variety of resistance mechanisms coexist in each metastasis tumor, and those subclones with higher selective advantage outgrow the others. Citation Format: Xinmeng Jasmine Mu, Eli Van Allen, Nikhil Wagle, Curtis Chong, Mohit Butaney, Deborah Farlow, Gad Getz, Pasi A. Jänne, Levi A. Garraway. Heterogeneity of resistance mechanisms in lung adenocarcinoma patients with acquired resistance to EGFR inhibitors. [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 2371. doi:10.1158/1538-7445.AM2014-2371

Prospective Molecular Evaluation of Small Cell Lung Cancer (Sclc) Utilizing the Comprehensive Mutation Analysis Program (Map) at Memorial Sloan Kettering Cancer Center (Mskcc)

ABSTRACT Aim: Recent studies using next generation sequencing (NGS) on resected SCLC specimens have led to insights into the molecular heterogeneity of this disease. However, comprehensive, prospective molecular profiling of patients (pts) with advanced SCLC using the biopsy specimens available in clinical practice has not been performed. Methods: Utilizing an IRB-approved protocol, we prospectively are evaluating SCLC tumors (SCLC-MAP) of pts in active treatment. These biopsies are evaluated by: fluorescence in situ hybridization (FISH) for FGFR1 and MET copy number; point mutation genotyping for known oncogenes by a mass spectrometry based assay (Sequenom); and NGS with a panel of 300 cancer-related genes. We first tested the feasibility of this approach in a series of pts with SCLC identified retrospectively, with matched tumor and normal pairs, and performed NGS, confirming the findings by FISH. Results: In the feasibility cohort, 21 pts with SCLC had FFPE samples available. After histologic review and DNA extraction, 10 pts had adequate material for NGS. We observed recurrent mutations in RB1 (N=7) and TP53 (N=8) and amplifications of FGFR1 (N=2) and MET (N=1), using as little as 15 nanograms of DNA. FISH confirmed FGFR1 and MET amplification in the identified cases. Since 2/2013, SCLC pts undergoing active treatment, with sufficient archived tissue, are providing consent for SCLC-MAP. Thus far, 36 pt samples have been tested. Sequenom (N=32) identified an AKT1 E17 mutation (N=1) and a PIK3CA E542K mutation (N=1). NGS (N=25) has yielded the following: loss of RB1 (N=18 mutations; N=4 deletions); mutations in TP53 (N=24), MLL3 (N=9), and EPHA 5 (N=9); and amplifications of CDKN2C (N=5), MYCL1 (N=3), SOX2 (N=2), and FGFR1 (N=1, confirmed by FISH). 4 pts had insufficient material. Conclusions: Comprehensive molecular evaluation of SCLC is feasible on clinical specimens. Prospective collection of SCLC tumor samples and mutational analyses continue. Such analyses will allow us to characterize the molecular diversity of SCLC, identify pts who will be candidates for targeted therapies, and ascertain clinical characteristics. Funded, in part, by the Lung Cancer Research Foundation. Disclosure: All authors have declared no conflicts of interest.

The Role of Targeted Therapy and Oncogene Dependence on the PIK3CA Mutation

Objectives: (1) Determine the role of oncogene dependence on one of the more common and targetable oncogenes in head and neck squamous cell carcinoma (HNSCC) PIK3CA. (2) Evaluate the consequence of this oncogene on the effectiveness of newly developed targeted therapies. Methods: A basic science study was performed to test the hypothesis that PI3KCA hot spot mutations confer increased sensitivity or oncogene addiction to PI3K and related inhibitors. The PI3KCA hotspot mutations—E545K and H1047R—were engineered within the HNSCC cell line SCC25. Cell viability was measured using high throughput microscopy to determine the survivability cell lines expressing the hotspot mutations compared with control cell lines when treated with increasing concentrations of newly developed targeted therapies 17-AAG, GDC-0941, and Trametinib. Results: Surprisingly, hotspot E545K and H1047R mutations conferred increased rather than reduced survivability when treated with increased concentrations of the respective HSP90, PI3K, and MEK inhibitors, 17-AAG, GDC-0941, and Trametinib compared to the SCC25 control cell lines. Conclusions: (1) The PIK3CA mutations within our engineered cell model did not lead to oncogene dependent cell death when treated with targeted therapy including direct inhibition of the Phosphatidylinositol 3-kinase (PI3K) enzyme. (2) Oncogene addiction to PIK3CA hot spot mutations, if it occurs, is likely to evolve in vivo in the context of additional molecular changes that remain to be identified.

The presence of B-RAF V600E and K601E mutations in our Ligurian population

The presence of B-RAF V600E and K601E mutations in our Ligurian population

Assessing the subcellular distribution of oncogenic phosphoinositide 3-kinase using microinjection into live cells.

Oncogenic mutations in PIK3CA lead to an increase in intrinsic phosphoinositide kinase activity, but it is thought that increased access of PI3Kα (phosphoinositide 3-kinase α) to its PM (plasma membrane) localized substrate is also required for increased levels of downstream PIP3/Akt [phosphoinositide-3,4,5-trisphosphate/also called PKB (protein kinase B)] signalling. We have studied the subcellular localization of wild-type and the two most common oncogenic mutants of PI3Kα in cells maintained in growth media, and starved or stimulated cells using a novel method in which PI3Kα is pre-formed as a 1:1 p110α:p85α complex in vitro then introduced into live cells by microinjection. Oncogenic E545K and H1047R mutants did not constitutively interact with membrane lipids in vitro or in cells maintained in 10% (v/v) FBS. Following stimulation of RTKs (receptor tyrosine kinases), microinjected PI3Kα was recruited to the PM, but oncogenic forms of PI3Kα were not recruited to the PM to a greater extent and did not reside at the PM longer than the wild-type PI3Kα. Instead, the E545K mutant specifically bound activated Cdc42 in vitro and microinjection of E545K was associated with the formation of cellular protrusions, providing some preliminary evidence that changes in protein–protein interactions may play a role in the oncogenicity of the E545K mutant in addition to the well-known changes in lipid kinase activity.

PIK3CA Mutations in Mucinous Cystic Neoplasms of the Pancreas

Mucinous cystic neoplasms (MCNs) are rare, potentially curable, mucin-producing neoplasms of the pancreas. We have previously reported PIK3CA (phosphoinositide-3-kinase catalytic subunit, p110α) mutations in intraductal papillary mucinous neoplasms, another mucin-producing neoplasm of the pancreas. In this study, we analyzed the presence of PIK3CA and AKT1/PKB (V-akt murine thymoma viral oncogene homolog 1) hot-spot mutations in MCN specimens. Using the genomic DNA sequencing of tumor tissues isolated by laser capture microdissection, we evaluated 15 well-characterized MCNs for the E542K, E545K (exon 9), and H1047R (exon 20) hot-spot mutations in the PIK3CA gene and the E17K mutation in the AKT1 gene. A hot-spot mutation (E545K) of the PIK3CA gene was detected in 1 of the 15 MCNs and further confirmed by a mutant-enriched method. Interestingly, this mutation was found to be present only in the high-grade but not in low-grade dysplastic epithelium obtained from this neoplasm and coexisted with a KRAS mutation. No mutations were identified in the AKT1 gene. Our data, when combined with previous reports on intraductal papillary mucinous neoplasms, indicate that oncogenic activation of the PI3K pathway involving PIK3CA gene mutations can contribute to the progression of mucin-producing neoplasms but not pancreatic intraepithelial neoplasia. PIK3CA status could be useful for understanding their progression to malignancy.

Abstract P2-08-03: Patterns of somatic mutations in metaplastic carcinoma of the breast

Abstract Introduction Metaplastic carcinoma of the breast (MCB) is a heterogeneous group of malignant tumors comprising less than 1% of all breast carcinomas encompassing tumors with purely epithelial, as well as epithelial and mesenchymal components, which may be entirely metaplastic or admixed with carcinoma. There is no agreed upon optimal treatment regime for these tumors, which overall have a worse prognosis than similar stage invasive ductal carcinoma (IDC). Research into MCB has been limited by the rarity of this tumor; however as the majority of these tumors do not express ER, PR or Her2 (triple negative) and response to traditional chemotherapy is limited, there is a need for further research to identify predictive biomarkers and new targets for treatment. We report our findings of multigene mutation profiling in a series of 21 MCB. Methods DNA was extracted from tumors of twenty one patients with MCB, genotyped using the Oncocarta v1.0 kit a sensitive, high throughput mass spectrometry based assay which detects a panel of 238 specific mutations in 19 oncogenes including KRAS, BRAF, PIK3CA, MET, AKT1 & ERBB2 and processed on the Sequenom compact MassARRAY platform. In the case with heterologous differentiation, separate cores were taken from the spindle cell, osteo- and chrondrosarcomatous areas. Results Histological review revealed three pure spindle cell cases with the remainder showing mixed patterns of spindle, squamous and IDC, no special type. A single case showed heterologous osteosarcomatous and chondrosarcomatous differentiation. Ten mutations were detected in these 21 MCB. PIK3CA gene mutations (six H1047L and one E545K mutation) were seen in seven patients; and KRAS gene mutations (two G12V and one G12A) were observed in three patients. No mutations were detected in the case with heterologous elements in the separate areas of spindle, osteo- and chondrosarcomatous differentiation. Conclusions PIK3CA mutations were seen in 33% and KRAS mutations were seen in 14% of MCB cases in this series. Given the general lack of effective chemotherapies in MCB, these findings suggest that the PI3K pathway may be a promising therapeutic target worthy of further investigation. We plan to also undertake detailed mutational analyses using next generation sequencing to identify further potential therapeutic targets in these rare and difficult to treat malignancies. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-08-03.

Loss of PTEN Expression Is Associated with Poor Prognosis in Patients with Intraductal Papillary Mucinous Neoplasms of the Pancreas

Previously, we reported PIK3CA gene mutations in high-grade intraductal papillary mucinous neoplasms (IPMN). However, the contribution of phosphatidylinositol-3 kinase pathway (PI3K) dysregulation to pancreatic carcinogenesis is not fully understood and its prognostic value unknown. We investigated the dysregulation of the PI3K signaling pathway in IPMN and its clinical implication. Thirty-six IPMN specimens were examined by novel mutant-enriched sequencing methods for hot-spot mutations in the PIK3CA and AKT1 genes. PIK3CA and AKT1 gene amplifications and loss of heterozygosity at the PTEN locus were also evaluated. In addition, the expression levels of PDPK1/PDK1, PTEN, and Ki67 were analyzed by immunohistochemistry. Three cases carrying the E17K mutation in the AKT1 gene and one case harboring the H1047R mutation in the PIK3CA gene were detected among the 36 cases. PDK1 was significantly overexpressed in the high-grade IPMN versus low-grade IPMN (P = 0.034) and in pancreatic and intestinal-type of IPMN versus gastric-type of IPMN (P = 0.020). Loss of PTEN expression was strongly associated with presence of invasive carcinoma and poor survival in these IPMN patients (P = 0.014). This is the first report of AKT1 mutations in IPMN. Our data indicate that oncogenic activation of the PI3K pathway can contribute to the progression of IPMN, in particular loss of PTEN expression. This finding suggests the potential employment of PI3K pathway-targeted therapies for IPMN patients. The incorporation of PTEN expression status in making surgical decisions may also benefit IPMN patients and should warrant further investigation.

Abstract P1-04-04: Towards methodology of detecting PIK3CA mutations on single cell level by massive parallel sequencing

Abstract Background: The oncogene PIK3CA encodes the p110α catalytic subunit of phosphatidylinositol 3-kinase. Gain-of-function mutations occur in 20 – 40% of the diagnosed breast cancer cases and may predict response to PI3K/AKT/mTOR inhibitors. We have developed a methodology to determine the PIK3CA exon 9 and 20 mutational status on single cell level, allowing analysis of low-input patient material such as circulating tumor cells (CTC). Methods: Single and groups of cultured breast cancer cells (BT20, MDA-MB-361 and MCF7) were isolated and recovered from spiked blood samples using the CellSearch and DEPArray platform respectively, and subjected to whole-genome amplification. Additionally, Formalin-fixed, paraffin-embedded (FFPE) tissue sections from 3 breast cancer patients were collected, of which two were mutant (MT) and one carried the wild-type (WT) genotype, as previously determined by external laboratories. Targeted PIK3CA PCR for exon 9 and 20 with unique multiplex identifiers (MIDs) allowed mixing of 32 samples and differentiation in the sequence data. Massive parallel sequencing was carried out using Titanium Amplicon chemistry (Lib-A) on a 454 GS Junior and data were analyzed using the amplicon variant analyzer (AVA) software. Results: A throughput of 120.464 high-quality reads with a mean coverage depth of 987 reads (range 288 – 5979) allowed the reliable assessment of variants, which were concordant with NCBI SNP/COSMIC databases and external laboratories. In BT20, the variants and frequencies were the missense P539R (48,83%) and H1047R (45,36%) mutations. Missense E545K mutation was detected in MDA-MB-361 (33,33%) and MCF7 (70,83%). Above mentioned mutations were detected in 73,3% and 100% of analyzed single and groups of tumor cells, respectively. Inability to determine the mutational status in some single cells was due to loss of amplicon during purification. In the MT FFPE tissue sections, two variants were detected and were concordant with previously obtained results. The variants and frequencies detected were E542K (14,12%) and E545K (21,38%), and correlated well with the tumor cell content of the actual sample (40% and 50%, respectively). Additionally, variant analysis showed 2 different SNPs in exon 20, which are classified as the synonymous mutations T1025T and A995A. In the WT sample no PIK3CA mutations were detected, suggesting a 100% specificity on the obtained 454 data. Conclusions: Our study delivers a semiautomated workflow using next-generation sequencing for the assessment of PIK3CA gain-of-function mutations on FFPE tissue sections and on single DEPArray purified cells from CellSearch pre-enriched whole blood samples. We are now implementing this workflow in the analysis of circulating tumor cells from patients with metastatic breast cancer. At the time of submitting, a cohort of 40 patients with luminal breast cancer was selected to assess the PIK3CA mutational status in the primary tumor, CTC and (if present) metastatic compartment. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-04-04.

Analysis of mutations in the PIK3CA and FGFR3 genes in verrucous epidermal nevus

Verrucous epidermal nevi are congenital hamartomas composed of keratinocytes and may occur alone or in association with developmental abnormalities. A close relationship between variations in the PIK3CA and FGFR3 genes and the appearance of nevi has been recently reported. Based on that, we performed molecular assays for the identification of E542K, E545G/K and H1047R mutations in the PIK3CA gene and of the R248C mutation in the FGFR3 gene. Interestingly, during the amplification process, we did not observe the PCR product of exon 9 of the PIK3CA gene, a region comprising amino acids 542-545. This strongly suggests the occurrence of a microdeletion of that region and indicates a possible allelic variant, which has not yet being described in the literature.

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.