PIK3CA Mutation Status Research Articles

Abstract PS4-07: Identification of novel molecules that enhance neratinib efficacy in triple-negative breast cancer by high-throughput RNA interference

Abstract Background: Neratinib is a potent, irreversible pan-HER inhibitor that inhibits the ErbB family members EGFR, HER2, and HER4 and downstream signal transduction of these receptors. Triple-negative breast cancer (TNBC) is a heterogeneous disease that lacks druggable levels of receptors for estrogen, progesterone and HER2 and therefore challenging to treat. There is evidence that some cases of TNBC have activated signaling pathways mediated by ErbB family members that may contribute to aggressive behavior. The purpose of this preclinical study was to identify and validate kinases whose targeting may enhance the antitumor activity of neratinib in TNBC cell lines. Methods: In vitro proliferation assays were used to evaluate the efficacy of neratinib in TNBC cell lines. Baseline and post-neratinib-treatment expression of EGFR and phosphorylated EGFR (phospho-EGFR) were assessed via Western blot analysis in 18 TNBC cell lines. Reverse-phase protein array (RPPA) was used to profile and validate the signaling networks induced by neratinib. To identify potential targets or pathways that may synergize with neratinib treatment, we performed high-throughput RNA interference (HT RNAi) screening using a 709-kinome library. CellTiter-Blue, sulforhodamine B, and soft-agar assays were performed to evaluate the antiproliferative effect of neratinib alone and with target inhibitor. Mammary fat pad xenograft models were used to evaluate the efficacy of neratinib alone or with inhibitor in vivo. Results: In vitro proliferation assays showed that the half-maximal inhibitory concentration (IC50) of neratinib in tested TNBC cell lines ranged from 0.16 µM to 1.25 µM. RPPA and Western blot analyses revealed that the efficacy of neratinib correlated with phospho-EGFR expression levels across the TNBC cell lines tested (R2 = 0.3245). Among the tested TNBC cell lines, SUM149 cells (PIK3CA wild-type) were selected for high throughput RNAi screening because this cell line has high EGFR expression and is moderately sensitive to neratinib (IC50 = 0.35 µM). We identified the 40 most relevant kinase targets by the sensitivity index analysis, and further pathway analysis identified PI3K/AKT/mTOR (drug: everolimus) and MAPK (drug: trametinib) as major canonical pathways whose targeting enhanced the cytotoxic effect of neratinib. Everolimus (mTOR inhibitor) produced a strong antiproliferative effect when combined with neratinib in most tested TNBC cell lines (12 of 15 cell lines; combination index [CI] values, 0.1-0.5) and was more effective in PIK3CA-mutated compared to wildtype cell lines. Trametinib (MEK inhibitor) showed a moderate antiproliferative effect (effective in 10 of 15 cell lines; CI values, 0.2-0.9). Synergistic antitumor effects of neratinib combined with everolimus or with trametinib were also observed in anchorage-independent growth conditions (P < 0.05). In vivo experiments demonstrated that neratinib plus everolimus and neratinib plus trametinib combinations inhibited tumor growth in the SUM149 xenograft model for than single drug (neratinib, 42.3% growth inhibition; everolimus, 29.7%; trametinib, 47.1%; neratinib plus everolimus, 69.7%; neratinib plus trametinib, 77.7%; P < 0.0001). Conclusion: Combining neratinib with everolimus or with trametinib enhanced the antitumor effects of these drugs in TNBC regardless of PIK3CA mutation status, and clinical investigations evaluating these combination regimens for the treatment of TNBC are warranted. Citation Format: Jangsoon Lee, Troy Pearson, Huey Liu, Jon A. Fuson, Toshiaki Iwase, Irmina Diala, Alshad S. Lalani, Lisa D. Eli, Debu Tripathy, Bora Lim, Naoto T. Ueno. Identification of novel molecules that enhance neratinib efficacy in triple-negative breast cancer by high-throughput RNA interference [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-07.

Abstract PS5-35: Detection of PI3K pathway activation in circulating tumor cells in PIK3CA mutated metastatic breast cancer as a putative predictive biomarker for PI3K inhibitor therapies

Abstract Background: Somatic genomic alterations that activate the phosphatidylinositol-3-kinase (PI3K) pathway signaling are found in 30-50% of breast cancers, and the p110a-specific inhibitor alpelisib is approved for use in combination with fulvestrant in PIK3CA mutated hormone receptor-positive (HR+) metastatic breast cancer. However, preclinical and clinical data has demonstrated significant variability in response to alpelisib and other PI3K inhibitors even in the presence of hotspot PIK3CA activating mutations, likely reflecting functional differences between somatic alterations, bypass signaling mechanisms, and intratumor clonal heterogeneity. Thus, there is an ongoing need for novel predictive biomarkers to help guide patient selection for these therapies. Circulating tumor cells (CTCs) represent an accessible source of tumor derived analytes that allow for the interrogation of protein level readouts of PI3K pathway activation, may better reflect the biologic heterogeneity of metastatic disease than single site solid tumor biopsy, and are amenable to longitudinal analysis. Here, we report the development of an assay to evaluate the expression of activated AKT (AKT pS473) in CTCs as a putative biomarker of sensitivity to PI3K inhibitor therapies in metastatic breast cancer. Methods: Peripheral blood for CTC isolation was collected serially from patients with metastatic breast cancer and known somatic PI3K pathway mutation status. Samples were processed using VERSA (Versatile Exclusion-based Rare Sample Analysis), a microfluidic technology that integrates CTC capture and downstream analysis. Following fixation, CTCs were captured immunomagnetically with antibodies for EpCAM and Trop2, permeabilized and stained on chip for pan-AKT plus AKT pS473. Quantitative fluorescent microscopy was used to enumerate CTCs, defined as cytokeratin positive cells with intact nuclei and negative for a group of normal blood cell markers (CD45/CD11b/CD34/CD66b), and to quantify activated AKT protein expression in CTCs and matched peripheral blood mononuclear cells (PBMCs). Results: 100% of patients with somatic PIK3CA mutations in our pilot cohort had CTCs with detectable phospho-AKT expression, and in 50% of patients, the median phospho-AKT/pan AKT ratio was higher in CTCs compared to matched peripheral blood cells, suggestive of increased PI3K pathway activity. All patients demonstrated heterogeneity in phospho-AKT expression and phospho-AKT/pan-AKT ratio among individual CTCs at a single timepoint and across timepoints in longitudinal analysis. Conclusion: We report here the feasibility of quantitative and longitudinal detection of activated AKT in EpCAM/Trop2 captured CTCs from metastatic breast cancer with somatic PIK3CA mutations. Future work will prospectively evaluate multiple clinical applications of this assay in patients receiving alpelisib plus endocrine therapy for PIK3CA mutated HR+ metastatic breast cancer, as well as expanding the assay to include the detection of additional phospho-protein readouts of PI3K pathway activity in CTCs. In addition to the potential to complement solid biopsy PIK3CA mutation status as a predictive biomarker of sensitivity to PI3K therapies, this assay has the unique potential to provide a pharmacodynamic assessment of PI3K inhibitor activity in CTCs while on treatment, which may serve as an early biomarker of clinical response or resistance. Citation Format: Marina N Sharifi, Jamie M Sperger, Cole Gilsdorf, Serena K Wolfe, Amanda Parkes, Kari B Wisinski, Ruth M O'Regan, Joshua M Lang. Detection of PI3K pathway activation in circulating tumor cells in PIK3CA mutated metastatic breast cancer as a putative predictive biomarker for PI3K inhibitor therapies [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-35.

Clinicopathological Correlates of γδ T Cell Infiltration in Triple-Negative Breast Cancer.

Simple SummaryThe prognostic impact of the different tumor-infiltrating lymphocyte (TIL) subpopulations remains debated in solid cancers. We investigated the clinicopathological correlates and prognostic impact of TILs, particularly of γδ T cells, in 162 triple-negative breast cancer (TNBC) patients. A high γδ T cell density was significantly associated with younger age, higher tumor histological grade, adjuvant chemotherapy, BRCA1 promoter methylation, TIL density, and PD-L1 and PD-1 expression. In multivariate analyses, γδ T cell infiltration was an independent prognostic factor. However, this prognostic impact varied according to the tumor PIK3CA mutational status. High γδ T cell infiltration was associated with better survival in patients with PIK3CA wild-type tumors, without significant difference in the PIK3CA-mutated tumor subgroup. Altogether, these data suggest that high γδ T cell infiltrate is correlated with immune infiltration and might represent a prognostic tool in TNBC patients.The prognostic impact of the different tumor-infiltrating lymphocyte (TIL) subpopulations in solid cancers is still debated. Here, we investigated the clinicopathological correlates and prognostic impact of TILs, particularly of γδ T cells, in 162 patients with triple-negative breast cancer (TNBC). A high γδ T cell density (>6.625 γδ T cells/mm2) was associated with younger age (p = 0.008), higher tumor histological grade (p = 0.002), adjuvant chemotherapy (p = 0.010), BRCA1 promoter methylation (p = 0.010), TIL density (p < 0.001), and PD-L1 (p < 0.001) and PD-1 expression (p = 0.040). In multivariate analyses, γδ T cell infiltration (cutoff = 6.625 γδ T cells/mm2) was an independent prognostic factor (5-year relapse-free survival: 63.3% vs. 89.8%, p = 0.027; 5-year overall survival: 73.8% vs. 89.9%, p = 0.031, for low vs. high infiltration). This prognostic impact varied according to the tumor PIK3CA mutational status. High γδ T cell infiltration was associated with better survival in patients with PIK3CA wild-type tumors, but the difference was not significant in the subgroup with PIK3CA-mutated tumors. Altogether, these data suggest that high γδ T cell infiltrate is correlated with immune infiltration and might represent a candidate prognostic tool in patients with TNBC.

High SEC61G expression predicts poor prognosis in patients with Head and Neck Squamous Cell Carcinomas.

Background: Overexpression of the membrane protein SEC61 translocon gamma subunit (SEC61G) has been observed in a variety of cancers; however, its role in head and neck squamous cell carcinomas (HNSCC) is unknown. This study aimed to elucidate the relationship between SEC61G and HNSCC based on data from The Cancer Genome Atlas (TCGA) database.Methods: Data for HNSCC patients were collected from TCGA and the expression level of SEC61G was compared between paired HNSCC and normal tissues using the Wilcoxon rank-sum test. The relationship between clinicopathologic features and SEC61G expression was also analyzed using the Wilcoxon rank-sum test and logistic regression. Receiver operating characteristic (ROC) curves were generated to evaluate the value of SEC61G as a binary classifier using the area under the curve (AUC value). The association of clinicopathologic characteristics with prognosis in HNSCC patients was assessed using Cox regression and the Kaplan-Meier methods. A nomogram, based on Cox multivariate analysis, was used to predict the impact of SEC61G on prognosis. Functional enrichment analysis was performed to determine the hallmark pathways associated with differentially expressed genes in HNSCC patients exhibiting high and low SEC61G expression.Results: The expression of SEC61G was significantly elevated in HNSCC tissues compared to normal tissues (P < 0.001). The high expression of SEC61G was significantly correlated with the T stage, M stage, clinical stage, TP53 mutation status, PIK3CA mutation status, primary therapy outcome, and cervical lymph node dissection (all P < 0.05). Meanwhile, ROC curves suggested the significant diagnostic ability of SEC61G for HNSCC (AUC = 0.923). Kaplan-Meier survival analysis showed that patients with HNSCC characterized by high SEC61G expression had a poorer prognosis than patients with low SEC61G expression (hazard ratio = 1.95, 95% confidence interval 1.48-2.56, P < 0.001). Univariate and multivariate analyses revealed that SEC61G was independently associated with overall survival (P = 0.027). Functional annotations indicated that SEC61G is involved in pathways related to translation and regulation of SLITs/ROBOs expression, SRP-dependent co-translational protein targeting to the membrane, nonsense-mediated decay, oxidative phosphorylation, and Parkinson's disease.Conclusion: SEC61G plays a vital role in HNSCC progression and prognosis; it may, therefore, serve as an effective biomarker for the prediction of patient survival.

The prognostic significance of tumor-infiltrating lymphocytes in cervical cancer.

ObjectiveTo predict the prognosis of cervical cancer, we constructed a novel model with 5 specific cell types and identified a potential biomarker.MethodsWe employed CIBERSORT and xCell method to evaluate the abundances of 23 cells types in tumor microenvironment. Five specific cell types were filtrated to determine different immunotypes by applying least absolute shrinkage and selection operator (LASSO) Cox regression method. The expression of immune checkpoints (ICPs) and effectors were validated by immunohistochemistry. Correlation analysis was performed to examine the relevance between PIK3CA mutational status and ICPs.ResultsUnsupervised clustering of patients on the basis of tumor infiltrating lymphocytes and fibroblasts identified patients with shorter overall survival (OS) (hazard ratio [HR]=3.0729; 95% confidence interval [CI]=1.5103–6.2522; p=0.0118). An immunoscore (IS) signature consisting of 5 immune cell types infiltrating in tumor core (CD8T, activated NK cells, neutrophils, activated mast cells, macrophages) was constructed using LASSO Cox regression analysis. Receiver operating characteristic curves confirmed that the area under the curve of IS was significantly higher to that of International Federation of Gynecology and Obstetrics staging alone (0.637 vs. 0.55). Survival analysis revealed patients in high IS group exhibited a poorer OS (HR=3.0113; 95% CI=1.8746–4.8373; p<0.0001). The multivariate analysis indicated the IS was an independent prognostic factor. In addition, the lower IS related to higher expression of ICPs and neoantigen load.ConclusionsThe identification of IS in cervical cancer tissues could facilitate patient risk stratification and selection of immunotherapeutic responses, but more prospective studies are needed to assess its reliability.

Prognostic and predictive parameters in breast pathology: a pathologist's primer

The pathologist's role in the breast cancer treatment team has evolved from rendering a diagnosis of breast cancer, to providing a growing list of prognostic and predictive parameters such that individualized treatment decisions can be made based on likelihood of benefit from additional treatments and potential benefit from specific therapies. In all stages, ER and HER2 status help segregate breast cancers into treatment groups with similar outcomes and treatment response rates, however, traditional pathologic parameters such as favorable histologic subtype, size, lymph node status, and Nottingham grade also have remained clinically relevant in early stage disease decision-making. This is especially true for the most common subtype of breast cancer; ER positive, HER2 negative disease. For this same group of breast cancers, an ever-expanding list of gene-expression panels also can provide prediction and prognostication about potential chemotherapy benefit beyond standard endocrine therapies, with the 21-gene Recurrence Score, currently the only prospectively validated predictive test for this purpose. In the more aggressive ER-negative cancer subtypes, response to neoadjuvant therapy and` the extent of tumor infiltrating lymphocytes (TILs) are more recently recognized powerful prognostic parameters, and clinical guidelines now offer additional treatment options for those high-risk patients with residual cancer after standard neoadjuvant therapy. In stage four disease, predictive tests like germline BRCA status, tumor PIK3CA mutation status (in ER+ metastatic disease) and PDL-1 status (in triple negative metastatic disease) are now used to determine additional new treatment options. The objective of this review is to describe the latest in prognostic and predictive parameters in breast cancer as they are relevant to standard pathology reporting and how they are used in breast cancer clinical treatment decisions.

Phase III randomized study of taselisib or placebo with fulvestrant in estrogen receptor-positive, PIK3CA-mutant, HER2-negative, advanced breast cancer: the SANDPIPER trial

The phase III SANDPIPER study assessed taselisib (GDC-0032), a potent, selective PI3K inhibitor, plus fulvestrant in estrogen receptor-positive, HER2-negative, PIK3CA-mutant locally advanced or metastatic breast cancer. Postmenopausal women with disease recurrence/progression during/after an aromatase inhibitor were randomized 2 : 1 to receive taselisib (4 mg; taselisib arm) or placebo (placebo arm) plus fulvestrant (500 mg). Stratification factors were visceral disease, endocrine sensitivity, and geographic region. Patients with PIK3CA-mutant tumors (central cobas® PIK3CA Mutation Test) were randomized separately from those without detectable mutations. The primary endpoint was investigator-assessed progression-free survival (INV-PFS) in patients with PIK3CA-mutant tumors. Secondary endpoints included objective response rate, overall survival, clinical benefit rate, duration of objective response, PFS by blinded independent central review (BICR-PFS), safety, and time to deterioration in health-related quality of life. The PIK3CA-mutant intention-to-treat population comprised 516 patients (placebo arm: n= 176; taselisib arm: n= 340). INV-PFS was significantly improved in the taselisib {7.4 months [95% confidence interval (CI), 7.26-9.07]} versus placebo arm (5.4 months [95% CI, 3.68-7.29]) (stratified hazard ratio [HR] 0.70; 95% CI, 0.56-0.89; P= 0.0037) and confirmed by BICR-PFS (HR 0.66). Secondary endpoints, including objective response rate, clinical benefit rate, and duration of objective response, showed consistent improvements in the taselisib arm. Safety was assessed in all randomized patients who received at least one dose of taselisib/placebo or fulvestrant regardless of PIK3CA-mutation status (n= 629). Serious adverse events were lower in the placebo versus taselisib arm (8.9% versus 32.0%). There were more discontinuations (placebo arm: 2.3%; taselisib arm: 16.8%) and dose reductions (placebo arm: 2.3%; taselisib arm: 36.5%) in the taselisib arm. SANDPIPER met its primary endpoint; however, the combination of taselisib plus fulvestrant has no clinical utility given its safety profile and modest clinical benefit.

Identification of novel prognosis-related genes in the endometrial cancer immune microenvironment.

The incidence of endometrial cancer is increasing each year, and treatment effects are poor for patients with advanced and specific subtypes. Exploring immune infiltration-related factors in endometrial cancer can aid in the prognosis of patients and provide new immunotherapy targets. We downloaded immune metagene and functional data of patients with different subtypes of endometrial cancer from The Cancer Genome Atlas database and selected the lymphocyte-specific kinase (LCK) metagene as a representative genetic marker of the immune microenvironment in endometrial cancer. The results showed that LCK metagene expression is related to the prognosis of patients with endometrioid endometrial adenocarcinoma subtypes and highly correlated with the PTEN and PIK3CA mutational status. A search for LCK-related modules returned seven independent genetic predictors of survival in patients with endometrial cancer. The TIMER algorithm showed that the expression of these seven genes was positively correlated with the infiltration levels of six types of immune cells. The diagnostic value of these markers was validated using real-time quantitative PCR and immunohistochemical methods. Our results identified CD74, HLA-DRB5, CD52, HLA-DPB1 and HLA-DRB1 as possible valuable genetic markers for the diagnosis and prognosis of endometrial cancer and provided a theoretical basis for immunotherapy targets for its clinical treatment.

PI3K inhibition sensitizes EGFR wild‑type NSCLC cell lines to erlotinib chemotherapy

Tyrosine kinase inhibitors (TKIs) bring significant benefits for patients with cancers harboring epidermal growth factor receptor (EGFR) mutations. However, after treatment for a certain period, most patients ultimately acquire resistance. Numerous studies indicated that PI3K has an important role in tumor cell growth and drug sensitivity. Furthermore, inhibition of PI3K may lead to sensitization of non-small cell lung cancer (NSCLC) cells to EGFR-TKIs. The aim of the present study was to explore whether LY294002, an inhibitor of PI3K, is able to improve the sensitivity of NSCLC cell lines with wild-type EGFR to the EGFR-TKI erlotinib. An MTT assay was used to examine the effect of combined treatment with LY294002 and erlotinib on cell survival of two EGFR wild-type NSCLC cell lines, NCI-H661 and NCI-H460. Furthermore, flow cytometry was used to assess apoptosis in NCI-H661 and NCI-H460 cells after treatment with erlotinib and LY294002. In addition, the expression of downstream proteins was detected by western blot analysis. The results indicated that the number of viable NCI-H661 and NCI-H460 cells was dose-dependently reduced by erlotinib or LY294002. Compared to treatment with erlotinib alone, the cell apoptosis was enhanced if combined treatment of erlotinib and LY294002 was performed in NCI-H661 cells. Furthermore, combination treatment of erlotinib and LY294002 resulted in a significant reduction of phosphorylated p70S6K levels in NCI-H661 [PI3K catalytic subunit alpha (PI3KCA) wild-type] cells. However, this phenomenon was not observed in the NCI-H460 cell line (PIK3CA mutant-type). In conclusion, the present study indicated that inhibition of PI3K may have the potential to improve the sensitivity of NSCLC cells to an EGFR-TKI. However, the therapeutic effect may depend on the mutation status of PIK3CA.

An evaluation of buparlisib for the treatment of head and neck squamous cell carcinoma

ABSTRACT Introduction Five-year overall survival for head and neck squamous cell carcinoma (HNSCC) is relatively poor at around 50–66%, and there has been little improvement over the past several decades. PIK3CA alterations are common in HNSCC and offer a promising therapeutic target. Areas covered The authors discuss the PIK3 pathway and the use of PIK3 inhibitors in cancer, with a particular focus on HNSCC. A summary of the safety and efficacy of buparlisib, a class I pan-PI3K inhibitor, from several phase I and phase II HNSCC trials is provided. Expert opinion With a maximum tolerated dose of 100 mg/day and an acceptable toxicity profile, buparlisib may be effective in HNSCC, irrespective of PIK3CA mutational status. On-going clinical trials will help determine the developmental strategy of buparlisib while novel combinatory strategies including combination with immune checkpoint inhibitors should be considered. Importantly, biomarker strategies, including wider use of tumor sequencing and circulating tumor DNA, should be utilized to improve patient selection.

Relating PIK3CA Mutation Status With Clinicopathologic Factors and Outcome in Patients With Cervical Cancer Treated With Primary Surgery With or Without Post-Operative Adjuvant Chemoradiotherapy

Relating PIK3CA Mutation Status With Clinicopathologic Factors and Outcome in Patients With Cervical Cancer Treated With Primary Surgery With or Without Post-Operative Adjuvant Chemoradiotherapy

PI3K mutations detected in liquid biopsy are associated to reduced sensitivity to CDK4/6 inhibitors in metastatic breast cancer patients

BackgroundPI3K pathway hyperactivation due to PIK3CA mutations contributes to endocrine resistance, and PIK3CA is one of the most frequently mutated genes in breast cancer (BC), occurring approximately 40 % of HR+, HER2- advanced BC (ABC). Cyclin-dependent kinase 4 and 6 inhibitors (CDK4/6i) have changed the treatment landscape of HR+, HER2- ABC. Putative mechanisms of resistance to CDK4/6i have been identified, but limited data are available on PI3K deregulation. The present study evaluates the impact of PIK3CA mutations on CDK4/6i plus hormone therapy and evaluates potential characteristics that may suggest for a PI3K screening in patients with ABC. MethodsABC patients were enrolled, and 12 mL of blood were collected in EDTA tubes at baseline prior to CDK4/6i plus hormone therapy. Plasma was separated and circulating free DNA (cfDNA) was extracted. PIK3CA mutation analysis was performed on a ddPCR. Selected and analyzed mutations included: p.C420R, p.E542 K, p.E545A, p.E545D, p.E545G, p.E545K, p.Q546E, p.Q546R, p.H1047L, p.H1047R, p.H1047Y. Statistical analysis were performed to investigate the predictive power of such mutations and any association with clinical factors. ResultsThirty patients were enrolled. PIK3CA mutation status at baseline was independently associated with shorter median PFS (7.44 vs 12.9 months, p = 0.01) in subject receiving CDK4/6i plus hormone therapy. PIK3CA mutations were found to be associated to Ki67 expression in primary lesions (p = 0.006). Moreover, the probability to find a PI3K mutation improved considering also the therapeutic management in previous lines of treatment (McFadden's R2 = 0.415, p = 0.004; AUC of the ROC curve = 0.914). ConclusionThe findings of this pilot study suggest that the presence of a PI3K mutation in liquid biopsy correlates with a worse PFS in patients with ABC receiving CDK4/6i, and that liquid biopsy is a useful tool to suggests a better tailored pharmacological intervention.

Abstract B24: Phase II trial of AKT inhibitor MK-2206 in patients with advanced breast cancer who have tumors with PIK3CA or AKT mutations, and/or PTEN loss/PTEN mutation

Abstract Background: The PI3K/AKT pathway is activated through PIK3CA or AKT1 mutations and PTEN loss in breast cancer. We conducted a phase II trial with an allosteric AKT Inhibitor MK-2206 in patients with advanced breast cancer who had tumors with PIK3CA/AKT1 mutations and/or PTEN loss/mutation. Methods: The primary endpoint was objective response rate (ORR). Secondary endpoints were 6-month progression-free survival (6m PFS), predictive and pharmacodynamic markers, safety and tolerability. Patients had pretreatment and on-treatment biopsies as well as collection of peripheral blood mononuclear cells (PBMC) and platelet-rich plasma (PRP). Next-generation sequencing, immunohistochemistry and reverse phase protein arrays (RPPA) were performed. Results: Twenty-seven patients received MK-2206. Eighteen patients were enrolled into the PIK3CA/AKT1 mutation arm (cohort A): 13 had PIK3CA mutations, four had AKT1 mutations and one had a PIK3CA mutation as well as PTEN loss. ORR and 6m PFS were both 5.6% (1/18), with one patient with HR+ breast cancer and a PIK3CA E542K mutation experiencing a partial response (on treatment for 36 weeks). Nine patients were enrolled on the PTEN loss/mutation arm (cohort B). ORR was 0% and 6m PFS was 11% (1/9), observed in a patient with triple-negative breast cancer and PTEN loss. The study was stopped early due to futility. The most common adverse events were fatigue (48%) and rash (44%). On pretreatment biopsy, PIK3CA and AKT1 mutation status was concordant with archival tissue testing. However, two patients with PTEN loss based on archival testing had PTEN expression on the pretreatment biopsy. MK-2206 treatment was associated with a significant decline in pAKT S473 and pAKT T308 and PI3K activation score in PBMC and PRPs, but not in tumor biopsies. By IHC, there was no significant decrease in median pAKT S473 or Ki-67 staining, but a drop was observed in both responders. Conclusions: MK-2206 monotherapy had limited clinical activity in advanced breast cancer patients selected for PIK3CA/AKT1 or PTEN mutations or PTEN loss. This may, in part, be due to inadequate target inhibition at tolerable doses in heavily pretreated patients with pathway activation, as well as tumor heterogeneity and evolution in markers such as PTEN conferring challenges in patient selection. Citation Format: Yan Xing, Nancy U. Lin, Mathew Mauer, Huiqin Chen, Armeen Mahvash, Aysegul Sahin, Argun Akcakanat, Yisheng Li, Vandana Abraham, Jennifer Litton, Mariana Chavez-McGregor, Vicente Valero, Sarina A. Piha-Paul, David Hong, Kim-Anh Do, Emily Tarco, Dianna Riall, Karina Agda Eterovic, Lewis Cantley, Gordon B. Mills, L. Austin Doyle, Eric Winer, Gabriel Hortobagyi, Ana Maria Gonzalez-Angulo, Funda Meric-Bernstam. Phase II trial of AKT inhibitor MK-2206 in patients with advanced breast cancer who have tumors with PIK3CA or AKT mutations, and/or PTEN loss/PTEN mutation [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr B24.

Targeting HER2 heterogeneity in early-stage breast cancer.

HER2-positive (HER2+) breast cancer is clinically and biologically a heterogenous disease and not all patients benefit to the same extent from current anti-HER2 therapies. Among HER2+ breast cancer, molecular intrinsic subtypes, PIK3CA mutation status, levels of HER2 gene/protein, immune infiltration, or intratumor heterogeneity modulate HER2-treatment sensitivity. HER2-enriched carcinomas, with high levels of HER2 and tumor-infiltrating lymphocytes (TILs) are highly sensitive to anti-HER2 therapies, regardless of chemotherapy. Luminal A/B tumors are more estrogen receptor-dependent than HER2-dependent, harbor higher rates of PIK3CA mutations, and are less responsive to anti-HER2 treatment. HER2 intratumoral heterogeneity that exists in approximately 10% of HER2+ disease may also cause treatment resistance. Early changes occur during neoadjuvant anti-HER2 therapy that can predict response. Importantly, HER2 expression is not a binary but rather a continuous variable. Overall, 34-63% of HER2-negative breast cancers express HER2, and HER2-low tumors have become a new entity, for which novel targeted therapies may be effective. Although much of what is discussed currently remains investigational, it is clear that HER2+ breast cancer is a complex disease comprising different entities. Future strategies to escalate or de-escalate treatment in early-stage HER2+ disease should consider other biomarkers beyond HER2 and estrogen receptor status, including intrinsic subtype, HER2 levels, and TILs; and evaluate different treatment strategies among patients with estrogen receptor-positive/HER2+ and estrogen receptor-negative/HER2+ diseases.

PI3K p110α Blockade Enhances Anti-Tumor Efficacy of Abemaciclib in Human Colorectal Cancer Cells.

Simple SummaryColorectal cancer (CRC) is the third most common cancer and the second highest cause of cancer related mortality worldwide. Especially, the survival of advanced CRC patients who were failed to achieve durable remission after the anti-angiogenic and anti-epithelial growth factor receptor agents are still poor. The aim of our study was to investigate the anti-tumor activity of the CDK4/6 inhibitor, abemaciclib, as a single agent and to identify an optimal combination agent with abemaciclib in CRC cell lines. We confirmed that abemaciclib monotherapy showed anti-tumor activity and combination therapy with abemaciclib and BYL719 demonstrated synergistic effects in CRC cell lines. Moreover, our study suggested that PIK3CA mutation could be a predictive marker for efficacy of abemaciclib and BYL719 combination therapy. These findings provide novel insight into a possible therapeutic strategy for patients with relapsed and refractory CRC.Targeting cell cycle regulation in colorectal cancer has not been fully evaluated. We investigated the efficacy of the CDK4/6 inhibitor, abemaciclib, and confirmed a synergistic interaction for PI3K p110α and CDK dual inhibition in colorectal cancer cell lines. Caco-2 and SNU-C4 cell lines were selected to explore the mechanism of action for and resistance to abemaciclib. In vitro and in vivo models were used to validate the anti-tumor activity of abemaciclib monotherapy and BYL719 combination therapy. Abemaciclib monotherapy inhibited cell cycle progression and proliferation in Caco-2 and SNU-C4 cells. CDK2-mediated Rb phosphorylation and AKT phosphorylation appeared to be potential resistance mechanisms to abemaciclib monotherapy. Abemaciclib/BYL719 combination therapy demonstrated synergistic effects regardless of PIK3CA mutation status but showed greater efficacy in the PIK3CA mutated SNU-C4 cell line. Growth inhibition, cell cycle arrest, and migration inhibition were confirmed as mechanisms of action for this combination. In an SNU-C4 mouse xenograft model, abemaciclib/BYL719 combination resulted in tumor growth inhibition and apoptosis with tolerable toxicity. Dual blockade of PI3K p110α and CDK4/6 showed synergistic anti-tumor effects in vivo and in vitro in human colorectal cancer cell lines. This combination could be a promising candidate for the treatment of patients with advanced colorectal cancer.

Abstract 4330: HPV double capture NGS method in cervical cancer: Identification of MACROD2 gene as HPV hot spot integration site and viral load status as a prognostic factor

Abstract BACKGROUND: Cervical cancer (CC) remains a leading cause of gynaecological cancer-related mortality worldwide and constitutes the second most common malignancy in women. CC exhibits differences in clinical behavior; infection by high-risk Human Papilloma Virus (HPV) remains an important initiating event in tumorigenesis and the most important risk factors for CC. As HPV integration types may be specific biomarkers for prediction of clinical outcomes, we analyzed the association between the different viral integration signatures and clinic-pathological parameters in CC patients. EXPERIMENTAL DESIGN: Patients included in this study were enrolled in the EU-funded RAIDs Network (Rational Molecular Assessment and Innovative Drug Selection, www.raids-fp7.eu) prospective CC BioRAIDs study [NCT02428842]. HPV double capture method followed by NGS was used to define the different integration signatures. Correlations between HPV integration signatures and clinical, biological and molecular features were assessed in a large series of 272 CC BioRAIDs patients. RESULTS: The distribution of HPV signatures differed from that previously described in HPV-positive anal squamous cell carcinoma (p&amp;lt;0.001). We identified more than 250 HPV insertions sites (inter-genic or intra-genic) and reported MACROD2 gene as the most frequent integration site followed by the MIPOL1/TTC6, TP63, ERBB2, KLF12. The seven patients with HPV integration in the MACROD2 gene tend to have a worse progression free survival (PFS) compared to other patients. HPV integration signatures were not associated with treatment type, histological subtype or FIGO staging, but were associated with PIK3CA mutational status (p=0.023) and HPV genotype status (p&amp;lt;0.0001). HPV45 and HPV18 genotypes were always integrated in our patients' population, as compared to other HPV genotypes. There was no significant association between the different HPV integration signatures and PFS. High viral load was associated with longer PFS (p=0.011). CONCLUSION: This is the first study assessing the prognostic value of HPV integration in a prospectively annotated CC patient cohort, which reports a hot-spot of HPV integrations at the MACROD2 gene, known to be implicated in impaired PARP1 activity and chromosome instability. Citation Format: Maud Kamal, Sonia Lameiras, Adeline Morel, Charlotte Lecerf, Sophie Vacher, Celia Dupain, Emmanuelle Jeannot, Marc Deloger, Nicolas Servant, Elodie Girard, Sylvain Baulande, Gemma Kenter, Ekaterina Jordanova, Els Berns, Roman Rouzier, Wulfran Cacheux, Christophe Le Tourneau, Alain Nicolas, Suzy Scholl, Ivan Bieche. HPV double capture NGS method in cervical cancer: Identification of MACROD2 gene as HPV hot spot integration site and viral load status as a prognostic factor [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4330.

Abstract 1433: Role of the E3 ubiquitin ligase NEDD4 in the regulation of PTEN and MDM2 in colorectal cancer

Abstract Introduction: NEDD4 (neural precursor cell-expressed developmentally down-regulated 4), a member of the HECT (homologous to E6AP C-terminus) family of E3 ubiquitin ligases, has been shown to be an important regulator of multiple proteins involved in cancer development, including the tumor suppressor PTEN and the proto-oncogene MDM2. Furthermore, NEDD4 has been shown to be overexpressed and act as an oncogene in multiple cancer types. We have previously shown that NEDD4 is overexpressed in colorectal cancer (CRC) and that it can promote growth of colon cancer cells independently of PTEN and PI3K/AKT signaling. In the present study, we investigated the role of NEDD4 in regulating the PTEN/PI3K pathway and the MDM2/p53 axis in CRC. Materials and Methods: The CRISPR/Cas9 system was applied to generate a Caco2 NEDD4 knockout cell line, and NEDD4 knockdown was performed in LS174T and SW480 cells. Gene expression profiles of 412 primary CRCs, 51 normal mucosa samples, 38 CRC cell lines, as well as the Caco2 parental and NEDD4 knockout cell lines, were generated using exon-resolution Affymetrix Human Exon Arrays or Human Transcriptome Arrays. The NEDD4 expression level was correlated with the mutation status of KRAS, PTEN, TP53, PIK3CA, BRAF and NRAS. Western blotting was used to detect and quantify NEDD4, PTEN and MDM2 protein levels. Results and Discussions: Gene expression analysis of the patient material confirmed our previous study that NEDD4 is significantly upregulated in CRC as compared to normal colonic mucosa. There was no correlation between NEDD4 expression and mutations in KRAS, BRAF, NRAS, PTEN, PIK3CA and TP53. CRISPR/Cas9-mediated NEDD4 knockout in Caco2 cells resulted in a reduction (P &amp;lt;0.01) in both the PTEN and MDM2 protein levels as compared to control cells. Gene set analysis showed that PI3K/AKT/MTOR signaling was upregulated in the NEDD4 knockout cells as compared to control cells. siRNA-mediated depletion of NEDD4 in LS174T cells was associated with reduced levels of MDM2, but did not affect the PTEN protein level. In SW480 cells, depletion of NEDD4 affected neither the MDM2 nor the PTEN protein level. By analyzing the expression of NEDD4, PTEN and MDM2 in 38 CRC cell lines by Western blotting, a positive correlation (P&amp;lt;0.01) was observed between NEDD4 and PTEN protein, while there was no significant correlation between NEDD4 and MDM2. Conclusion: The data show that NEDD4 is significantly upregulated in CRC, and that NEDD4 expression correlates with PTEN expression. The data further suggest that NEDD4 has the ability to regulate the PTEN and MDM2 protein levels in colon cancer cells in a cell line-specific manner. Citation Format: Lars M. Knudsen, Anita Sveen, Christer A. Andreassen, Christian H. Bergsland, Ina A. Eilertsen, Nikoline L. Rasmussen, Max Z. Totland, Peter W. Eide, Jarle Bruun, Ragnhild A. Lothe, Edward Leithe. Role of the E3 ubiquitin ligase NEDD4 in the regulation of PTEN and MDM2 in colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1433.

PI3K-targeting strategy using alpelisib to enhance the antitumor effect of paclitaxel in human gastric cancer

PIK3CA mutations are frequently observed in various human cancers including gastric cancer (GC). This study was conducted to investigate the anti-tumor effects of alpelisib, a PI3K p110α-specific inhibitor, using preclinical models of GC. In addition, the combined effects of alpelisib and paclitaxel on GC were evaluated. Among the SNU1, SNU16, SNU484, SNU601, SNU638, SNU668, AGS, and MKN1 GC cells, three PIK3CA-mutant cells were predominantly sensitive to alpelisib. Alpelisib monotherapy decreased AKT and S6K1 phosphorylation and induced G0/G1 phase arrest regardless of PIK3CA mutational status. The alpelisib and paclitaxel combination demonstrated synergistic anti-proliferative effects, preferentially on PIK3CA-mutant cells, resulting in increased DNA damage response and apoptosis. In addition, alpelisib and paclitaxel combination potentiated anti-migratory activity in PIK3CA-mutant cells. Alpelisib partially reversed epithelial–mesenchymal transition markers in PIK3CA-mutant cells. In a xenograft model of MKN1 cells, the alpelisib and paclitaxel combination significantly enhanced anti-tumor activity by decreasing Ki-67 expression and increasing apoptosis. Moreover, this combination tended to prolong the survival of tumor-bearing mice. Our data suggest promising anti-tumor efficacy of alpelisib alone or in combination with paclitaxel in PIK3CA-mutant GC cells.

PIK3CA mutation and CNV status and post-chemoradiotherapy survival in patients with cervical cancer

PurposeThis study aimed to describe the prognostic value of PI3K/AKT pathway mutations in a large cohort of patients with cervical cancer. Experimental designPatients with pre-treatment archival specimens, diagnosed with FIGO stages IB-IVA cervical cancer between 1998 and 2014 and treated with radical, curative intent chemoradiotherapy (CRT) at a single center were identified. Mutational status was determined by next generation sequencing and PIK3CA copy number (CNV) was assessed by digital PCR. Results190 patients with available pre-treatment tumor specimens were identified. Median OS and PFS were 57.4 and 46.0 months, respectively.A total of 161 tumors were successfully sequenced; 60 (37.3%) had PI3K/AKT pathway mutations, with 50 (30.1%) having PIK3CA hotspot mutations. PIK3CA CNV gain was noted in 79 (59.2%) of the 154 successfully analyzed. On univariate analysis, PIK3CA mutation was associated with poor OS (HR 1.73; 95% CI: 1.03–2.92; p = .037) but not PFS (HR 1.38; 0.84–2.28; p = .204). Absence of any PI3K/AKT pathway mutation was associated with improved OS (HR 1.68; 1.01–2.81; p = .046) but not PFS (HR 1.50; 0.93–2.43; p = .202). Associations were not maintained when adjusting for clinical factors. On univariate analysis, PIK3CA mutation positive, CNV normal tumors were associated with poorer OS (HR 2.55; 1.18–5.50; p = .017) and trend to worse PFS (HR 1.87; 0.90–3.83; p = .094) when compared to those with CNV gain and wildtype PIK3CA. ConclusionsPI3K/AKT pathway mutations are common in cervical cancer. Consideration of PIK3CA mutational status with CNV status may be important in predicting outcome in cervical cancer patients.

Mutation analysis and genomic imbalances of cells found in effusion fluids from patients with ovarian cancer.

Ovarian carcinomas and carcinosarcomas often cause malignant effusions, an accumulation within serous cavities of fluid containing cancer cells. Few studies have focused on the molecular alterations and genetic mechanisms behind effusion formation. The present study investigated the mutation status of TP53, PIK3CA, KRAS, HRAS, NRAS and BRAF in effusion fluids from 103 patients with ovarian cancer. In addition, array Comparative Genomic Hybridization (aCGH) analysis was performed on 20 effusions from patients with high-grade serous carcinoma (10 cases positive for TP53 mutation and 10 with TP53 wild-type). TP53 mutations, two of which were novel: c.826_830delCCTGT and c.475_476GC>TT, were identified in 44% of the cases. Mutations in KRAS, HRAS, and PIK3CA were identified in two, two and four cases, respectively. None of the effusions analysed showed NRAS or BRAF mutations. The aCGH analysis revealed highly imbalanced genomes similar to those described in primary ovarian carcinomas. No specific profile was indicated to distinguish tumors with TP53 mutations from those without. The molecular profiling of cells found in effusion fluids from patients with ovarian cancer thus showed considerable molecular heterogeneity. TP53 seems to be the most frequently mutated gene in these cells and may serve a leading role in the metastatic process.