PIK3CA Alterations Research Articles

Improved outcomes in PI3K-pathway-altered metastatic HPV oropharyngeal cancer.

While it has been recognized that human papillomavirus-associated (HPV-associated) oropharyngeal cancer (OPC) portends an improved prognosis, distinct patterns of disease recurrence have emerged. Molecular characterization of this subset of HPV patients remains unexplored. We evaluated 52 metastatic HPV+ OPC patients from our institution and paired massively parallel sequencing data with clinical parameters and survival outcomes in 81% of patients. Genomic data were then compared with 2 molecularly defined, curable HPV+ cohorts. Metastatic HPV+ OPC patients with pulmonary-only metastases demonstrated worse outcomes. Nonexclusive somatic alterations in KMT2D and PIK3CA were most frequent, with PRKDC alterations occurring at higher frequency when compared with all sequenced HPV+ OPC patients. PI3K pathway alterations were associated with improved outcomes among metastatic HPV+ OPC patients. We demonstrate subtle differences in the mutational landscape between curable and metastatic HPV+ OPC populations, with a trend towards more frequent DNA repair protein alterations in the latter. We demonstrate improved outcomes when PI3K pathway alterations are present in these patients. We provide molecular insights for this important HPV+ subgroup that have significant therapeutic implications.

PIK3CA mutation is a favorable prognostic factor in esophageal cancer: molecular profile by next-generation sequencing using surgically resected formalin-fixed, paraffin-embedded tissue

BackgroundPractical and reliable genotyping procedures with a considerable number of samples are required not only for risk-adapted therapeutic strategies, but also for stratifying patients into future clinical trials for molecular-targeting drugs. Recent advances in mutation testing, including next-generation sequencing, have led to the increased use of formalin-fixed paraffin-embedded tissue. We evaluated gene alteration profiles of cancer-related genes in esophageal cancer patients and correlated them with clinicopathological features, such as smoking status and survival outcomes.MethodsSurgically resected formalin-fixed, paraffin-embedded tissue was collected from 135 consecutive patients with esophageal cancer who underwent esophagectomy. Based on the assessment of DNA quality with a quantitative PCR-based assay, uracil DNA glycosylase pretreatment was performed to ensure quality and accuracy of amplicon-based massively parallel sequencing. Amplicon-based massively parallel sequencing was performed using the Illumina TruSeq® Amplicon Cancer Panel. Gene amplification was detected by quantitative PCR-based assay. Protein expression was determined by automated quantitative fluorescent immunohistochemistry.ResultsData on genetic alterations were available for 126 patients. The median follow-up time was 1570 days. Amplicon-based massively parallel sequencing identified frequent gene alterations in TP53 (66.7%), PIK3CA (13.5%), APC (10.3%), ERBB4 (7.9%), and FBXW7 (7.9%). There was no association between clinicopathological features or prognosis with smoking status. Multivariate analyses revealed that the PIK3CA mutation and clinical T stage were independent favorable prognostic factors (hazard ratio 0.34, 95% confidence interval: 0.12–0.96, p = 0.042). PIK3CA mutations were significantly associated with APC alterations (p = 0.0007) and BRAF mutations (p = 0.0090).ConclusionsOur study provided profiles of cancer-related genes in Japanese patients with esophageal cancer by next-generation sequencing using surgically resected formalin-fixed, paraffin-embedded tissue, and identified the PIK3CA mutation as a favorable prognosis biomarker.

Frequency of actionable alterations in epidermal growth factor receptor (EGFR) wild type non-small cell lung cancer: experience of the Wide Catchment Area of Romagna (AVR).

Molecular diagnostics for non-small cell lung cancer (NSCLC) has become the standard of care for personalized treatment. Epidermal growth factor receptor (EGFR) mutation and EML4-ALK translocation represent the two most important alterations in first-line treatment decision-making. However, other potentially targetable alterations are also present. One thousand consecutive NSCLC patients with EGFR wild type (wt) tumors diagnosed by routine molecular analysis were considered. KRAS, BRAF, ERBB2, PIK3CA, NRAS, ALK, MAP2K1, RET and DDR2 gene mutations were analyzed using the multiparametric Sequenom MassARRAY® platform. EML4-ALK and ROS1 rearrangements were also assessed by fluorescent in situ hybridization. HER4 status was determined by direct sequencing. Three hundred and forty-eight (34.8%), 31 (3.1%), 39 (4.4%), 14 (1.8%), 6 (0.7%), 16 (1.8%), 5 (0.6%) and 9 (0.9%) patients showed an alteration in KRAS, BRAF, ALK, ROS1, NRAS, PIK3CA, MAPK1/2 and HER2 genes, respectively. Of the 657 patients for whom all markers were determined, 318 (48%) patients had at least one alteration. Eight patients showed overlapping mutations, 4 KRAS mutation/EML4-ALK translocation, one KRAS mutation/ROS1 rearrangement, 2 KRAS/PIK3CA mutations, and one BRAF/PIK3CA mutations. About 50% of our patients had a potentially targetable alteration, confirming the usefulness of a multiparametric approach for routine molecular diagnostics aimed at identifying potential therapeutic targets.

Heterogeneous responses and resistant mechanisms to crizotinib in ALK-positive advanced non-small cell lung cancer.

BackgroundALK‐tyrosine kinase inhibitors (TKIs) have been proven effective for treating ALK‐positive non‐small cell lung cancer (NSCLC), although patients present with variable responses and disease progression courses. The detailed underlying molecular mechanisms require further investigation to yield a better prognosis.MethodsTargeted next‐generation sequencing (NGS) mutation profiling was performed on samples from 42 NSCLC patients confirmed positive for ALK rearrangements by fluorescence in situ hybridization or immunohistochemistry who experienced disease progression after crizotinib treatment.Results ALK rearrangements were not confirmed in six patients (14%) with other potential oncogenic drivers identified by NGS, who therefore did not respond to crizotinib and had significantly shorter overall survival (OS) compared to NGS ALK ‐positive patients. Fifteen ALK activating mutations were detected in 8 out of 26 post‐treatment samples (31%), among which ALK L1196M and G1269A were the most common acquired mutations detected in half of the patients with ALK activating mutations. Dynamic monitoring of the genetic evolution in one patient revealed both spatial and temporal heterogeneity of resistant mechanisms during different ALK‐TKI treatment courses. Activation of ALK downstream or bypass pathways was detected in patients without ALK activating mutations, such as genetic alterations in PIK3CA, MET, and KRAS. Interestingly, we identified two patients with acquired mutations in the DNA mismatch repair gene POLE, which resulted in a dramatically increased tumor mutation burden, and might contribute to the poor response to crizotinib.ConclusionsHeterogeneous resistant mechanisms have been identified and correlate to diverse responses to crizotinib. Comprehensive and dynamic mutation profiling is required to better predict clinical outcomes.

Abstract 2582: A novel PI3K/Akt-pathway activation biomarker using comprehensive genomic profiling (CGP) for clinical trial assay

Abstract Introduction Patients with PI3K/Akt-pathway activation may be sensitive to selective Akt-inhibitors that are currently under development. We have developed a novel next-generation sequencing (NGS)-based composite biomarker assay that identifies patients with PI3K/Akt-pathway activated tumors by identifying activating PIK3CA and AKT1 alterations, and inactivating alterations in PTEN. This assay was analytically validated, and applied to triple-negative breast cancer (TNBC) patients in the LOTUS trial (NCT02162719), a placebo-controlled phase II clinical trial to assess the safety and efficacy of adding ipatasertib to paclitaxel treatment in patients with metastatic TNBC (Kim et al., 2017). Methods DNA extracted from FFPE tumor tissue underwent whole-genome shotgun library construction and hybridization-based capture, followed by sequencing using Illumina HiSeq 4000. Sequence data were processed using a proprietary analysis pipeline designed to detect base substitutions, indels, copy number alterations, genomic rearrangements, microsatellite instability, and tumor mutational burden. The assay further evaluated the PI3K/Akt-pathway activation biomarker status that consists of six features: 1-2) AKT1 and PIK3CA activating mutations, 3) PTEN homozygous deletion, 4) PTEN heterozygous deletion (HE), 5) PTEN dominant negative mutations, and 6) bi-allelically inactivated (BI) PTEN mutations defined as mutation plus loss of heterozygosity (LOH). We evaluated the limit of detection (LoD) and the precision of the biomarker for two novel genomic features: HE and BI, with the other four features previously validated. Results Analytical validation of novel biomarker features: The LoD of detecting PTEN HE and BI was determined to be 30%, the lowest tumor content at which the features can be detected at 90% probability. In the precision study, 100% (81/81) agreement was achieved across different replicates within the same sequencer run and across different sequencer runs for biomarker positive samples, demonstrating high reproducibility in calling PTEN HE and BI. Conclusions We developed and analytically validated an NGS-based assay that identifies complex and novel genomic alterations (heterozygous deletion and bi-allelic inactivation) in PTEN that is part of a composite PI3K/Akt-pathway activation biomarker. This assay identified patients that appeared to derive greater benefit in the Phase II LOTUS study as compared to using PTEN IHC to only identify patients with PTEN protein loss (Kim et al., 2017). This assay could be generalized to identify other biomarkers with similar types of genetic alterations. It also demonstrates that NGS-based CGP can broaden the intent to treat population to be more specifically related to the mechanism of action of a drug, while also being more selective to patients with potential to respond. Citation Format: Yuting He, Matthew Wongchenko, Joel Skoletsky, Christine Burns, Yali Li, Paula Maness, Doris Kim, Doron Lipson, Philip Stephens, Vincent Miller, Jeffrey Ross, Steven Gendreau, James Sun. A novel PI3K/Akt-pathway activation biomarker using comprehensive genomic profiling (CGP) for clinical trial assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2582.

Abstract 2973: A comprehensive panel of patient-derived xenografts representing the molecular heterogeneity and diversity of triple-negative breast cancer

Abstract Purpose: Triple-negative breast cancer (TNBC) is a heterogeneous disease. Patients diagnosed with TNBC have a poor prognosis and identification of new biomarkers and therapeutic agents is a high priority. Patient-derived xenografts (PDX) are clinically relevant models that have emerged as important tool for the analysis of drug activity and predictive biomarker discovery. The purpose of this work was to analyze the molecular heterogeneity of a large panel of TNBC PDX with the perspective to test targeted therapy and to identify biomarkers of response. Experimental Design: PDX of early-stage TNBC established from 2003 to 2016 (N=60) were analyzed by high-resolution array CGH and gene expression profiling (Cytoscan HD and Human Gene 1.1 ST arrays). Subtypes were identified with the tool TNBCtype (Chen et al., 2012) based on transcriptomic data. A targeted next-generation sequencing (NGS) of 100 genes (the top frequently mutated genes in breast cancer) was performed on Illumina HiSeq2500 sequencer. COSMIC, Tumorportal and cBioportal databases were used for the interpretation of genomic variants. Immunohistochemistry (IHC) and morphologic analysis of PDX were performed as compared to the corresponding patients' tumors. PDX carrying targetable genomic alterations in the PI3K/AKT/mTOR and MAPK signaling pathways were treated by specific inhibitors (selumetinib, BAY 80-6946 and PF-04691502). Results: At the gene expression level, TNBC PDX represent all the different TNBC subtypes identified by the Lehmann classification. The frequency of the different TNBC subtypes was similar to the TGCA TNBC, except for the immunomodulatory subtype, underrepresented in PDX. Somatic pathologic mutations and copy number alterations were similar in PDX and TCGA TNBC patients. Among the top altered genes are TP53 and oncogenes and tumor suppressors of the PI3K/AKT/mTOR and MAPK pathways (including PIK3CA, AKT1, NF1 and NRAS/KRAS). At the histologic level, TNBC PDX were mainly composed of invasive ductal carcinoma of no special type, with some tumors being classified as apocrine or metaplastic carcinomas. Comparison with the original tumors show similar patterns (based on IHC analysis of CK5, CK8/18, CK14 and AR, FOXA1, EGFR and Ki67). In vivo efficacy experiments with PI3K and MAPK pathways inhibitors showed marked antitumor activity in PDX carrying genomic alterations of PIK3CA, AKT1 and NRAS, NF1 genes. Drug combination experiments are currently ongoing in PDX with simultaneous genomic alterations of PI3KCA and MAPK related genes. Conclusions: TNBC PDX reproduce the molecular heterogeneity and diversity of TNBC patients. This large collection of PDX is a clinically relevant platform for drug testing, biomarker discovery and translational research. Citation Format: Florence Coussy, Virginie Bernard, Marion Lavigne, Anais Boulai, Sophie Chateau-Joubert, Ahmed Dahmani, Elodie Montaudon, Cécile Reyes, Rania El Botty, Gaëlle Pieron, Cécile Laurent, Samia Melaabi, Anne Vincent Salomon, Ivan Bièche, Elisabetta Marangoni. A comprehensive panel of patient-derived xenografts representing the molecular heterogeneity and diversity of triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2973.

Molecular profiles and tumor mutational burden analysis in Chinese patients with gynecologic cancers

The goal of this work was to investigate the tumor mutational burden (TMB) in Chinese patients with gynecologic cancer. In total, 117 patients with gynecologic cancers were included in this study. Both tumor DNA and paired blood cell genomic DNA were isolated from formalin-fixed paraffin-embedded (FFPE) specimens and blood samples, and next-generation sequencing was performed to identify somatic mutations. TP53, PTEN, ARID1A, and PIK3CA alterations were significantly different in various types of gynecologic cancers (p = 0.001, 1.15E-07, 0.004, and 0.009, respectively). The median TMB of all 117 gynecologic tumor specimens was 0.37 mutations/Mb, with a range of 0–41.45 mutations/Mb. Despite the lack of significant difference, endometrial cancer cases had a higher median TMB than cervical and ovarian cancer cases. Younger gynecologic cancer patients (age <40 years) had a significantly lower TMB than older patients (age ≥40 years) (p = 0.04). In addition, TMB was significantly increased with increasing clinical stage of disease (p = 0.001). PTEN alterations were commonly observed in patients with a moderate to high TMB (n = 8, 38.10%, p = 9.95E-04). Although limited by sample size, all of the patients with TSC2 (n = 3, p = 3.83E-11) or POLE (n = 2, p = 0.005) mutations had a moderate to high TMB. Further large-scale, prospective studies are needed to validate our findings.

Prognostic role of PIK3CA and TP53 in human papillomavirus-negative oropharyngeal cancers.

Human papilloma virus (HPV)-negative oropharyngeal squamous cell carcinomas (OPCs) have a poorer prognosis and best management is an unmet need. We studied the prognostic role of epidermal growth factor receptor (EGFR) and PIK3CA amplifications and TP53 functional status. Between 1992 and 2000, 90 consecutive patients with OPCs were treated with surgery, followed by radiotherapy in case of high-risk pathologic features. Of those, 73 cases were HPV-negative and therefore were selected for molecular analysis ( PIK3CA and EGFR fluorescent in situ hybridization [FISH] analysis and TP53 mutation analysis). FISH analyses of EGFR and PIK3CA were successfully conducted on 69 and 63 of 73 tumor samples, respectively. EGFR alterations were detected in 43% of patients but just 7% showed amplification. Seven cases (11%) carried PIK3CA amplification and 18 (29%) gene gain or high polysomy. TP53 was detected as nonfunctional in 24 of 67 (36%) successfully analyzed cases. Both univariable and multivariable analysis showed statistically significantly worse disease-free survival (DFS) for patients with PIK3CA disomy compared to those with gene gain or high polysomy. No differences in overall survival or DFS for EGFR and TP53 alteration were evident. The combined evaluation of PIK3CA and TP53 showed that PIK3CA gene copy number gain separated a population with better outcome, defining an overall worse prognosis population (disomy) now clearly further divided according to TP53 functional status. PIK3CA gene copy number increase is associated with a favorable clinical outcome in HPV-negative OPCs treated with surgery ± postoperative radiotherapy. In patients without PIK3CA alteration, TP53 nonfunctional mutations are associated with poor prognosis.

Gastric Carcinomas With Lymphoid Stroma

Gastric carcinoma with lymphoid stroma is an uncommon variant enriched for mutually exclusive Epstein-Barr virus (EBV) positivity and mismatch repair (MMR) deficiency. We performed this study to evaluate molecular alterations in this morphologically homogeneous subtype and compare them with 295 conventional gastric cancers analyzed in The Cancer Genome Atlas study. We identified 31 study cases and subjected them to in situ hybridization for EBV-encoded RNAs and assessment for MMR status. Immunostains for PD-L1, β-catenin, and HER2 were performed; extracted DNA was sequenced with a Comprehensive Cancer Panel. Most study patients were older adult men with stage I or II disease (76%). Tumors were classified as EBV/MMR-proficient (MMR-P) (n=7), EBV/MMR deficient (n=12), and EBV/MMR-P (n=12). EBV/MMR-P tumors were usually located in the proximal stomach (83%) and showed heterogenous growth patterns with glandular differentiation (83%). Tumors in all groups showed numerous tumor infiltrating lymphocytes and PD-L1 expression, infrequent nuclear β-catenin accumulation (10%), and lacked both membranous HER2 staining and HER2 amplification. EBV/MMR-deficient tumors showed significantly higher tumor mutation burden (P=0.001) and KRAS alterations (56%) compared with EBV/MMR-P tumors (9%, P=0.05). TP53 variants were more common among EBV/MMR-P tumors (82%) compared with EBV/MMR proficient (0%, P=0.01) and EBV/MMR-deficient (11%, P<0.01) tumors. Alterations in KRAS, ARID1A, PIK3CA, and TP53 followed similar patterns of distribution compared with The Cancer Genome Atlas dataset. We conclude that gastric carcinomas with lymphoid stroma show a spectrum of molecular changes and frequent PD-L1 expression, raising the possibility that this subgroup of tumors may be susceptible to checkpoint inhibitors and/or agents that target receptor tyrosine kinase-mediated signaling.

Overexpression of PIK3CA in head and neck squamous cell carcinoma is associated with poor outcome and activation of the YAP pathway

ObjectivesPhosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) is commonly altered in many human tumors, leading to the activation of p110α enzymatic activity that stimulates growth factor-independent cell growth. PIK3CA alterations such as mutation, gene amplification and overexpression are common in head and neck squamous cell carcinoma (HNSCC) and. We aim to explore how these alterations and clinical outcome are associated, as well as the molecular mechanisms involved. Material and methodsMutation and copy-number variation in PIK3CA, and whole-genome expression profiles, were analyzed in primary HNSCC tumors from The Cancer Genome Atlas (TCGA) cohort (n = 243). The results were validated in an independent cohort form the University Hospital of A Coruña (UHAC, n = 62). Expression of the PIK3CA gene protein product (PI3K p110α) and nuclear YAP were assessed in tissue microarrays in a cohort from the University Hospital 12 de Octubre (UH12O, n = 91). ResultsOnly high expression of the PIK3CA gene was associated with poor clinical outcome. The study of gene expression, transcription factor and protein signatures suggested that the activation of the Hippo-YAP pathway, involved in organ size, stem cell maintenance and tumorigenesis, could underlie tumor progression in PI3KCA overexpressing tumors. Tissue arrays showed that PI3K p110α levels correlated with YAP nuclear localization in HNSCC tumors. ConclusionsHigh expression of PIK3CA in HNSCC primary tumors identifies patients at high risk for recurrence. In these tumors, progression could rely on the Hippo-YAP pathway instead of the canonical Akt/mTOR pathway. This observation could have important implications in the therapeutic options for patients.

Pan-cancer Alterations of the MYC Oncogene and Its Proximal Network across the Cancer Genome Atlas.

Although the MYC oncogene has been implicated in cancer, a systematic assessment of alterations of MYC, related transcription factors, and co-regulatory proteins, forming the proximal MYC network (PMN), across human cancers is lacking. Using computational approaches, we define genomic and proteomic features associated with MYC and the PMN across the 33 cancers of The Cancer Genome Atlas. Pan-cancer, 28% of all samples had at least one of the MYC paralogs amplified. In contrast, the MYC antagonists MGA and MNT were the most frequently mutated or deleted members, proposing a role astumor suppressors. MYC alterations were mutually exclusive with PIK3CA, PTEN, APC, or BRAF alterations, suggesting that MYC is a distinct oncogenic driver. Expression analysis revealed MYC-associated pathways in tumor subtypes, such as immune response and growth factor signaling; chromatin, translation, and DNA replication/repair were conserved pan-cancer. This analysis reveals insights into MYC biology and is a reference for biomarkers and therapeutics for cancers with alterations of MYC or the PMN.

Abstract PD3-12: PIK3CA alterations and benefit with neratinib after trastuzumab-based adjuvant therapy in early-stage HER2+ breast cancer: Correlative analyses of the phase III ExteNET trial

Abstract Background: Neratinib is a pan-HER tyrosine kinase inhibitor that blocks the PI3K/Akt and MAPK signaling pathways downstream from HER2. The international, randomized, placebo-controlled phase III ExteNET trial showed that a 1-year course of neratinib after trastuzumab-based adjuvant therapy significantly improved 2-year invasive disease-free survival (iDFS) in early-stage HER2+ breast cancer (HR 0.67; 95% CI 0.50–0.91; p=0.0091) [Chan et al. Lancet Oncol 2016]. Furthermore, the effects of neratinib on iDFS were shown to be durable at 5 years' follow-up (HR 0.73; 95% CI 0.57–0.92; p=0.008) [Martin et al. ESMO 2017]. PIK3CA alterations are common in HER2+ breast cancers, and in general are associated with a worse prognosis. We sought to assess the prognostic and predictive significance of PIK3CA alterations in an exploratory substudy of the ExteNET trial. Methods: ExteNET is an international, multi-center, randomized, double-blind, placebo-controlled phase III trial (Clinicaltrials.gov: NCT00878709). Patients received oral neratinib 240 mg/day or placebo for 1 year. Of the intent-to-treat (ITT) population (n=2840), primary formalin-fixed paraffin-embedded (FFPE) tumor specimens were available from 991 patients for PIK3CA mutation testing by RT-PCR for two hot-spot mutations in exon 9 (E542K, E545K/D) and one hot-spot mutation in exon 20 (H1047R). 702 FFPE tumor slides underwent FISH analysis for PIK3CA amplification with a ratio of ≥2.2 considered as amplified. Primary endpoint: iDFS. iDFS events were tested by 2-sided log-rank tests, and HR (95% CI) were estimated using Cox proportional-hazards models. Data cut-off: March 2017. Results: Baseline demographics and disease characteristics between treatment arms of the correlative cohort (n=1201) were balanced. Overall, 21.2% (n=210) of primary tumors harbored one of the specified PIK3CA mutations, and 8.7% (n=61) were PIK3CA FISH-amplified. Patients with PIK3CA-altered tumors (i.e. PIK3CA mutations or FISH-amplified) had fewer iDFS events with neratinib compared with placebo (HR 0.41; 95% CI 0.17-0.90, p=0.028). The interaction test was not significant (p=0.1842). Results of the various correlative analyses within treatment arms are shown in the table. NeratinibPlacebo iDFS iDFS 2-sidedPopulationnevents, nnevents, nHR (95% CI)P valueaITT142011614201630.73 (0.57–0.92)b0.008bCorrelative cohort59345608700.67 (0.45–0.96)0.0317PIK3CA-mutation positive1047106170.43 (0.17–1.01)0.056PIK3CA-mutation negative38527396420.66 (0.40-1.06)0.089PIK3CA-amplified3312840.20 (0.01-1.33)0.106PIK3CA-non-amplified31629325360.85 (0.52-1.39)0.521PIK3CA-altered1308132200.41 (0.17-0.90)0.028a. Log-rank test; b. Stratified analysis Conclusions: One year of neratinib treatment after trastuzumab-based adjuvant therapy significantly improves iDFS after 5 years in patients with early-stage HER2+ breast cancer. From this modest-sized exploratory cohort, it appears that PIK3CA may be a biomarker for differential sensitivity to neratinib after 1 year of trastuzumab in the adjuvant setting.These exploratory results should be validated in a larger subset. Citation Format: Chia SKL, Martin M, Holmes FA, Ejlertsen B, Delaloge S, Moy B, Iwata H, von Minckwitz G, Mansi J, Barrios CH, Gnant M, Tomašević Z, Denduluri N, Šeparović R, Kim S-B, Hugger Jakobsen E, Harvey V, Robert N, Smith II J, Harker G, Lalani AS, Zhang B, Eli LD, Buyse M, Chan A. PIK3CA alterations and benefit with neratinib after trastuzumab-based adjuvant therapy in early-stage HER2+ breast cancer: Correlative analyses of the phase III ExteNET trial [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD3-12.

Abstract P2-06-01: Molecular characterization of human malignant phyllodes tumors reveals potential targeted approaches

Abstract Malignant phyllodes tumor (MPT) which belong to the fibroepithelial neoplasm spectrum is a rare type of breast malignancy, and currently there is no effective targeted approach available for MPT. In this study, we tried to identify key genomic alterations and biologic pathways in MPT by whole exome and RNA sequencing of nine MPT tissues. Whole exome sequencing revealed somatic alterations in EGFR, MED12, PIK3CA, PIK3R1, PDGFRA, PDGFRB, PTEN, and TP53. Transcriptome sequencing showed dysregulation of ECM-receptor interaction, focal adhesion, and PI3K-Akt signaling in MPTs when compared to normal breast or invasive breast cancer tissues. Based on the transcriptome profiles, the MPTs were classified into two subtypes; fibrous subtype with upregulation of stromal genes such as collagens and epithelial subtype with upregulation of E-cadherin and Claudins. The molecular classification of fibrous and epithelial subtypes was validated in 28 paraffin-embedded MPT tissues. The fibrous subtype showed higher mitotic index and increased risk for recurrence when compared to the epithelial subtype. We established a patient-derived xenograft model from one fibrous subtype MPT which harbored somatic mutation in PIK3R1 and PDGFRB. In that model, targeted treatment against PIK3CA/mTOR and PDGFR pathways effectively suppressed the tumor growth in vivo. Our data provide insights on the biologic understanding of MPT and suggest a clinically relevant molecular classification. Furthermore, we show that developing effective targeted approaches in MPT can be possible with genomic profiles and patient-derived xenograft models. The clinical efficacy of targeting PDGFR and PIK3CA/mTOR pathways in MPT should be tested in future clinical trials. Citation Format: Moon H-G, Yun J, Hong BS, Lee E, Lee H-B, Han W, Kim J-I, Noh D-Y, Heo W, Hur S, Kang W, Lee C. Molecular characterization of human malignant phyllodes tumors reveals potential targeted approaches [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-06-01.

Abstract PD8-05: Genomic profiling of metastatic invasive lobular carcinoma reveals unique genomics and therapeutic opportunities

Abstract Background Invasive lobular carcinoma (ILC) is a common breast cancer histological subtype comprising ˜10-15% of all cases. ILC possesses many unique features when compared to invasive ductal carcinoma (IDC). First, ILC has distinct genomic alterations expanding beyond the defining event of CDH1 loss to other genes such as TBX3, FOXA1, and AKT signaling related genes. Second, ILC responds differently to chemotherapeutics and endocrine therapies despite similar clinical staging. Third, ILC tumors spread to a distinct set of organs compared to IDC tumors, commonly forming distant metastases in the ovary, colon, omentum, and stomach. However, the genomics of metastatic ILC have yet to be fully explored. Methods Comprehensive hybrid-capture based genomic analysis of 286-395 cancer related genes was performed on 5523 histologically defined ILC (n=613) and IDC (n=4910) tumors. Of these, 29% and 21% were from distant metastatic sites for ILC and IDC, respectively. Additionally, histology based ER-status was available for a subset of tumors allowing a subgroup of ER-positive, HER2-negative IDC (ER-IDC) samples to be identified (n=655). Results We examined the genetic differences between ILC and IDC in the context of both local and metastatic disease. Overall, the genomic profiles of ILC are enriched for alterations in CDH1, TBX3, PIK3CA, and RUNX1 in agreement with previous studies. Alterations in genes involved in AKT signaling (PIK3CA, PTEN, and AKT1) are also enriched in ILC (64% v. 49%; p&amp;lt;10-7). Interestingly, NF1 loss of function alterations are enriched in metastatic ILC compared to ER-IDC (12.2% v. 3.6%, p&amp;lt;0.001) but not in local disease (4.8% v. 4.1%, p=0.72). NF1 is a negative regulator of RAS-cyclic AMP pathway and suggests that NF1 driven RAS signaling is an important driver of metastasis in ILC. We next examined metastatic ILC samples for alterations enriched at specific metastatic tissue sites. Two metastatic sites were exclusive to ILC samples compared to ER-IDC: GI (19.4%) and the female reproductive tract (11.7%). Within metastatic ILC, alterations in ESR1 showed strong tissue site bias towards liver metastases with 29% harboring an alteration in ESR1 (range: 8-13% in other sites, excluding ovary). Interestingly, ESR1 alterations were never observed in 14 ovary metastases, potentially reflecting an effect of local estrogen production on ILC ovarian metastases. In support of this, ILC ovarian metastases occur in younger women with a median age of 53.5 compared to 63.5 across all other sites. Lastly, high tumor mutation burden (TMB) is strongly associated with metastatic ILC with 8.9% of metastatic ILC classified as TMB-high (320 mutations/Mb) compared to 2.1% of ILC in the breast. A similar but less pronounced finding was also observed for ER-IDC (1.6% versus 0.8%). This suggests that checkpoint blockage therapies may be a more common option in metastatic ILC than previously appreciated. Conclusions Genomic profiling of metastatic ILC reveals numerous potential therapeutic options enriched in this disease. Inhibition of RAS signaling driven by NF1 loss and TMB-high directed immunotherapeutics may be potential therapeutic options for a substantial portion of metastatic ILC patients. Citation Format: Sokol ES, Basudan A, Lee AV, Stephens PJ, Frampton GM, Oesterreich S, Hartmaier RJ. Genomic profiling of metastatic invasive lobular carcinoma reveals unique genomics and therapeutic opportunities [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD8-05.

Abstract P2-02-13: Utilization of cell-free circulating tumor DNA for management of breast cancer: Practices in academic and community oncology

Abstract Background: Next-generation sequencing (NGS) of cell-free DNA (cfDNA) is increasingly being utilized to assess somatic genomic alterations in patients with breast cancer. We investigated the clinical use of such testing in breast cancer care in a major healthcare system with both academic and community-based practices. We also explored the observed genomic landscape in the analyzed patient cohort and whether treatment plans were modified based on the results. Methods: A retrospective review of cfDNA NGS results (Guardant360) ordered at the University of Pittsburgh Medical Center for patients with breast cancer from 7/2015-3/2017 was performed. Test ordering patterns, the landscape of genomic alterations identified, and clinical use of select results were assessed. Results: During this period 95 samples were submitted, 73 (77%) ordered by academic center providers and 22 (25%) from community providers. Alterations were detected in 88 samples (93%) with a median of 3 alterations per test. Five patients had serial samples ordered assessing dynamic cfDNA across clinical treatment and progressions, leaving 84 unique patients in the dataset. The average patient age was 57, and 95% of patients were female. Patients were most often observed to have alterations in TP53 (51%), PIK3CA (44%), and ESR1 (26%). Additional clinical data were collected for 48 patients with mutations or amplifications in PIK3CA, ESR1, and/or ERBB2 (HER2) to assess for clinical use of genomic information. Results were used to change clinical care in 13 (27%) of these cases. Community providers were more likely to use genomic results to guide clinical management in these cases (9/16, 56%) than academic providers (4/32, 12.5%), p=0.001. Of this patient subset, those with tests ordered by an academic provider had more lines of prior therapy at the time of testing vs. those in the community (average 5.9 vs 3.4 respectively, p=0.019). Conclusions: CfDNA NGS analysis for somatic genomic alterations in breast cancer is being ordered clinically by both academic and community practices within this healthcare system. Results for a subset of clinically annotated patients were acted on more frequently by community-based ordering providers, which may be related to patients tested at academic sites having had more lines of prior treatment. Citation Format: Thomas RA, Klar N, Kiedrowski L, Nagy RJ, Lee AV, Brufsky A. Utilization of cell-free circulating tumor DNA for management of breast cancer: Practices in academic and community oncology [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-02-13.

Mutational signatures and chromosome alteration profiles of squamous cell carcinomas of the vulva

Vulvar squamous cell carcinoma (SCC) consists of two different etiologic categories: human papilloma virus (HPV)-associated (HPV (+)) and HPV-non-associated (HPV (−)). There have been no genome-wide studies on the genetic alterations of vulvar SCCs or on the differences between HPV (+) and HPV (−) vulvar SCCs. In this study, we performed whole-exome sequencing and copy number profiling of 6 HPV (+) and 9 HPV (−) vulvar SCCs and found known mutations (TP53, CDKN2A and HRAS) and copy number alterations (CNAs) (7p and 8q gains and 2q loss) in HPV (−) SCCs. In HPV (+), we found novel mutations in PIK3CA, BRCA2 and FBXW7 that had not been reported in vulvar SCCs. HPV (−) SCCs exhibited more mutational loads (numbers of nonsilent mutations and driver mutations) than HPV (+) SCCs, but the CNA loads and mutation signatures between HPV (+) and HPV (−) SCCs did not differ. Of note, 40% and 40% of the 15 vulvar SCCs harbored PIK3CA and FAT1 alterations, respectively. In addition, we found that the SCCs harbored kataegis (a localized hypermutation) in 2 HPV (+) SCCs and copy-neutral losses of heterozygosity in 4 (one HPV (+) and 3 HPV (−)) SCCs. Our data indicate that HPV (+) and HPV (−) vulvar SCCs may have different mutation and CNA profiles but that there are genomic features common to SCCs. Our data provide useful information for both HPV (+) and HPV (−) vulvar SCCs and may aid in the development of clinical treatment strategies.

Association between PIK3CA alteration and prognosis of gastric cancer patients: a meta-analysis.

BackgroundIncreasing evidence suggests that dysregulation of phosphatidylinositol-4, 5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) plays an important role in carcinogenesis. However, the relationship between PIK3CA expression and gastric cancer (GC) prognosis remains controversial.MethodsWe searchedPubMed, Embase, Web of Science, and the Cochrane Library databases for relevant studies up to June 30, 2017. Primary outcomes were hazard ratio (HR), odds ratio (OR), and 95% confidence intervals (CI) for association with overall survival and clinicopathological features.ResultsEleven studies comprising 2481 GC patients were analyzed. Pooled analysis showed that PIK3CA upregulation was significantly associated with worse overall survival (HR = 1.79, 95% CI 1.42–2.27, p< 0.001) at the protein (HR = 1.94, 95% CI 1.52–2.47, p< 0.001) but not the gene (HR = 1.57, 95% CI 0.92–2.69, p= 0.097) level. PIK3CA gene mutation did not correlate with overall survival (HR = 1.05, 95% CI 0.83–1.34, p= 0.666) but was significantly associated with poor tumor differentiation (OR = 0.37, 95% CI 0.17–0.76, p= 0.011).ConclusionHigh PIK3CA protein expression predicted poor prognosis in GC, whereas PIK3CA gene amplification or mutation did not. Moreover, PIK3CA mutation was an indicator of poorly differentiated tumors.

Abstract A029: Biomarker analysis in HR+, HER2-, locally advanced, or metastatic breast cancer patients treated with buparlisib: results from BELLE-3

Abstract Background: Endocrine therapy resistance in hormone receptor-positive breast cancer often leads to activation of phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin pathway. In the BELLE-3 study the progression-free survival (PFS) improvement in buparlisib (BUP) vs placebo (PBO) arm was greater in patients (pts) with PIK3CA-mutant than PIK3CA-wildtype (Wt) tumors, based on circulating tumor DNA. In the current study we report the additional exploratory biomarker results from BELLE-3. Methods: The relationship between PFS and frequently mutated genes (PIK3CA, CCND1, ESR1 and 8q11.23 region) and pathways (PI3K and CDK pathways) was explored using NGS in tumor and pts were classified as altered (Alt) vs Wt. Genes and pathways with at least 10% frequency in both arms were carried forward to correlative analyses with PFS. In addition, ESR1 mutations were detected by BEAMing technology in baseline cell free DNA (cfDNA) for 167 pts. Results: NGS data were available for 185 pts and the NGS panel covered 500+ gene mutations, short insertions/deletion, copy number alterations, and translocations. PIK3CA was altered in 41% of pts and PI3K pathway was altered in 64% of pts. Benefit of BUP treatment was observed in PIK3CA altered pts, but did not extend to other alterations in the PI3K pathway. CCND1 was altered in 19% of pts and the CDK pathway was altered in 34% of pts and improved benefit of BUP was observed in altered pts in both the groups (HR = 0.31 and 0.38 respectively). Alterations in the 8q11.23 region (ZNF703, WHSC1L1 and FGFR1) were observed in 21% of pts with the benefit of BUP observed only among wildtype. In cfDNA, a higher treatment effect was observed in the ESR1 mutant subgroup (HR = 0.76 [Wt] and 0.27 [Alt]). Subgroup analyses of ESR1 mutations revealed a greater improvement in median PFS among pts with D538G mutation (BKM vs PBO = 8.25 vs 1.51; HR = 0.18) than among pts with Y537N/S/C mutation (BKM vs PBO = 4.17 vs 2.76; HR = 0.45). The PFS of different groups analyzed by NGS is mentioned in the table. Conclusions: A trend for stronger treatment effect was observed among pts with alterations in PIK3CA, CDK pathway, CCND1 (tumor), and ESR1 (ctDNA). The possibility of correlation among identified biomarkers is currently being explored. Table: Median PFSGroupTreatmentNEventsMedian PFS, moHR (95% CI)PIK3CAWtPBO34271.480.73 (0.46 - 1.16)BUP75602.83AltPBO23191.40.44 (0.25 - 0.79)BUP53344.11PI3K pathway (excluding PIK3CA Alt pts)WtPBO19171.450.51 (0.28 - 0.93)BUP47402.83Alt*PBO15104.671.1 (0.51 - 2.39)BUP28202.86CCND1WtPBO47371.480.61 (0.41 - 0.92)BUP103733.35AltPBO1091.430.31 (0.13 - 0.76)BUP25212.1CDK pathwayWtPBO40321.480.63 (0.4 - 0.98)BUP83583.42AltPBO17141.430.38 (0.19 - 0.75)BUP45362.68q11.23 regionWtPBO48391.450.49 (0.33 - 0.73)BUP98704.11AltPBO971.410.9 (0.38 - 2.13)BUP30241.48ESR1 mutation statusWtPBO29202.70.76 (0.43 - 1.31)BUP56372.8AltPBO23202.60.27 (0.14 - 0.51)BUP59246.0 * To assess the impact of genes in the PI3K pathway other than PIK3CA, the altered group includes patients who are PIK3CA wildtype but have an alteration in a different gene in the PI3K pathway. Citation Format: Nadia Solovieff, Ying A. Wang, Banu Sankaran, Nicolas Scheuer, Mona El-Hashimy, Denis Weber, Dalila Sellami, Angelo Di Leo. Biomarker analysis in HR+, HER2-, locally advanced, or metastatic breast cancer patients treated with buparlisib: results from BELLE-3 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A029.

Abstract A087: Empowering precision medicine in metastatic colorectal cancer: preliminary results from the FUNNEL platform

Abstract Background: Metastatic colorectal cancer still has a dismal 5-yr survival of less than 10%. Chemotherapy, targeted therapies, and immune-checkpoint inhibitors are effective in only a fraction of patients, and even in responders, long-term remission are rare. To harness insight into the evolving heterogeneity of mCRC, and to combat it therapeutically, we have established the logistic research matrix FUNNEL that integrates the topographic acquisition of patient samples with the clinical validation of hypothesis-driven trials in primary or secondary resistance disease. Methods: FUNNEL will follow 1000 sequential cases from diagnosis to death, acquiring biologic specimens, imaging, and clinical data at critical steps along the disease continuum. At diagnosis of metastatic disease, patients are genotyped with a CRC-specific panel designed to map the landscape of primary resistance to anti-EGFR therapies (Bertotti A. et al,, Nature 2015). Paraffin samples are centrally processed and tested using Sequenom® (with a 5% detection sensitivity for 168 mutations in KRAS, BRAF, NRAS, PIK3CA, EGFR, HER2, IDH1, MEK1) and NanoString® technologies [measuring gene copy number variation (CNV) for RAF1, EGFR, FGRF1/2/3, HER2, IGF1/2, IGF1R, KRAS, MET, and NF1]. Genes are considered amplified with a CNV ≥ 10 cut-off validated by FISH. Dynamic data of centralized disease assessments imaging coupled with longitudinal liquid biopsies are also collected at significant clinical events (baseline, response to therapy, and progression of sequential treatment’s lines). Proteomics and neoantigenic profile of cancer cells in parallel with function and tumor specificity of infiltrating T cells will also be determined in retrospectively selected patients. Results: From 7/2015 to 6/2017, 428 patients have been successfully genotyped. Overall we found 52% mutated and 6% amplified tumors (2% both amplified and mutated). Of these, 81% (N 180) had a single gene mutation in KRAS (67%), BRAF (13%), PIK3CA (11%), or NRAS (9%). Remaining cases had a double gene alteration in PIK3CA (N 38) and KRAS (81%) or BRAF (7%) or NRAS (5%), or BRAF (N3: KRAS, NRAS, IDH1 ). 29% of the 24 amplified tumors showed concomitant mutations, while 16% had multiple amplifications. Interestingly, 2 out of 13 HER2-amplified tumors also harbored KRAS (G12D) and PIK3CA mutations (E542K and E545A). Conclusion: Upfront genotyping detected 44% multiple wild type and 9% patients with potentially actionable drivers including HER2, FGFR1 and 3, EGFR, MET, IGF1 and 2, and IGF1R. Two embedded trials for HER2-amplified (HERACLES - EudraCT 2012-002128-33, NCT03225937) and multiple wild type patients (CHRONOS EudraCT 2016-002597-12, NCT03227926) are ongoing. Beyond "molecular triage" purposes, FUNNEL is also building an integrated repository of lifetime clinical and molecular information, that all together will allow a comprehensive insight of mCRC clonal evolution, laying the groundwork to design further innovative trials, and, eventually, inform patient care. Funded by AIRC Grant 9970; Italian Ministry of Health grant NET-2011-02352137. Citation Format: Cosimo Martino, Enrico Berrino, Carmine Dell'Aglio, Laura Casorzo, Mara Panero, Salvatore Siena, Andrea Sartore-Bianchi, Andrea Cassingena, Vittorina Zagonel, Sara Lonardi, Fotios Loupakis, Giuseppe Tonini, Patrizia Racca, Livio Trusolino, Andrea Bertotti, Ilaria Depetris, Antonella Balsamo, Anna Sapino, Silvia Marsoni. Empowering precision medicine in metastatic colorectal cancer: preliminary results from the FUNNEL platform [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A087.

Abstract A183: Blood-based genomic profiling of cell-free tumor DNA (ctDNA) in patients with biliary tract cancer

Abstract Background: Targeted therapies are increasingly under evaluation in clinical trials for the treatment of biliary tract cancer (BTC), and cell-free tumor DNA assays are an emerging technology for the detection of actionable alterations. Methods: ctDNA analysis was performed in patients with BTC using the CLIA-certified Guardant360 liquid biopsy assay as part of routine clinical care. The assay screens up to 73 genes for single-nucleotide variants (SNVs), 23 for insertions or deletions (INDELs), 18 for copy number variations (CNVs), and 6 for fusions. Results: 855 samples were analyzed from 751 unique BTC patients. Median age was 64 years (range, 29-89), 53% were female, and 631 (84%) had at least one alteration in their first sample. In the 751 patients, the most common genes altered were TP53 (39%), KRAS (15%), PIK3CA (13%), ARID1A (13%), EGFR (11%), FGFR2 (11%), ERBB2 (11%), NF1 (10%), IDH1 (10%), APC (9%), BRAF (9%), MYC (8%), MET (7%), CCNE1 (7%), and FGFR1 (7%). Alterations in DNA damage response genes were also seen, including BRCA2 (6%), BRCA1 (5%), ATM (4%), and MLH1 (1%). The median number of alterations per sample was 3 (range, 0-40). In a clinically annotated dataset from the Massachusetts General Hospital Cancer Center, 112 samples were obtained from 61 unique patients. Among these, 52 (85%) had intrahepatic cholangiocarcinoma (ICC), 3 (5%) had extrahepatic cholangiocarcinoma, and 6 (10%) had gallbladder cancer. 20/61 (33%) patients were treated with targeted therapy. Among 20 patients with both ctDNA and tumor sequencing results, the targetable mutation was found in both ctDNA and tumor tissue in 7/20 (35%), solely in ctDNA in 1/20 (5%), and solely in tissue in 12/20 (60%) patients. The patient with the alteration detected solely in ctDNA had insufficient tissue for analysis; she was found to have an FGFR2 F276C mutation and benefited for 8.5 months from an FGFR inhibitor on a clinical trial. Of the 12 patients whose targetable mutation was not detected in plasma, 10 (83%) had an FGFR2 fusion, 1 had an INDEL in FGFR2 (Leu262_Ala266delinsAsp), and 1 had an FGFR3 splice site mutation. 15/20 (75%) patients treated with targeted therapy were followed with serial ctDNA analysis, and a potential resistance mechanism was identified in 5/15 (33%). All 5 patients had FGFR2-fusion positive ICC and developed mutations in the FGFR2 kinase domain as a potential resistance mechanism to FGFR inhibition, including the FGFR2 V564F gatekeeper mutation in 4/5 patients. Conclusions: In patients with BTC, ctDNA-based genomic profiling can detect alterations in IDH, FGFR, PIK3CA, ERBB2, BRAF, and MET. The technology is still evolving for the detection of FGFR2 fusions, which is hindered in capture-based methods in plasma by the large number of unique breakpoints and fusion partners. ctDNA analysis may also provide a noninvasive mechanism to study resistance to targeted therapy and clonal dynamics in BTC. Citation Format: Lipika Goyal, Robin Kate Kelley, Lesli Kiedrowski, Daniel Catenacci, Kabir Mody, Rachna Shroff, Aparna Parikh, Stephanie Reyes, Jordan R. Maurer, Ryan B. Corcoran, Paul Fanta, Mike Cusnir, Becky Nagy, Richard Lanman, Michael Morse, Benjamin Tan, Milind Javle, Andrew Zhu. Blood-based genomic profiling of cell-free tumor DNA (ctDNA) in patients with biliary tract cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A183.