Activating Mutations In PIK3CA Research Articles

Abstract PO5-21-10: Efficacy and Safety of Alpelisib in PIK3CA-mutated, hormone receptor-positive advanced breast cancer after a CDK4/6 inhibitor: An Open-label, Multi-centre, Prospective, Single Arm Clinical Trial

Abstract Introduction: Currently, patients with luminal metastatic breast cancer receive a CDK4/6 inhibitor in combination with endocrine therapy as a first or second line of treatment. When tumors become resistant to the CDK4/6 inhibitors, treatment with alpelisib (PI3K inhibitor) in combination with an endocrine agent can be a next treatment option if an activating PIK3CA mutation is confirmed (cfr. SOLAR-1 trial). However, limited data is available after treatment with CDK4/6 inhibitors (cfr. BYLieve trial) and even less about alpelisib in later lines of therapy. Therefore, we aim to investigate the therapeutic efficacy and safety of alpelisib in combination with an endocrine agent in PIK3CA-mutated advanced breast cancer patients in later lines after prior CDK4/6i treatment. Methods: This is an open-label, prospective, multi-centre, single arm clinical trial enrolling patients from both the University Hospital of Leuven (UHL) and Ghent University Hospital (GUH). For each patient, metastatic tumor was tested for PIK3CA mutations using the UHL 96 gene panel. Our endpoints are clinical benefit rate, progression-free survival, time to treatment discontinuation (defined as the date of starting alpelisib to the date of treatment discontinuation or death) and safety. Descriptive statistics were used. Results: Between June 18th 2019 and August 23rd 2021, 38 patients have been included with confirmed PIK3CA hotspot mutations. All patients had CDK4/6 inhibitor in an earlier treatment line. Median age at alpelisib initiation was 64 years (range: 39-82 years). Included patients had a median of four lines of systemic therapy for advanced disease prior to starting alpelisib (range: 1- 11). Clinical benefit rate (patients receiving ³ 6 months of treatment) was 26.3% (10/38). The median progression-free-survival as well as time to treatment discontinuation were 3 months (range: 1 – 18). Dose reduction due to toxicity occurred in 11 patients (29%), of which 7 due to hyperglycemia grade 3, 2 due to diarrhea grade 3, 1 due to rash grade 2 and 1 due to anorexia. Finally, 84% of patients (32/38) stopped alpelisib treatment because of progressive disease, 8% (3/38) because of intolerance, one patient died because of cerebral hemorrhage during treatment, one patient withdrew their informed consent and one patient is still ongoing with the treatment. Conclusion: Our trial demonstrates activity of alpelisib in patients with PIK3CA mutated advanced breast cancer in later lines of treatment after treatment with CDK4/6 inhibitors with a clinical benefit rate of 26.3%. Discontinuation of therapy due to toxicity was seen in only 8% of patients, although toxicity induced dose reduction was needed in 29% of patients. These findings support the results of the BYLieve trial and additionally show efficacy of alpelisib in later lines of treatment for hormone receptor-positive advanced breast cancer. Citation Format: Rik Van Severen, Anne-Sofie De Crem, Hava Izci, Laurence Slembrouck, Isabelle Vanden Bempt, Hans Wildiers, Kevin Punie, Eline Naert, Ingeborg Hilderson, Ann Smeets, Ines Nevelsteen, Anne Deblander, Nynke Willers, Patrick Berteloot, Ignace Vergote, Sileny Han, Adriaan Vanderstichele, Giuseppe Floris, Christine Desmedt, Hannelore Denys, Patrick Neven. Efficacy and Safety of Alpelisib in PIK3CA-mutated, hormone receptor-positive advanced breast cancer after a CDK4/6 inhibitor: An Open-label, Multi-centre, Prospective, Single Arm Clinical Trial [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-21-10.

Abstract 1250: Activating PIK3CA mutations and hedgehog signaling may confer resistance to KRAS inhibition in colorectal cancer

Abstract Background: Treatment with KRASG12C inhibitors results in an objective response in only small fraction of patients with KRASG12C mutant colorectal cancer (CRC) (7.1% and 22% objective response rates (ORR) in two prospective trials), in contrast to lung cancer where the ORR exceeds 35%. We hypothesize that co-mutation of other oncogenes and/or transcriptomic factors may contribute to the resistance of KRASG12C CRC to KRAS inhibitors. Methods: Mutation profiles of CRC patients were obtained from the AACR Project-GENIE v14 CRC cohort (N = 9441). The impact of KRAS knockout in 59 CRC cell lines was queried from the DepMap database. KRASG12C mutant CRC cell lines SW837 and SW1463 were treated with Sotorasib (KRASG12C inhibitor), Vismodegib and Taladegib (Hedgehog inhibitors), and cell viability was measured up to 96 hours after treatment. Results: In the AACR Project-GENIE CRC (N = 9441) cohort, KRASG12C tumors (2.6%, 245/9441) were significantly co-mutated with PIK3CAmut (19.2%, 47/245; Odds ratio = 1.7 (95% CI: 1.2 - 2.4), Chi-Square test, p-value = 0.002). Dependence on KRAS in CRISPR knockout viability assays was higher in KRASmut vs. wildtype CRC cell lines (median CERES score -1.3 vs -0.5, p-value < 0.001). Interestingly, KRASmut cell lines with PIK3CA co-mutation (n=10) were more resistant to KRAS knockout than PIK3CAwt cells (n=22) (median -1.0 vs -1.4, p-value = 0.03). The SW837 and SW1463 cell lines initially displayed high sensitivity to Sotorasib (IC50: 0.92 µM and 0.90 µM). However, overexpression of PIK3CAE545K mutant induced resistance to the same treatment (IC50: 5.18 µM and 6.45 µM). Furthermore, gene set enrichment analysis (GSEA) showed that Hedgehog signaling was significantly enriched in KRAS knockout resistant KRASmut CRC cell lines. SW837 cell line treated with Sotorasib (S) and a fixed dosage of 25 µM of Vismodegib (V) or Taladegib (T) showed higher sensitivities in combination treatment assays (IC50 decreased: ~11 folds in S+V vs S; and ~15 folds in S+T vs S) as compared to Sotorasib (S) treatment alone. Similar results were seen for the SW1463 cell line (IC50 decreased: ~18 folds in S+V vs S; and ~15 folds in S+T vs S). Conclusion: Resistance in CRC to KRASG12C inhibitors may be attributed to activated PIK3CA mutations and the Hedgehog signaling pathway. The future study will focus on detailed investigations involving comprehensive transcriptome and exome profiles in tumors and PDX models treated with anti-KRAS drugs in monotherapy and combination therapy. Citation Format: Saikat Chowdhury, Jibran Ahmed, Valsala Haridas, David S. Hong, Scott Kopetz, John Paul Shen. Activating PIK3CA mutations and hedgehog signaling may confer resistance to KRAS inhibition in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1250.

Abstract C157: Targeting PIK3CA and CDKN2A alterations in Esophageal Adenocarcinoma with Alpelisib and Palbociclib

Abstract Our study investigates the efficacy of targeted therapies for Esophageal Adenocarcinoma (EAC) tumors harboring an activating PIK3CA mutation and inactivating CDKN2A mutation. We tested the small molecule inhibitors alpelisib, which targets the Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit (PIK3CA), and palbociclib, which targets Cyclin D kinase 4/6 (CDK4/6), in EAC in vitro organoid and in vivo mouse models. EAC and Gastric cancer (GC) organoid models were treated with single agent alpelisib or palbociclib on a log scale to determine their IC50 values. An EAC organoid harboring activating PIK3CA E545K and inactivating CDKN2A D74A mutations demonstrated more significant growth inhibition when treated with each agent alone versus standard of care chemotherapy. Subsequent experiments involved combination treatment of our EAC-mutant organoid with alpelisib and palbociclib as well as a representative GC organoid and EAC organoids lacking these specific mutations as controls. We confirmed PIK3CA downstream target engagement after alpelisib treatment by qPCR, western blot analysis and immunofluorescence analysis. Once in vitro efficacy was confirmed, EAC-mutant organoids were transplanted into mice by either subcutaneous or orthotopic injections and treated with chemotherapy, alpelisib and palbociclib alone or in combination. Our data demonstrate that a PIK3CA/CDKN2A mutated patient-derived EAC organoid is sensitive to alpelisib and palbociclib in a dose dependent manner, and analysis of PIK3CA downstream targets by western and qPCR was consistent with growth inhibition of treated organoids. These findings offer support for the use of alpelisib and palbociclib in the treatment of EAC patients with PIK3CA/CDKN2A mutations. Citation Format: Naryan Rustgi, Thomas Ryan, Jin Cho, Ryan Moy, Sam Yoon, Sandra Ryeom, Changhwan Yoon. Targeting PIK3CA and CDKN2A alterations in Esophageal Adenocarcinoma with Alpelisib and Palbociclib [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C157.

CBF-Beta Mitigates PI3K-Alpha-Specific Inhibitor Killing through PIM1 in PIK3CA-Mutant Gastric Cancer.

Loss of either NEDD9 or BCL-XL confers hypersensitivity to PI3K-alpha inhibition whereas loss of CBFB confers resistance through a CBFB/PIM1 signaling axis.

Expanding the Clinicopathologic and Molecular Spectrum of Lipoblastoma-Like Tumor in a Series of 28 Cases

Lipoblastoma-like tumor (LLT) is a rare adipocytic neoplasm with a predilection for the vulva. Since 2002, <30 cases have been reported, characterizing it as an indolent tumor that may sometimes recur locally. Diagnosis can be challenging due to its rarity and morphologic overlap with other adipocytic tumors. Thus far, there are no specific molecular or immunohistochemical features to aid in the diagnosis of LLT. Recent case reports have described LLT arising at other sites, including the spermatic cord and gluteal region, suggesting wider anatomical distribution. We present a large series of LLT to further characterize its clinicopathologic and molecular features. Twenty-eight cases of LLT were retrieved from departmental and consult archives (including 8 from a prior series). The cohort comprised 28 patients (8 males, 20 females) with a median age of 28 years (range: 1-80 years). There were 17 primary LLT of the vulva. Other anatomical sites included the scrotum (n = 3), spermatic cord (n = 2), inguinal region (n = 2), limbs (n = 2), pelvis (n = 1), and retroperitoneum (n = 1). Median tumor size was 6.0 cm (range: 1.8-30.0 cm). The tumors had a lobulated architecture and were typically composed of adipocytes, lipoblasts, and spindle cells in a myxoid stroma with prominent thin-walled vessels. Using immunohistochemistry, a subset showed loss of Rb expression (12/23 of samples). Follow-up in 15 patients (median: 56 months) revealed 8 patients with local recurrence and 1 patient with metastases to the lung/pleura and breasts. Targeted DNA sequencing revealed a simple genomic profile with limited copy number alterations and low mutational burden. No alterations in RB1 were identified. The metastatic LLT showed concurrent pathogenic PIK3CA and MTOR activating mutations, both in the primary and in the lung/pleural metastasis; the latter also harbored TERT promoter mutation. One tumor had a pathogenic TSC1 mutation, and one tumor showed 2-copy deletion of CDKN2A, CDKN2B, and MTAP. No biologically significant variants were identified in 8 tumors. No gene fusions were identified by RNA sequencing in 4 tumors successfully sequenced. This study expands the clinicopathologic spectrum of LLT, highlighting its wider anatomical distribution and potential for occasional metastasis. Molecularly, we identified activating mutations in the PI3K-MTOR signaling pathway in 2 tumors, which may contribute to exceptional aggressive behavior.

Incidence and outcomes among patients with HR+/HER2- metastatic breast cancer (mBC) with co-occurring ESR1 and PIK3CA mutations detected by ctDNA and treated with alpelisib: A retrospective review.

e13068 Background: ESR1 and PIK3CA mutations inform the use of targeted agents such as alpelisib and elacestrant in patients (pts) with HR+/HER2- metastatic breast cancer (mBC) following progression. Each of these mutations predict a worse prognosis and shorter response to endocrine therapy (ET). The prevalence of PIK3CA and ESR1 mutations detected via cell-free tumor DNA (ctDNA) in mBC is about 35% and 33%, respectively, and the co-occurrence rate ranges from 10-15%. Here, we evaluate the incidence and outcomes of pts with mBC HR+/HER2- harboring co-occurring ESR1 and PIK3CA mutations as detected by ctDNA in a real-world tertiary care center with a predominantly Hispanic patient population. Methods: We analyzed pts with HR+/HER2- mBC with ctDNA testing (Guardant360) between Jan 1, 2020 and Jan 31, 2023 at Miami Cancer Institute. We determined the rate of co-occurrence of activating PIK3CA and ESR1 mutations (co-mutation group). In these pts, demographics, disease characteristics, response to alpelisib + ET, and overall survival (OS) after diagnosis of mBC were collected. For comparison, we analyzed pts with PIK3CA mutations but no ESR1 mutation detected who had received alpelisib + ET ( PIK3CA only group). Results: 372 patients with a diagnosis of mBC who underwent ctDNA testing during the study period, 80 (21.5%) had at least one activating PIK3CA mutation, and 37 (9.95%) had a co-occurring ESR1 mutation. Median age was 70 vs 69 years in the PIK3CA only group compared to the co-mutation group, with 82% vs 92% being post-menopausal, respectively. In the PIK3CA only group, 36.4% were of Hispanic ethnicity, while 70.3% of those in the co-mutation group were Hispanic. Most pts received prior CDK4/6 inhibitor (91% in PIK3CA only group vs 97% with co-mutation). 16 (43.2%) of the 37 patients in the co-mutation group were treated with alpelisib + ET after detection of the co-occurring mutation via ctDNA, and outcomes were compared to the PIK3CA only group. Pts in the PIK3CA only group were treated with alpelisib + ET for a median of 7.23 months vs 4.24 months in the co-mutation group (p=0.6023). Reason for discontinuation, most commonly progressive disease (63.6% in PIK3CA only vs 62.5% in co-mutation) was similar between groups. A partial response or better was seen in 30% of the PIK3CA only group vs 20% of the co-mutation group. OS was 138 months for PIK3CA only vs 98 months for the co-mutation group (p=0.96). Conclusions: The incidence of co- PIK3CA and ESR1 mutations in this predominantly Hispanic patient population was consistent with previously reported literature. We identified a trend towards worse OS and shorter duration on treatment with alpelisib + ET in the co-mutation group as compared to the PIK3CA alone group. Further studies are needed to understand the optimal sequencing of therapy in pts with co-mutation in ESR1 and PIK3CA.

First results from the RETgistry: A global consortium for the study of resistance to RET inhibition in RET-altered solid tumors.

9065 Background: Rearranged during transfection ( RET) gene alterations are the oncogenic driver in diverse tumor types, including RET fusions identified in 1-2% of non-small cell lung cancers (NSCLC). While RET-selective tyrosine kinase inhibitors (TKIs) selpercatinib and pralsetinib are effective, acquired drug resistance remains a challenge. Here, we report the initial results from the RETgistry, an international consortium aimed at elucidating mechanisms of resistance to RET TKIs across RET-altered solid tumors. Methods: This was a retrospective analysis performed across 16 institutions. Patients (pts) were eligible if they had advanced solid tumor harboring an oncogenic RET alteration, received ≥1 RET TKI with disease progression, and underwent resistant tumor or liquid biopsy for next-generation sequencing (NGS). Results: A total of 103 time-distinct biopsies (62 tissue, 30 plasma, 11 paired tissue/plasma) were included in analysis, obtained from 88 pts with progression on a RET-selective TKI [selpercatinib (n = 70), pralsetinib (n = 14), selpercatinib and pralsetinib sequentially (n = 4)]. Pts had the following tumor types: 72 NSCLC (69% KIF5B-RET, 21% CCDC6-RET, 10% other RET fusion), 13 medullary thyroid cancer (TC) (54% RET M918T, 46% other RET mutation), and 2 papillary and 1 anaplastic TC (all, RET fusion). Median age was 58 (range, 21-86); 48% were male with 89% never/light smokers. Median duration of RET TKI preceding biopsies [first-line, n = 32 (31%); second-line, n = 42 (41%); third-/greater-line, n = 29 (28%)] was 16.5 months (mos) (95% CI, 14.0-19.6); median PFS was 14.1 mos (95% CI, 9.3-17.0). Resistant biopsies were obtained at median 15.0 mos from TKI initiation (range, 1.8-58.8). Acquired RET mutations were detected in 14 (14%), most common being G810 substitutions in 12 (12%). Potential off-target resistance gene alterations identified in 43 cases (42%) included MET amplification (14%), BRAF V600E or fusion (2%), KRAS gain or mutation (5%), ERBB2 amplification (2%), EGFR amplification (3%), ROS1 fusion (1%), and activating PIK3CA mutation or PTEN loss (4%). No resistant lung cancer biopsy demonstrated small cell transformation. The duration of TKI therapy (HR 0.64, p = 0.11) or PFS (HR 0.75, p = 0.33) did not differ according to the presence of on-target vs off-target resistance. NGS analyses of pre-RET-selective TKI tumors (n = 92) revealed frequent co-occurring alterations in TP53 (29%) and CDKN2A/B (12%). Conclusions: On-target resistance to RET inhibition due to acquired RET mutations was less common than off-target resistance, identified in 14%. The majority of RET TKI resistance is mediated by off-target mechanisms such as bypass receptor tyrosine kinase activation. Further studies are warranted to enable the development of therapeutic strategies to address resistance in pts with RET-altered tumors. The RETgistry accrual and data analyses continue.

Cribriform Morular Thyroid Carcinoma - Ultimobranchial Pouch-Related? Deep Molecular Insights of a Unique Case.

A 44-year-old female patient with a familial adenomatous polyposis (FAP) was diagnosed with a cribriform morular thyroid carcinoma (CMTC). We observed within the very necrotic tumor a small but distinct poorly differentiated carcinomatous component. As expected, next generation sequencing of both components revealed a homozygous APC mutation and in addition, a TERT promoter mutation. A TP53 mutation was found exclusively in the CMTC part, while the poorly differentiated component showed a clonal evolution, harboring an activating PIK3CA mutation and copy number gains of BRCA2, FGF23, FGFR1, and PIK3CB-alterations which are typically seen in squamous cell carcinoma. The mutational burden in both components was low, and there was no evidence for microsatellite instability. No mutations involving the mitogen-activated protein kinase (MAPK) pathway, typically seen in papillary thyroid carcinomas, were detected. Immunohistochemically, all tumor parts were negative for thyroglobulin, providing further evidence that this entity does not belong to the follicular epithelial cell-derived thyroid carcinoma group. CD5 was negative in the poorly differentiated component, making a relation to intrathyroidal thymic carcinoma rather unlikely. However, since this marker was seen in the morules, a loss in the poorly differentiated component and a relation to the ultimobranchial body cannot be excluded either. After total thyroidectomy and radioiodine ablation, the patient was disease-free with no residual tumor burden on 2-year follow-up.

Patient-derived head and neck cancer organoids allow treatment stratification and serve as a tool for biomarker validation and identification

Organoids are invitro three-dimensional structures that can be grown from patient tissue. Head and neck cancer (HNC) is a collective term used for multiple tumor types including squamous cell carcinomas and salivary gland adenocarcinomas. Organoids were established from HNC patient tumor tissue and characterized using immunohistochemistry and DNA sequencing. Organoids were exposed to chemo- and radiotherapy and a panel of targeted agents. Organoid response was correlated with patient clinical response. CRISPR-Cas9-based gene editing of organoids was applied for biomarker validation. A HNC biobank consisting of 110 models, including 65 tumor models, was generated. Organoids retained DNA alterations found in HNC. Comparison of organoid and patient response to radiotherapy (primary [n= 6] and adjuvant [n= 15]) indicated potential for guiding treatment options in the adjuvant setting. In organoids, the radio-sensitizing potential of cisplatin and carboplatin could be validated. However, cetuximab conveyed radioprotection in most models. HNC-targeted treatments were tested on 31 models, indicating possible novel treatment options with the potential for treatment stratification in the future. Activating PIK3CA mutations did not predict alpelisib response in organoids. Protein arginine methyltransferase 5 (PRMT5) inhibitors were identified as a potential treatment option for cyclin-dependent kinase inhibitor 2A (CDKN2A) null HNC. Organoids hold potential as a diagnostic tool in personalized medicine for HNC. Invitro organoid response to radiotherapy (RT) showed a trend that mimics clinical response, indicating the predictive potential of patient-derived organoids. Moreover, organoids could be used for biomarker discovery and validation. This work was funded by Oncode PoC 2018-P0003.

215P Impact of HER2 low on survival in patients treated with standard therapies in advanced breast cancer

Breast cancer (BC) pioneered precision medicine with immunohistochemistry (IHC) defined subtypes and early advent of targeted therapies. Trastuzumab deruxtecan (T-DXd) has expanded traditional HER2 definitions to include HER2-low (H2L) tumors, defined as HER2 IHC score of 1+ (>10% cells stained), or as 2+ (>10%) and negative in-situ hybridization (ISH) assay. Studies suggest H2L BC is not a separate clinical entity, strongly determined by hormone receptor (HR) status. We sought to understand the impact of H2L on current standards of care (SOC). Retrospective analysis was performed on 24,824 advanced BC with available genomic, transcriptomic, subtyping, and treatment data. Tumors were tested at Caris Life Sciences via NextGen DNA Sequencing (592 gene panel or whole exome) and RNA (whole transcriptome). BC subtypes were defined by IHC/ISH per accepted definitions. PD-L1 was determined by IHC (SP142 immune cells [IC] ≥ 1%). CODEai, a real-world evidence database was used to access clinical outcomes from insurance claims data, with Kaplan–Meier estimates for overall survival (OS) and 95% CI calculated from time of first treatment to last contact. Patients with HR positive (HR+)/H2L tumors treated with CDK4/6 inhibitors (n=1,348) had similar OS compared to pts with HR+/HER2 0 (n=3,027; 1,179 vs 1,105 days; HR=0.89, CI 0.82–0.98, p=0.012). 27.2% of HR+/H2L pts had activating PIK3CA mutations. Among HR+ PIK3CA mutated tumors, H2L pts treated with alpelisib showed no difference in OS (n=27; 342 days) vs. HER2 0 alpelisib treated pts (n=42; 566 days, HR=1.23, CI 0.66 – 2.29, p=0.517), with limited sample size. 13.9% of H2L TNBC pts were PDL-1+. Pts with H2L PD-L1+ TNBC BC treated with immune checkpoint inhibitors (ICI; atezolizumab, pembrolizumab, nivolumab; n=49) showed improved OS vs. HER2 0 (n=233; 991 vs 524 days; HR=0.61, CI 0.37–0.99, p=0.046). HR+/H2L BC pts treated with SOC endocrine therapy demonstrate no difference in OS compared to HER2 0, and thus, should exhaust endocrine therapy options before initiating T-DXd or chemotherapy until indicated by prospective trials. Combinations of T-DXd plus ICI in PDL1+ H2L BC may be promising opportunities for further investigation.

Abstract 5778: Breast cancer mutations HER2V777L and PIK3CAH1047R activate the p21-CDK4/6 -Cyclin D1 axis driving tumorigenesis and drug resistance

Abstract In metastatic breast cancer, HER2 activating mutations frequently co-occur with mutations in the PIK3CA, TP53, or E-cadherin genes. Of these co-occurring mutations, HER2 and PIK3CA mutations are the most prevalent gene pair, with approximately 40% of HER2 mutated breast cancers also having activating mutations in PIK3CA. To study the effects of co-occurring HER2 and PIK3CA mutations, we bred genetically engineered mice with the (loxP-STOP-loxP) HER2V777L; PIK3CAH1047R transgenes (HP mice) and studied the resulting breast cancers both in vivo as well as ex vivo using breast cancer organoids. HP mice rapidly developed invasive mammary adenocarcinoma at a median time of 2.1 weeks after adenoviral Cre injection into the mammary gland. Organoids from these breast cancers showed increased number of buddings in branching morphogenesis assay and increased migration and invasion in vitro. In vivo, HP breast cancers are resistance to the pan-HER tyrosine kinase inhibitor, neratinib, but are effectively treated by the combination of neratinib plus trastuzumab deruxtecan (T-DXd). Ex vivo, we found strong synergy between neratinib and T-DXd in HP organoids. Proteomic and RNA-seq analysis of HP breast cancers showed increased gene expression of CCND1 (cyclin D1) and CDKN1A (which encodes p21WAF1/Cip1) and changes in cell cycle markers. An increase in p-p53, p-p27, and p-PDK1 in HP organoids was seen. The GSEA analysis showed that the mTOR pathway and the MYC target signature were significantly upregulated in the HP organoid group. As p21 stabilizes the cyclin D1-CDK4/6 complex to further activate CDK4/6, we found CDK4/6 inhibitors inhibit cell proliferation in HP mice-derived organoids. Combining neratinib with CDK4/6 inhibitors was another effective strategy for HP breast cancers with neratinib plus palbociclib showing a statistically significant reduction in mouse HP tumors as compared to either drug alone. We validated both the neratinib plus T-DXd and neratinib plus palbociclib combinations using a human breast cancer patient-derived xenograft that has HER2 and PIK3CA mutations very similar to our transgenic mouse. This study provides valuable preclinical evidence for these drug combinations, which are being tested in phase 1 clinical trials. Citation Format: Xiaoqing Cheng, Yirui Sun, Maureen Highkin, Nagalaxmi Vemalapally, Xiaohua Jin, Brandon Zhou, Julie L. Prior, Ashley R. Tipton, Shunqiang Li, Anton Iliuk, Samuel Achilefu, Ian S. Hagemann, John Edwards, Ron Bose. Breast cancer mutations HER2V777L and PIK3CAH1047R activate the p21-CDK4/6 -Cyclin D1 axis driving tumorigenesis and drug resistance. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5778.

Abstract 4928: Gedatolisib, a well-tolerated pan-PI3K/mTOR inhibitor, exhibits potent therapeutic effects on endometrial cancer models regardless of their PI3K pathway mutational status

Abstract Background: The PI3K, AKT, and mTOR (PAM) pathway is one of the most commonly activated oncogenic pathways in gynecological cancers. Loss of PTEN function and PIK3CA activating mutations are especially frequent in endometrial cancer (EC). Targeting the PAM pathway, in combination with other cooperative oncogenic pathways (e.g., the estrogen pathway), is a promising EC treatment strategy. Currently, everolimus, an mTORC1 inhibitor, in combination with letrozole, an aromatase inhibitor, is one of the available hormonal therapy options for patients with advanced endometrioid EC. Most PAM inhibitors (PAM-i) selectively spare (or weakly inhibit) one or more key PAM pathway components, which can lead to drug resistance. To minimize drug resistance, a more comprehensive inhibition of the PI3K isoforms and downstream mTOR complexes may be required. We hypothesized that gedatolisib, which potently inhibits all Class I PI3K isoforms, mTORC1, and mTORC2, can potentially be an effective and well tolerated therapy for patients with EC. Methods: A panel of endometrial, ovarian and cervical cancer cell lines with different PI3K pathway mutational status were assayed for their sensitivity to gedatolisib and other PAM inhibitors (PI3K-α: alpelisib, pan-PI3K: copanlisib; AKT: capivasertib; mTORC1: everolimus; pan-PI3K/mTOR: gedatolisib). Cell viability, cell proliferation, and flow cytometry analytical assays were conducted. Xenograft studies evaluating gedatolisib in EC cell lines were also performed. Results: Gedatolisib strongly inhibited PAM pathway activation, and reduced cell cycle progression and cell viability in the endometrial, ovarian, and cervical cancer cell lines. Compared to the other PAM-i, Gedatolisib exhibited superior anti-proliferative potency and efficacy in almost all the cell lines tested, regardless of the PI3K pathway mutational status or cancer type. In EC xenografts, gedatolisib substantively inhibited tumor growth irrespective of the cell line models’ PI3K pathway mutational status. Conclusions: We demonstrated that, gedatolisib induces superior anti-proliferative activity compared to the other PAM pathway inhibitors, regardless of the PI3K pathway mutational status of the cancer cell lines evaluated. Gedatolisib as a single agent and in combination with other therapies has previously reported promising preliminary clinical efficacy and safety in various solid tumor types, including EC. In summary, nonclinical and preliminary clinical data supports the development of gedatolisib in combination with hormonal therapy for treatment of patients with EC. Citation Format: Stefano Rossetti, Aaron Broege, Ian MacNeil, Ben Rich, Jhomary Molden, Brian Sullivan, Lance Laing. Gedatolisib, a well-tolerated pan-PI3K/mTOR inhibitor, exhibits potent therapeutic effects on endometrial cancer models regardless of their PI3K pathway mutational status. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4928.

Abstract 1557: Defining modulators of response to samuraciclib, a CDK7 inhibitor for breast cancer

Abstract Breast cancer (BC) is the most prevalent cancer in women worldwide. Despite initial sensitivity to endocrine therapies in hormone receptor positive BC, high mortality is driven by treatment-resistant disease. While several highly effective targeted treatments have been developed, resistance is common in the metastatic setting, highlighting the need for new therapies. Cyclin-dependent kinases (CDKs) have emerged as important targets for cancer treatment due to their integral role in driving the cell cycle. CDK4/6 inhibitors have advanced strongly in ER+ BC and are now becoming standard-of-care. However, resistance is a major problem. To address this, we have targeted CDK7, a master regulator of the cell cycle CDKs and transcription processes, both commonly dysregulated in cancer. CDK7 inhibition selectively inhibits cancer cell growth due to their reliance on aberrant gene expression and/or unregulated cell cycle progression. ICEC0942 (Samuraciclib), discovered in our lab at Imperial College London, has recently progressed to Phase 2 clinical trials. Considering the current clinical development of this and other CDK7 inhibitors (CDK7i), it is important to determine which patients will best respond to Samuraciclib. Towards identifying modulators of response to Samuraciclib, we performed a genome-wide CRISPR/Cas9-knockout screen in MCF7 cells. The mTOR/PI3K pathway was identified as important for response to CDK7i. Knockout of mTOR activators reduced sensitivity to Samuraciclib, while knockout of mTOR repressors increased sensitivity. Next, we studied the role of mutations in the catalytic subunit of PI3K (PIK3CA), which occur in nearly 30% of BC cases, in response to Samuraciclib. We utilized an isogenic panel of MCF10A cell lines, which were either wild-type for PIK3CA or had a heterozygous knock-in of either of the two most common mutations (E545K or H1047R). Cell growth was monitored following long-term (42-day) treatment with Samuraciclib. The drug was washed out (day 15) to determine the role of PIK3CA mutations in drug-induced growth arrest. We find that although Samuraciclib effectively inhibits growth of all cell lines, regardless of PIK3CA mutation status, the longevity of growth arrest post-washout is promoted by activating PIK3CA mutations. Finally, we assessed the role of mTOR and its downstream effectors in Samuraciclib response. Cell proliferation of MCF7 cells treated with Samuraciclib or in combination with mTOR/PI3K/AKT inhibitors was measured via flow cytometry. Samuraciclib treatment arrested cell proliferation; however, proliferation was reverted in combination with an mTOR/PI3K/AKT inhibitor. In conclusion, our findings highlight the importance of an active mTOR/PI3K pathway in promoting response to Samuraciclib. This may help guide patient treatments and development of effective combination therapies. Citation Format: Kaste Jurgaityte, Georgina Sava, Chun Fui Lai, Hailing Fan, Van Nguyen, Laki Buluwela, Simak Ali. Defining modulators of response to samuraciclib, a CDK7 inhibitor for breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1557.

Abstract 3418: Alterations in PIK3CA/PTEN as resistance mechanisms in lung cancer patients progressing on first-line next generation EGFR/ALK tyrosine kinase inhibitors

Abstract Introduction: Advent of targeted therapies has deeply changed treatment of advanced oncogene-addicted non-small cell lung cancer (NSCLC), but development of resistance remains a main issue. Given the efficacy of next generation tyrosine kinase inhibitors (TKIs) against on-target mutations, their first-line administration could increase the relevance of off-target resistance mechanisms. Activating PIK3CA mutations and PTEN loss have been described as putative off-target resistance mechanisms across generations of EGFR TKIs. The role of these alterations in the development of resistance to ALK TKIs has been less described. MATCH-R trial (NCT02517892) is an ongoing prospective study whose objective is to understand mechanisms of acquired resistance to cancer therapies and develop strategies to overcome it. Materials and methods: We collected clinical and molecular data of patients (pts) with NSCLC enrolled in the molecular target group (MTG) of MATCH-R trial. We identified pts with EGFR mutations or ALK rearrangements who developed PIK3CA/PTEN alterations in tissue biopsy and/or circulating tumor DNA upon progression to first-line osimertinib/alectinib. We established patient-derived cell lines (PDCL) from the identified MATCH-R pts and we engineered commercial cell lines (CCL) harboring EGFR mutation (PC9) or ALK fusion (H3122) to constitutively express PIK3CA mutations and/or to have a permanent loss of PTEN. Cell viability assays and Western blot studies with different EGFR/ALK/PI3K/mTOR inhibitors were performed. Results: Of the 186 pts with advanced NSCLC included in the MTG of MATCH-R, 110 (59.1%) harbored EGFR mutations (EGFR+) and 27 (14.5%) ALK rearrangements (ALK+). Among them, 19 received first-line osimertinib and seven first-line alectinib. Five and two pts developed PIK3CA/PTEN alterations in the EGFR+ and ALK+ groups, respectively. In the EGFR+ group, PIK3CA E545K (n=2), R108H, N345K and R357Q mutations were detected. PTEN mutations (F437fs*5 and Y27C) were concomitant with E545K and R357Q mutations, respectively. In both ALK+ pts, a PIK3CA E545K mutation was identified, together with PTEN exon 5 splicing in one case. We confirmed on CCL that PIK3CA E545K mutation, alone or in combination with PTEN loss, confers resistance to second-/third-generation EGFR/ALK TKIs. PTEN loss alone had a moderate impact on TKIs sensitivity. Overcoming strategies combining EGFR/ALK TKIs with PIK3CA/mTOR inhibitors are being evaluated and will be disclosed at the meeting. Conclusions: Activating PI3KCA mutations and molecular events leading to PTEN loss are frequent events at resistance to first-line EGFR/ALK TKIs, and can be detected concomitantly. Functional assays confirmed that these alterations act as resistance mechanisms. These observations are of interest as PI3KCA/mTOR inhibitors may have a role in overcoming resistance. Citation Format: Giorgia Guaitoli, Francesco Facchinetti, Juan David Flórez-Arango, Floriane Braye, Hayato Mizuta, Santiago Ponce-Aix, Damien Vasseur, Ken André Olaussen, Stefan Michiels, Mihaela Aldea, Jordi Remon, Fabrice Barlesi, Benjamin Besse, David Planchard, Luc Friboulet. Alterations in PIK3CA/PTEN as resistance mechanisms in lung cancer patients progressing on first-line next generation EGFR/ALK tyrosine kinase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3418.

Abstract OT3-09-01: Clinical Trial of Alpelisib and Tucatinib in Patients with PIK3CA-Mutant HER2-Positive Metastatic Breast Cancer

Abstract Phosphatidylinositol 3-kinase (PI3K) pathway plays a key role in resistance to the drugs targeting human epidermal growth factor receptor 2 (HER2). Activating mutations in the gene encoding alpha catalytic subunit of PI3K (PIK3CA) are present in approximately 30% of HER2+ tumors. PIK3CA mutations are linked to drug resistance and decreased survival in patients with HER2+ breast cancer. To overcome this resistance mechanism, we designed a phase IB/II clinical trial to evaluate the combination of HER2 small molecule inhibitor tucatinib with PI3K inhibitor alpelisib in patients with HER2+ metastatic breast cancer (NCT05230810). This multicenter clinical trial is conducted through the Academic Breast Cancer Consortium (ABRCC), with the University of Colorado Cancer Center as the lead site. Target enrollment: 40 patients. This is a run-in phase IB/roll-over phase II study. Phase IB will follow Time-to-Event Bayesian Optimal Interval design and enroll from 9 to 19 patients to find the maximum tolerated doses (MTDs) of tucatinib and alpelisib. From 21 to 31 patients will be enrolled in phase II part, for a total of 40 patients in the final efficacy analysis. Main inclusion criteria: 1. Women and men ≥ 18 years old 2. Eastern Cooperative Oncology Group (ECOG) performance status 0-1 3. Presence of activating PIK3CA mutation in the tumor 4. Patients with HR-/HER2+ or HR+/HER2+ breast cancer may enroll; ovarian suppression is mandatory for premenopausal patients with HR+/HER2+ disease 5. HR+/HER2+ patients should be agreeable to concomitant treatment with fulvestrant 6. Prior treatment with at least two FDA-approved HER2-targeted agents 7. Measurable or evaluable disease. Bone only disease is allowed. 8. Subjects with untreated central nervous system (CNS) metastases not needing immediate local therapy, and subjects with previously treated stable or progressive brain metastases may enroll, provided that there is no indication for immediate re-treatment. For patients with treated CNS metastases: time from treatment of CNS disease until the first dose of study drugs should be as follows: WBRT ≥ 21 days, surgical resection ≥ 14 days, SRS ≥ 7 days. 9. Adequate organ and marrow function Main exclusion criteria: 1. Contraindications to undergo contrast brain MRI 2. Leptomeningeal disease 3. Poorly controlled seizures 4. Diabetes mellitus type I, or uncontrolled diabetes mellitus type II 5. Acute pancreatitis within 1 year of screening, or history of chronic pancreatitis 6. History of severe cutaneous hypersensitivity reactions 7. Toxicities of prior cancer therapies that have not resolved to grade 1 or less, except peripheral neuropathy, which must have resolved to grade 2 or less, and alopecia 8. Previous treatment with EGFR or HER2 tyrosine kinase inhibitors, or PI3K/mTOR/AKT inhibitors. 9. Systemic anti-cancer therapy, palliative radiation to extracranial sites, or surgery within 2 weeks of the first dose of study drugs 10. Active bacterial, fungal, or viral infections, hepatitis B, C, or HIV 11. Clinically significant cardio-vascular disease Primary objectives: • Phase IB: safety and tolerability of combination therapy • Phase II: efficacy by progression free survival Exploratory assessment of biomarkers will be performed in the liquid biopsy samples. Study contact: Elena Shagisultanova, MD, PhD, elena.shagisultanova@cuanschutz.edu Citation Format: Elena Shagisultanova, Kari B. Wisinski, Chelsea D. Gawryletz, Farrah M. Datko, Diana Medgyesy, Jennifer R. Diamond, Virginia F. Borges, Peter Kabos. Clinical Trial of Alpelisib and Tucatinib in Patients with PIK3CA-Mutant HER2-Positive Metastatic Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT3-09-01.

Endothelial hyperactivation of mutant MAP3K3 induces cerebral cavernous malformation enhanced by PIK3CA GOF mutation.

Cerebral cavernous malformations (CCMs) refer to a common vascular abnormality that affects up to 0.5% of the population. A somatic gain-of-function mutation in MAP3K3 (p.I441M) was recently reported in sporadic CCMs, frequently accompanied by somatic activating PIK3CA mutations in diseased endothelium. However, the molecular mechanisms of these driver genes remain elusive. In this study, we performed whole-exome sequencing and droplet digital polymerase chain reaction to analyze CCM lesions and the matched blood from sporadic patients. 44 of 94 cases harbored mutations in KRIT1/CCM2 or MAP3K3, of which 75% were accompanied by PIK3CA mutations (P = 0.006). AAV-BR1-mediated brain endothelial-specific MAP3K3I441M overexpression induced CCM-like lesions throughout the brain and spinal cord in adolescent mice. Interestingly, over half of lesions disappeared at adulthood. Single-cell RNA sequencing found significant enrichment of the apoptosis pathway in a subset of brain endothelial cells in MAP3K3I441M mice compared to controls. We then demonstrated that MAP3K3I441M overexpression activated p38 signaling that is associated with the apoptosis of endothelial cells in vitro and in vivo. In contrast, the mice simultaneously overexpressing PIK3CA and MAP3K3 mutations had an increased number of CCM-like lesions and maintained these lesions for a longer time compared to those with only MAP3K3I441M. Further in vitro and in vivo experiments showed that activating PI3K signaling increased proliferation and alleviated apoptosis of endothelial cells. By using AAV-BR1, we found that MAP3K3I441M mutation can provoke CCM-like lesions in mice and the activation of PI3K signaling significantly enhances and maintains these lesions, providing a preclinical model for the further mechanistic and therapeutic study of CCMs.

Development of a multiplex allele-specific qPCR approach for testing PIK3CA mutations in patients with colorectal cancer

Phosphatidylinositol 3-kinases (PI3Ks) are lipid kinases involved in cellular growth and division. Somatic mutations in one of the PI3K catalytic subunit genes, PIK3CA, are frequently found in numerous malignancies, including colorectal cancer (CRC). Several PIK3CA inhibitors are approved for the treatment of breast cancer and lymphoma. Activating mutations in PIK3CA tend to occur in exons 9 and 20, with mutations in other exons 1, 4, and 7 being less common. Most test systems for PIK3CA mutation screening are designed to detect mutations in exons 9 and 20, leaving exons 1–7 overlooked. We have developed a multiplex AS-PCR to screen for PIK3CA mutations in exons 1, 4, 7, 9, and 20. Validation was performed on 515 CRC samples of patients from Siberia and the Far East of Russia. The assay sensitivity was 0.05–0.5% of mutant DNA, and the overall PIK3CA mutation frequency was 13.01%, with 9.32% of mutations in exon 9, 1.94% in exon 20, and 1.74% in exons 1–7. The assay designed is suitable for the analysis of activating PIK3CA mutations in formalin-fixed paraffin-embedded tissue samples. The present work is the first study characterizing the PIK3CA mutation frequency in CRC patients from the eastern part of Russia.

WIN Symposium 2022 - Abstract Poster Presentations.

WIN Symposium 2022 - Abstract Poster Presentations.

Abstract 3098: The first circulating tumor cell-derived explant (CDX) model of a Merkel cell carcinoma

Abstract NeuroEndocrine Carcinomas (NECs) are rare yet lethal diseases; they are frequently metastatic and have limited treatment options. Whilst progress has been made in understanding the biology of Small Cell Lung Cancer (SCLC), the most studied NEC, research on NECs from other anatomical sites lags behind. In SCLC, circulating tumor cells (CTCs) can be used to generate patient-relevant mouse models; termed CTC-Derived eXplants (CDX) which are faithful in biology and chemotherapy response to donor patients1,2. A pilot prospective study is on-going to assess the potential utility of cell-free DNA (cfDNA) and/or CellSearch (CS) CTCs (EpCAM/cytokeratin positive) as liquid biopsies and feasibility of CDX generation from patients with advanced stage extrapulmonary NECs undergoing palliative chemotherapy. CS CTCs are enumerated before and during treatment and marker-independent RosetteSep-enriched CTCs from parallel blood samples are implanted in immunodeficient mice to attempt CDX generation. Twenty-seven patients were recruited from Jun-19 to Nov-21. CS-CTCs were present in 19/27; 70% pre-treatment samples (mean 57, median 2, range 0-685 CTCs per 7.5mL of blood). CDX generation was successful from a pre-treatment CTC sample from a patient originally diagnosed with a NEC of unknown origin who responded to Platinum/Etoposide. Whole-exome sequencing of this CDX and matched pre-treatment cfDNA from the donor revealed largely overlapping copy number changes and common mutations including an activating PIK3CA mutation. In addition, there was no evidence of TP53 or RB1 loss (hallmarks of SCLC) in neither the CDX nor the donor’s cfDNA. Immunohistochemical (IHC) analysis of this CDX showed a small cell NEC morphology, 90% Ki-67 positive cells, expression of EpCAM, pan-cytokeratins and neuroendocrine (NE) diagnostic markers, concordant with the donor’s biopsy. Further IHC and RNA sequencing revealed expression of the NE transcription factor atonal homolog 1 (ATOH1)2, CK20 and abundant Merkel cell polyomavirus (MCPyV) transcripts. These characteristics prompted reclassification of the original patient diagnosis as Merkel cell carcinoma (MCC). There was only 1 CS-CTC in the parallel pre-treatment blood sample implying that EpCAM-negative CTCs may have given rise to the CDX. The CDX showed in vivo sensitivity to Platinum/Etoposide, mirroring treatment response of the donor. Ex vivo cultures from CDX-derived cells grow as suspension clusters similar to ‘classic’ SCLC and MCPyV-positive MCC cell lines. In summary, this first Merkel Cell Carcinoma CDX recapitulates the biology of the donor’s tumor. As the donor remains alive 25 months from diagnosis of advanced disease, their CDX can serve as an avatar to guide future treatment. 1. Hodgkinson et al., Nature Med 2014, 20: p. 897-903. 2. Simpson et al., Nature Cancer 2020, 1: p. 437-451. Citation Format: Melissa Frizziero, Elaine Kilgour, Kathryn L. Simpson, Dominic Rothwell, Kristopher Frese, Melanie Galvin, Yitao Chen, Alastair Kerr, Sam Humphrey, Mukarram Hossain, Juan W. Valle, Mairéad G. McNamara, Caroline Dive. The first circulating tumor cell-derived explant (CDX) model of a Merkel cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3098.

The onset of PI3K-related vascular malformations occurs during angiogenesis and is prevented by the AKT inhibitor miransertib.

Low‐flow vascular malformations are congenital overgrowths composed of abnormal blood vessels potentially causing pain, bleeding and obstruction of different organs. These diseases are caused by oncogenic mutations in the endothelium, which result in overactivation of the PI3K/AKT pathway. Lack of robust in vivo preclinical data has prevented the development and translation into clinical trials of specific molecular therapies for these diseases. Here, we demonstrate that the Pik3caH1047R activating mutation in endothelial cells triggers a transcriptome rewiring that leads to enhanced cell proliferation. We describe a new reproducible preclinical in vivo model of PI3K‐driven vascular malformations using the postnatal mouse retina. We show that active angiogenesis is required for the pathogenesis of vascular malformations caused by activating Pik3ca mutations. Using this model, we demonstrate that the AKT inhibitor miransertib both prevents and induces the regression of PI3K‐driven vascular malformations. We confirmed the efficacy of miransertib in isolated human endothelial cells with genotypes spanning most of human low‐flow vascular malformations.