PIK3CA-mutated Tumors Research Articles

Associations of SEMA7A, SEMA4D, ADAMTS10, and ADAM8 with KRAS, NRAS, BRAF, PIK3CA, and AKT Gene Mutations, Microsatellite Instability Status, and Cytokine Expression in Colorectal Cancer Tissue.

Semaphorins (SEMAs), ADAM, and ADAMTS family members are implicated in various cancer progression events within the tumor microenvironment across different cancers. In this study, we aimed to evaluate the expression of SEMA7A, SEMA4D, ADAM8, and ADAMTS10 in colorectal cancer (CRC) in relation to the mutational landscape of KRAS, NRAS, BRAF, PIK3CA, and AKT genes, microsatellite instability (MSI) status, and clinicopathological features. We also examined the associations between the expression of these proteins and selected cytokines, chemokines, and growth factors, assessed using a multiplex assay. Protein concentrations were quantified using ELISA in CRC tumors and tumor-free surgical margin tissue homogenates. Gene mutations were evaluated via RT-PCR, and MSI status was determined using immunohistochemistry (IHC). GSEA and statistical analyses were performed using R Studio. We observed a significantly elevated expression of SEMA7A in BRAF-mutant CRC tumors and an overexpression of ADAM8 in KRAS 12/13-mutant tumors. The expression of ADAMTS10 was decreased in PIK3CA-mutant CRC tumors. No significant differences in the expression of the examined proteins were observed based on MSI status. The SEMA7A and SEMA4D expressions were correlated with the expression of numerous cytokines associated with various immune processes. The potential immunomodulatory functions of these molecules and their suitability as therapeutic targets require further investigation.

Characterizing multi-PIK3CA mutations across cancer types: Toward precision oncology.

PIK3CA mutations are implicated in various cancers, but the implications of multiple concurrent mutations and their orientations within the gene have not been fully explored. In this study, we analyzed multi-PIK3CA mutations across a diverse pan-cancer cohort comprising 3564 tumors. Multi-PIK3CA mutations were present in 10.3% of all PIK3CA-mutant tumors, predominantly occurring in breast and gynecological cancers. Notably, mutations within the helical domain (E542:E545) exclusively occurred in the trans-orientation, contrasting with mutations in the kinase ABD and C2 domains, which mainly appeared in the cis orientation. The distinct pattern of mutation orientations in PIK3CA suggests variable oncogenic potential, with helical domain mutations in the trans-orientation potentially being less oncogenic. These findings highlight the importance of mutation orientation in the PIK3CA gene as potential biomarkers for targeted therapy. This understanding is crucial for designing clinical trials that leverage PI3K inhibitors, aiming for more effective and precise cancer treatment.

APOA5 alleviates reactive oxygen species to promote oxaliplatin resistance in PIK3CA-mutated colorectal cancer.

Although platinum-based chemotherapy is the frontline regimen for colorectal cancer (CRC), drug resistance remains a major challenge affecting its therapeutic efficiency. However, there is limited research on the correlation between chemotherapy resistance and lipid metabolism, including PIK3CA mutant tumors. In this present study, we found that PIK3CA-E545K mutation attenuated cell apoptosis and increased the cell viability of CRC with L-OHP treatment in vitro and in vivo. Mechanistically, PIK3CA-E545K mutation promoted the nuclear accumulation of SREBP1, which promoted the transcription of Apolipoprotein A5 (APOA5). APOA5 activated the PPARγ signaling pathway to alleviate reactive oxygen species (ROS) production following L-OHP treatment, which contributed to cell survival of CRC cells. Moreover, APOA5 overexpression enhanced the stemness-related traits of CRC cells. Increased APOA5 expression was associated with PIK3CA mutation in tumor specimens and poor response to first-line chemotherapy, which was an independent detrimental factor for chemotherapy sensitivity in CRC patients. Taken together, this study indicated that PIK3CA-E545K mutation promoted L-OHP resistance by upregulating APOA5 transcription in CRC, which could be a potent target for improving L-OHP chemotherapeutic efficiency. Our study shed light to improve chemotherapy sensitivity through nutrient management in CRC.

INAVO122: A phase III study of maintenance inavolisib or placebo + pertuzumab + trastuzumab following induction with pertuzumab + trastuzumab + a taxane in patients (pts) with PIK3CA-mutated, HER2-positive advanced breast cancer (HER2+ aBC).

TPS1124 Background: The PI3K pathway plays a crucial role in HER2 signaling. Somatic mutations of PIK3CA, the gene that encodes the PI3K p110α subunit, may induce resistance to HER2-targeted therapies and are associated with poorer clinical outcomes (Swain SM, et al. SABCS 2022; P2-11-07). Inavolisib, a potent p110α-selective inhibitor that induces degradation of mutant p110α, has shown antitumor activity in PIK3CA-mutated HER2+ BC and long-term tolerability with early intervention for common on-class toxicities, including hyperglycemia, diarrhea, and stomatitis (Jhaveri KL, et al. SABCS 2023; GS03-13, Bedard P, et al. ASCO 2022; 1052). The current study will assess the efficacy and safety of maintenance inavolisib + fixed-dose combination of pertuzumab + trastuzumab for subcutaneous injection (PH FDC SC) after first-line (1L) induction treatment in pts with PIK3CA-mutated, HER2+ aBC. Methods: INAVO122 (NCT05894239) is a multicenter, randomized, international, double-blind, placebo-controlled study. Pts will be enrolled for: 1L induction treatment if they are receiving/will receive PH + a taxane; or for maintenance treatment if they completed induction treatment per standard of care. In the maintenance phase, pts will be randomized 1:1 to receive inavolisib (9 mg orally once daily; 21-day cycles) + PH FDC SC (every 3 weeks), or placebo + PH FDC SC. Study treatment will continue until disease progression (PD), unacceptable toxicity, death, consent withdrawal, or at the investigator’s (INV) discretion. Enrolled pts must have centrally determined HER2+, PIK3CA-mutated tumors, with documented hormone receptor status per local assessment, and be disease-free for ≥6 months from completion of neoadjuvant/adjuvant HER2-targeted treatment. They must not have received any non-hormonal treatment for aBC prior to induction, and, at screening, fasting glucose must be <126 mg/dL and HbA1c <6.4%. To be randomized, pts must have completed induction therapy without PD and show left ventricular ejection fraction ≥50%. The primary endpoint is INV-assessed progression-free survival. Secondary endpoints are overall survival; INV-assessed objective response rate, duration of response, clinical benefit rate, and time to second disease progression; health-related quality of life; safety; and pharmacokinetics. The primary endpoint analysis will utilize a two-sided stratified log-rank test at a two-sided significance level of 5%. A stratified Cox proportional hazards model will be used to estimate the hazard ratio between the two treatment arms and its 95% confidence intervals. An independent data monitoring committee will be in place for safety. Target randomization is ~230 pts; this study is currently recruiting (15 pts enrolled). Encore from SABCS 2023 (PO2-19-09). Clinical trial information: NCT05894239 .

First-in-human study of RLY-2608, a pan-mutant and isoform selective PI3Kα inhibitor, as a single agent in patients (pts) with advanced solid tumors and in combination with fulvestrant in pts with advanced breast cancer.

TPS1128 Background: Oncogenic mutations in PIK3CA occur in ~35% of HR+, HER2– breast cancer (BC) and are frequent in multiple solid tumors. The approval of alpelisib, a non-mutant selective PI3Kα inhibitor, validates PI3Kα as a therapeutic target. However, inhibition of wild-type PI3Kα leads to significant toxicity, including hyperglycemia and rash. RLY-2608, a novel, oral allosteric PI3Kα inhibitor, is designed to overcome these limitations via mutant- and isoform-selective PI3Kα inhibition for greater target coverage, improved tolerability, and anti-tumor activity. ReDiscover (NCT05216432) is the ongoing, first-in-human study to investigate the maximum tolerated dose (MTD), recommended phase 2 doses (RP2Ds), safety, pharmacokinetics (PK), pharmacodynamics (PD) and anti-tumor activity of RLY-2608 monotherapy in pts with PIK3CA-mutant advanced solid tumors; and of RLY-2608 plus fulvestrant with or without ribociclib in pts with PIK3CA-mutant, HR+, HER2– BC. To date RLY-2608 plus fulvestrant has completed dose escalation; 400 and 600 mg BID is being investigated in the combination regimens, based on safety, PK, PD, and anti-tumor activity. Methods: This global, multi-center, dose escalation/expansion study of RLY-2608 monotherapy includes adult pts with advanced solid tumors who are refractory or intolerant to, or have declined, standard therapy. RLY-2608 is also being investigated in combination with fulvestrant and in combination with fulvestrant and ribociclib, each in previously treated pts with HR+, HER2– advanced/metastatic BC. Eligibility criteria include presence of a PIK3CA mutation (blood or tumor) per local assessment, ECOG performance status 0–1, evaluable disease per RECIST 1.1, and no prior treatment with a PI3K pathway inhibitor. RLY-2608 is administered daily in 4-week cycles. Adverse events (AEs) per CTCAE v5, PK, biomarkers (pAKT, mutant ctDNA and insulin pathway markers) and anti-tumor activity are assessed serially. Dose escalation employs a Bayesian Optimal Interval design to identify the MTD. Following dose escalation, expansion cohorts are enrolling patients with select PIK3CA-mutated solid tumors for treatment with RLY-2608 monotherapy and patients with HR+, HER2– metastatic BC for treatment with RLY-2608 plus fulvestrant with or without ribociclib. Primary endpoints are MTD/RP2D and AE profile; key secondary endpoints are PIK3CA genotype in blood and tumor, PK, biomarkers, and overall response rate. All dose expansion cohorts in ReDiscover are now enrolling in the United States and Europe. Clinical trial information: NCT05216432 .

What Do German Molecular Tumor Boards Recommend in Patients with PIK3CA-Mutated Tumors? Launch and First Results from the German Transsectoral Molecular Tumor Board Exchange Platform Deutschland

Introduction: Comprehensive molecular tumor profiling is widely used in the management of patients with cancer. Molecular tumor boards devise treatment strategies based on testing results. In this setting, the Transsectoral Molecular Tumor Board exchange platform Deutschland (TEAM-D) aims to drive peer-to-peer exchange to connect experts in the field. Methods: During the first virtual TEAM-D meeting, participants from 16 German universities and 5 nonacademic institutions discussed five cases with PIK3CA hotspot mutations. Furthermore, an illustrative case vignette was presented. Results: Overall, German caregivers show restraint in administering off-label PIK3CA inhibitor and favor clinical trials in this setting. Conclusion: In the setting of precision oncology, TEAM-D enables virtual case discussion across the different sectors of the German healthcare system. Based on the example of PIK3CA hotspot mutations, TEAM-D demonstrated the value of integrating knowledge from different healthcare professionals.

Abstract PO2-19-09: INAVO122: a Phase III study of maintenance inavolisib or placebo + pertuzumab + trastuzumab following induction with pertuzumab + trastuzumab + a taxane in patients with PIK3CA-mutated, HER2-positive advanced breast cancer

Abstract Background The PI3K pathway plays a crucial role in HER2 signaling. Somatic mutations of PIK3CA, the gene that encodes the PI3K p110α subunit, may induce resistance to HER2-targeted therapies and are associated with poorer clinical outcomes (Swain SM, et al. SABCS 2022; P2-11-07). Inavolisib, a potent p110α-selective inhibitor that induces degradation of mutant p110α, has shown antitumor activity in PIK3CA-mutated HER2-positive breast cancer (HER2+ BC) and long-term tolerability with early intervention for common on-class toxicities, including hyperglycemia, diarrhea, and stomatitis (Bedard P, et al. ASCO 2022; 1052). The current study will assess the efficacy and safety of maintenance inavolisib + fixed-dose combination of pertuzumab + trastuzumab for subcutaneous injection (PH FDC SC) after first-line induction treatment in patients with PIK3CA-mutated, HER2+, advanced BC (aBC). Trial design INAVO122 is a multicenter, randomized, international, double-blind, placebo-controlled study. Patients will be enrolled for: first-line induction treatment if they are receiving/will receive PH + a taxane; or maintenance treatment completing induction treatment. In the maintenance phase, patients will be randomized 1:1 to receive inavolisib (9 mg orally once daily; Days 1–21 of 21-day cycles) + PH FDC SC (every 3 weeks), or placebo + PH FDC SC. Study treatment will continue until disease progression, unacceptable toxicity, death, consent withdrawal, or at the investigator’s discretion. ELIGIBILITY Enrolled patients must have centrally determined HER2+, PIK3CA-mutated tumors, with documented hormone receptor status per local assessment, and be disease-free for ≥ 6 months from completion of neoadjuvant/adjuvant systemic non-hormonal treatment. They must not have received any treatment for aBC prior to induction, and, at screening, fasting glucose must be < 126 mg/dL and HbA1c < 6.4%. To be randomized, patients must have completed induction therapy per standard of care without progression of disease and show left ventricular ejection fraction ≥ 50%. AIMS The primary endpoint is investigator-assessed progression-free survival. Secondary endpoints are overall survival; investigator-assessed objective response rate, duration of response, clinical benefit rate, and time to second disease progression; health-related quality of life; safety; and pharmacokinetics. STATISTICAL METHODS The primary endpoint analysis will utilize a two-sided stratified log-rank test at a two-sided significance level of 5%. A stratified Cox proportional hazards model will be used to estimate the hazard ratio between the two treatment arms and its 95% confidence intervals. ACCRUAL Target randomization is ~230 patients; recruitment began in July 2023. CONTACT INFORMATION For more information or to refer a patient, email global.rochegenentechtrials@roche.com or call 1-888-662-6728 (USA only). Clinicaltrials.gov number NCT05894239. Citation Format: Sandra Swain, Carlos Barrios, Reva Basho, Giuseppe Curigliano, Nadia Harbeck, Chiun-Shen Huang, Sherene Loi, Nicholas Turner, Jessica Chen, Volkmar Henschel, Simon Warburton, Fabiola Amair-Pinedo, Javier Cortés. INAVO122: a Phase III study of maintenance inavolisib or placebo + pertuzumab + trastuzumab following induction with pertuzumab + trastuzumab + a taxane in patients with PIK3CA-mutated, HER2-positive advanced breast cancer [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 PO2-19-09.

Abstract PO3-20-03: First-in-human global study of RLY-2608, a pan-mutant and isoform selective PI3Kα inhibitor, as a single agent in patients with advanced solid tumors and in combination with fulvestrant in patients with advanced breast cancer

Abstract Background: Oncogenic mutations in PI3Kα are common driver events in ER+ breast cancer and other solid tumors. Alpelisib, a non-mutant selective PI3Kα inhibitor, is effective in ER+ breast cancer thus validating PI3Kα as a therapeutic target. Inhibition of wild-type PI3Kα results in toxicity including hyperglycemia which limits tolerability and drug exposure. RLY-2608, a novel, oral allosteric PI3Kα inhibitor, is uniquely designed to overcome these limitations via mutant- and isoform-selective PI3Kα inhibition for greater target coverage, improved tolerability, and antitumor activity. Initiated in December 2021, ReDiscover is the ongoing, first-in-human study to define the maximum tolerated dose (MTD), safety, pharmacokinetics (PK), pharmacodynamics (PD) and anti-tumor activity of RLY-2608 monotherapy in PIK3CA-mutant advanced solid tumor patients (pts) and of RLY-2608 plus fulvestrant in pts with PIK3CA-mutant, HR+HER2-BC. At AACR 2023, dose escalation data for RLY-2608 was reported and demonstrated encouraging target inhibition and anti-tumor activity (including partial response), across PIK3CA genotypes with minimal impact on glucose homeostasis. To date, pts have received RLY-2608 at doses of 100–2000 mg/day, with no reported dose-limiting toxicities; the MTD has not been reached. These proof-of-mechanism data indicate that RLY-2608 is the first allosteric, pan-mutant selective PI3Kα inhibitor and that RLY-2608 has broad therapeutic potential in PIK3CA-driven cancers. Methods: Enrollment is ongoing in this global, multi-center, dose escalation/expansion study of RLY-2608 as a single agent in adults who have advanced solid tumors and are refractory, intolerant, or have declined standard therapy; and RLY-2608 in combination with fulvestrant in previously treated pts with HR+/HER2– MBC. Eligibility criteria include presence of PIK3CA mutation (blood or tumor) per local assessment, ECOG performance status 0–1, evaluable disease per RECIST 1.1 and no prior PI3K inhibitor (except in expansion combination group 2). RLY-2608 is administered on a continuous schedule with 4-week cycles. Adverse events (AEs) per CTCAE v5, PK, biomarkers (pAKT, mutant ctDNAs and insulin pathway markers) and anti-tumor activity are assessed serially. Dose escalation employs a Bayesian Optimal Interval design to identify the MTD and/or the recommended Phase 2 dose (RP2D). Following dose escalation, expansion cohorts will enroll patients with select PIK3CA-mutated solid tumors for treatment with RLY-2608 monotherapy and patients with HR+/HER2– MBC for treatment with RLY-2608 and fulvestrant. The primary endpoints are MTD/RP2D and AE profile for single agent and combination; key secondary endpoints are PIK3CA genotype in blood and tumor, PK, biomarkers, and overall response rate. ReDiscover (NCT05216432) is enrolling worldwide. For further information, contact clinicaltrials@relaytx.com. Funding source: This study is funded by Relay Therapeutics, Cambridge, MA, United States. Citation Format: Andreas Varkaris, Komal Jhaveri, Elena Garralda Cabanas, Lucia Sanz, Janice Kim, Cesar A. Perez, Erika Hamilton, Alexander I. Spira, Kari Wisinski, Angel Guerrero, Jason Henry, Jordi Rodon Ahnert, Gege Tan, Xiaoyan Li, Diana Hummel, Tamieka Hunter, Alison Timm, Ramin Samadani, Erika Puente-Poushnejad, Eunice Kwak, Brenton G. Mar, Alison M. Schram. First-in-human global study of RLY-2608, a pan-mutant and isoform selective PI3Kα inhibitor, as a single agent in patients with advanced solid tumors and in combination with fulvestrant in patients with advanced breast cancer [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 PO3-20-03.

Arachidonic acid released by PIK3CA mutant tumor cells triggers malignant transformation of colonic epithelium by inducing chromatin remodeling

Key gene mutations are essential for colorectal cancer (CRC) development; however, how the mutated tumor cells impact the surrounding normal cells to promote tumor progression has not been well defined. Here, we report that PIK3CA mutant tumor cells transmit oncogenic signals and result in malignant transformation of intestinal epithelial cells (IECs) via paracrine exosomal arachidonic acid (AA)-induced H3K4 trimethylation. Mechanistically, PIK3CA mutations sustain SGK3-FBW7-mediated stability of the cPLA2 protein, leading to the synthetic increase in AA, which is transported through exosome and accumulated in IECs. Transferred AA directly binds Menin and strengthens the interactions of Menin and MLL1/2 methyltransferase. Finally, the combination of VTP50469, an inhibitor of the Menin-MLL interaction, and alpelisib synergistically represses PDX tumors harboring PIK3CA mutations. Together, these findings unveil the metabolic link between PIK3CA mutant tumor cells and the IECs, highlighting AA as the potential target for the treatment of patients with CRC harboring PIK3CA mutations.

NETs unleashed: neutrophil extracellular traps boost chemotherapy against colorectal cancer.

Chemotherapy, which primarily acts on cancer cells, can influence the tumor microenvironment and the recruitment and behavior of stromal cells. In this issue of the JCI, Li et al. explored the potent anticancer effect of the combination of a glutaminase inhibitor (CB-839) and 5-FU against PIK3CA-mutant colorectal cancer tumors. This chemotherapy treatment strongly induced the recruitment of neutrophils that formed neutrophil extracellular traps in cancer, which actively killed cancer cells by inducing apoptosis. This study substantially advances our understanding of the multifaceted role of neutrophils and NETs in the outcome of anticancer treatment.

A New Wave of PI3Kα Inhibitors.

This is the first peer-reviewed report of an allosteric, mutant-selective PI3Kα inhibitor, STX-478, that reduces PIK3CA-mutant tumor growth in mice. However, in contrast to the FDA-approved PI3Kα isoform-selective inhibitor alpelisib, STX-478 does not induce hyperglycemia or other metabolic dysfunctions. See related article by Buckbinder et al., p. 2432 (7).

Mutational Landscape of ER Receptor Negative Endometrial Cancer Patients Categorized as No Specific Molecular Profile (NSMP)

The endometrial cancer (EC) molecular classification introduced by The Cancer Genome Atlas (TCGA) has initiated molecular-based classification with clear prognostic value. Four major tumor subtypes exist: 1) TP53 mutation (p53abn); 2) POLE mutation (POLEmut); 3) mismatch repair protein deficient (MMRd); and 4) no specific molecular profile (NSMP). Of these subtypes, p53abn patients exhibited the worst prognosis. Recently, a prognostic refinement of NSMP EC using estrogen receptor (ER) status, from PORTEC-III trial data, showed that (ER-) NSMP patients had a strikingly poor prognosis, similar to p53abn patients. Currently, drivers of NSMP (ER-) disease are unknown and require genomic characterization. In this study, we perform an integrated molecular analysis to characterize this aggressive and poorly understood cohort. RNAseq and mutational data was downloaded from the TCGA, PanCancer Atlas, Uterine Endometrial Carcinoma dataset via the genomic data commons (GDC). All patients in this cohort demonstrated endometrioid histology. P53abn, and POLE mutated patients were filtered using mutational data. MMRd and NSMP ESR1 gene low expressors (ER Low) were filtered using Z-score cutoffs of RNAseq expression data. Survival analysis was performed using the Survival R-package. Gene expression testing was performed using the EdgeR R-package. Pathway Enrichment analysis was conducted using EnrichR software. Forty-two patients out of 529 samples met our GDC NSMP (ER low) filtering criteria (7%), similar to the 5% of PORTEC-III patients classified as NSMP (ER -). Strikingly 83% percent of GDC NSMP (ER low) patients harbored a mutation in the PI3K-AKT-mTOR signaling pathway, with 48% of GDC NSMP (ER low) patients carrying a mutation in the p110 alpha (PIK3CA) gene. Interestingly, of the patients with PIK3CA mutant tumors, 2/20 patients died in 5 years (10%), compared to 9/22 patients whose tumors were PIK3CA WT (41%) (p = 0.02). Among GDC NSMP (ER low) patients with PIK3CA WT tumors gene expression signatures were enriched for MYC target genes (p = 1.1e-51), DNA replication (p = 4.3e-30), cell cycle (p = 9.32e-20), and cell cycle checkpoints (p = 6.4e-20) pathways. Additionally, strongly in PIK3CA WT tumors are mitochondrial membrane proteins (p = 1.63e-37) and ribosomal proteins (2.9e-24). In this analysis, we show that GDC NSMP (ER low) tumors nearly all harbor a mutation in the PI3K-AKT-mTOR signaling pathway. Further we demonstrate that the most common gene mutation in the cohort, PIK3CA, is counterintuitively a marker of improved survival. Additionally, we show that within that this subpopulation, PIK3CA WT patients exhibit robustly upregulated gene expression programs dedicated to energy production, cell cycling, and division.

Abstract CT017: Pan-mutant and isoform selective PI3Kα inhibitor, RLY-2608, demonstrates selective targeting in a first-in-human study of PIK3CA-mutant solid tumor patients, ReDiscover trial

Abstract BACKGROUND: Oncogenic activation of PI3Kα via mutation of PIK3CA is the most common kinase driver event across solid tumors, particularly breast cancer (BC). Alpelisib, an orthosteric inhibitor, validated mutant-PI3Kα as a therapeutic target; however, toxicity from non-selective inhibition of WT PI3Kα (hyperglycemia) and other PI3K isoforms limits its clinical activity. RLY-2608, a novel oral allosteric PI3Kα inhibitor, is designed to overcome these limitations via mutant- and isoform-selective PI3Kα inhibition. We initiated a first-in-human study (NCT05216432) to define the MTD, safety, pharmacokinetics (PK), and anti-tumor activity of RLY-2608 in PIK3CA-mutant solid tumor patients (pts) and of RLY-2608 plus fulvestrant (combo) in pts with PIK3CA-mutant, HR+HER2-BC, who had received prior CDK4/6 inhibitor and endocrine therapy. METHODS: Adult pts with no prior PI3Ki/mTORi therapy received RLY-2608 QD or BID on a 4-week (wk) cycle following a Bayesian Optimal Interval dose escalation. Eligible BC pts received fulvestrant. PK, pharmacodynamics (ex vivo pAKT inhibition; tumor markers; ctDNAs), treatment emergent adverse events (TEAEs), and anti-tumor activity per RECIST 1.1 were assessed. RESULTS: As of 31Jan23, 40 pts (21 BC combo pts) with PIK3CA mutations (12 kinase, 18 helical, 10 other) received RLY-2608 at doses of 100-1600 mg/day. Median number of prior treatments was 4 (1-11). An MTD has not been reached and dose escalation continues. RLY-2608 PK were favorable with BID dosing providing low steady-state peak-to-trough variability for mutant-selective inhibition. This enabled dose-dependent increase in exposure and target inhibition with doses ≥600 mg BID and pAKT suppression (≥80%) in the expected therapeutic range with minimal/no impact on glucose homeostasis. Across arms, safety was consistent: most frequent TEAEs (>15%) were low grade (≤Gr 2) nausea (30%), fatigue (22.5%), headache (22.5%), hypokalemia (22.5%), and diarrhea (17.5%). No DLTs were observed, and no pts discontinued due to related AEs. Initial anti-tumor activity was observed in BC combo pts including declines in tumor markers, PIK3CA/ESR1 ctDNAs and radiographic tumor reduction in 7 of 10 RECIST measurable pts. At 600 mg BID, 6/6 BC combo pts had disease stabilization and remain on treatment, including all RECIST-measurable pts with tumor reduction. Overall, 17 (81%) BC combo pts remain on treatment with median duration of 12 (1-40) wks. CONCLUSION: Across PIK3CA genotypes, RLY-2608 demonstrated target inhibition and anti-tumor activity with minimal impact on glucose homeostasis. These proof-of-mechanism data indicate that RLY-2608 is the first allosteric, pan-mutant selective PI3Kα inhibitor and that RLY-2608 has broad therapeutic potential in PIK3CA-driven cancers. Citation Format: Andreas Varkaris, Komal Jhaveri, Cesar A. Perez, Janice S. Kim, Jason T. Henry, Vivek Subbiah, Alexander I. Spira, Lucía Sanz Gómez, Angel I. Guerrero-Zotano, Kari B. Wisinski, Xiaoyan Li, Gege Tan, Ramin Samadani, Tamieka Lauz Hunter, Wei Guo, Alison Timm, Djuro Karanovic, Beni B. Wolf, Erika P. Hamilton, Alison M. Schram. Pan-mutant and isoform selective PI3Kα inhibitor, RLY-2608, demonstrates selective targeting in a first-in-human study of PIK3CA-mutant solid tumor patients, ReDiscover trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT017.

PIK3CA and PIK3R1 tumor mutational landscape in a pan-cancer patient cohort and its association with pathway activation and treatment efficacy

There is little data concerning the implications of PIK3CA mutations outside of the known hotspots described in ER+/HER2− metastatic breast cancer (mBC). Similarly, PIK3R1 mutations could also lead to activation of PI3K pathway, but are poorly described. We determined the incidence and type of all somatic PIK3CA and PIK3R1 mutations by whole exome sequencing (WES) in a pan-cancer cohort of 1200 patients. Activation of the PI3K pathway was studied using phospho-AKT immunohistochemistry. Associations between PIK3CA/PIK3R1 mutations and response to chemotherapy were studied in mBC cases. We found 141 patients (11.8%) with a PIK3CA and/or PIK3R1 mutation across 20 different cancer types. The main cancer subtype was mBC (45.4%). Eighty-four mutations (62.2%) occurred in the three described hotspots; 51 mutations occurred outside of these hotspots. In total, 78.4% were considered activating or probably activating. Among PIK3R1 mutations, 20% were loss of function mutations, leading to a constitutional activation of the pathway. Phospho-AKT quantification in tumor samples was in favor of activation of the PI3K pathway in the majority of mutated tumors, regardless of mutation type. In ER+/HER2− mBC, first line chemotherapy efficacy was similar for PIK3CA-mutated and PIK3CA-WT tumors, whereas in triple negative mBC, chemotherapy appeared to be more effective in PIK3CA-WT tumors. In this large, real-life pan-cancer patient cohort, our results indicate that PIK3CA/PIK3R1 mutations are widely spread, and plead in favour of evaluating the efficacy of PI3K inhibitors outside of ER+/HER2− mBC and outside of hotspot mutations.

Abstract PD18-04: Prognostic implications of PIK3CA mutation by hormone receptor status and intrinsic subtype in early stage HER2-positive breast cancer: a correlative analysis from CALGB 40601

Abstract Background PIK3CA mutations have been described in 20-25% of early-stage HER2-positive breast tumors [1], and are associated with reduced pathologic complete response (pCR) rate after chemotherapy and anti-HER2 agents [2]. However, the independence of this finding and association with long-term outcomes within HER2+ patients is still largely unknown. Here, we studied the prognostic implications of PIK3CA mutations by hormone receptor (HR) status and intrinsic subtype in patients with early stage HER2+ breast cancer enrolled in CALGB 40601. Method In CALGB 40601, gene expression profiling by RNA sequencing (RNAseq) with PAM50-determined intrinsic subtype and PIK3CA mutations from whole exome sequencing (WES) were obtained from 184/305 (60%) pretreatment core biopsies. We examined the association of PIK3CA mutations with pCR and event free survival (EFS) by HR status and intrinsic subtype using logistic and Cox regression analyses. Results PIK3CA mutations were found in 32 patients (32/184, 17%). The most frequent mutation was H1047R (12/32,38%), followed by E545K (7/32,22%) and E542K (5/32,16%). PIK3CA mutations were present in 20% and 15% of HR-positive and HR-negative BC subpopulations, respectively. Within Luminal-B, Luminal-A and HER2-Enriched breast tumors, PIK3CA mutations occurred in 36%, 10% and 19% respectively. In the overall population there was lower rate of pCR in mutated-PIK3CA patients than wild-type (34% vs 49%). Using only the subset of patients treated with neoadjuvant trastuzumab-based therapy as standard of care (excluding the lapatinib plus paclitaxel arm), we found a statistically significant lower pCR rate among PIK3CA-mutated tumors using logistic regression model (30% vs 54%, OR=0.30, p=0.045). At a median follow-up of 9.1 years, the presence of PIK3CA mutation was significantly associated with worse EFS in the overall study population (HR 2.58, 95% CI 1.24- 5.35, p=0.011). In a multivariable model including pCR status, HR status and intrinsic subtype (HER2-E vs. not), PIK3CA mutation was independently and significantly associated with worse EFS (HR 2.18, 95% CI 1.04- 4.56, p=0.039). The negative impact of PIK3CA mutation on EFS was statistically significant only in patients with HR-positive (HR 3.6, 95% CI 1.45-8.96, p=0.06) and luminal breast tumors (HR 4.84, 95% CI 1.08-21.7, p=0.039), but not in HR-negative and non-luminal subtypes. Conclusion In our study, the presence of PIK3CA mutation was significantly associated with lower pCR rates in patients treated with chemotherapy plus trastuzumab. Moreover, in uni- and multivariable Cox models, PIK3CA mutations were associated with worse long-term survival, which appeared to be driven by HR-positive and luminal HER2-positive breast tumors.

Abstract P5-14-06: Tumor Genomic Landscape in Older Women with Metastatic Breast Cancer (MBC)

Abstract Background. Patients (pts) who develop MBC at older ages are underrepresented in clinical trials, are less likely to be included in comprehensive biomarker characterization studies, and experience worse breast cancer-specific survival than their younger counterparts. Elucidating genomic underpinnings of MBC and possible therapeutic targets for older breast cancer patients are critical priorities. Methods. We identified pts age >70 years at MBC diagnosis and a younger cohort (ages 50-69; age < 50), who were treated for MBC at a single center and who had their metastatic (or if not available, the primary) tumor, assessed by a targeted, tumor-only next generation sequencing (NGS) platform (OncoPanel) between 2013-2020. The NGS panel included mutations, copy number variation, tumor mutational burden (TMB), and hypermutation (HM) status, with mutations classified as oncogenic using the OncoKB tool and additional annotation. Copy number events were selected as being “oncogenic” if a high amplification was called for an oncogene or a deep deletion for a tumor suppressor. We compared findings for older (age >70) vs. younger (age < 50 and ages 50-69) MBC pts using Chi-Square and Kruskal-Wallis tests. To determine genomic event enrichment, logistic regression (LR) models were used, controlling for age (continuous), background rate, and tumor subtype (those with unknown subtype [n=27] were excluded from models). False discovery rate (FDR) was used to correct for multiple hypothesis testing. Results. The final analytic cohort included 2,380 pts. The median age at MBC diagnosis was 54.1 years overall (range 18.5- 91.9) and 73.6 years for those age >70. A total of 137 metastatic and 76 primary tumors were sequenced in pts age >70; in those age < 70, 1383 metastatic and 784 primary tumors were sequenced (for age < 50 [n=857] and 50-69 [n=1310]). Older pts were more likely to present with HR+/HER2- tumors (70.9% v. 62.4% v. 52.4%), and less likely to present with HER2+ (9.4% v. 14.4% v. 22.8%) or triple-negative breast cancer (TNBC) (18.8% v. 21.9% vs. 24.0%) at MBC diagnosis (listed >70, 50-69, < 50; P=1e-7). Older pts had higher average TMB vs. younger pts (9.57 in pts > 70, 8.56 in ages 50-69, 7.34 in ages < 50; P=3.5e-5). This was due to older pts having a higher incidence of hypermutation status as defined as TMB >10: 26.3% in age >70, 23.2% in ages 50-69, 16.8% in age < 50. Using q=0.1 as the threshold of significance, the presence of CDH1, PIK3CA, MAP3K1, TET2, and AKT oncogenic mutations were also enriched in older pts, while the presence of oncogenic GATA3, BRCA2, and TP53 mutations, as well as any mutation in BRCA1 were enriched in younger pts (too few oncogenic BRCA1 mutations were present for accurate modeling). The frequency of oncogenic PIK3CA mutations in HR+/HER2- tumors was highest in the oldest pts (44.4% in pts age >70 v. 31.6% in age 50-69 v. 26.7% in age < 50). Of pts who had oncogenic BRCA1/2 mutations identified on tumor-only NGS testing and underwent clinical germline testing (n=7 v. 60 v. 67, oldest to youngest), older pts had the lowest incidence of germline BRCA pathogenic variants (14.3% vs. 47.2.% vs. 67.2%; p=0.01); most BRCA mutations identified on NGS testing in older patients were considered likely somatic. When assessing enrichment in copy number events, ERBB2, RAD21, and BRIP1 amplifications were all significantly less frequent in older pts (q< 0.1), even when accounting for tumor subtype. Conclusions. In a large cohort of pts with MBC, the mutational and copy number landscape for older pts differs from that in younger pts, even after controlling for tumor subtype. Key actionable findings include a higher proportion of high TMB and PIK3CA-mutated tumors, emphasizing the importance of genomic profile testing in this pt population and further exploration of efficacy and tolerability of relevant therapies in those age >70 years. Citation Format: Hersh V. Gupta, Rachel Freedman, Melissa E. Hughes, Yvonne Y. Li, Gregory Kirkner, Janet L. Files, Sarah Strauss, Ana C. Garrido-Castro, Lauren Buckley, Romualdo Barroso-Sousa, Brittany Bychkovsky, Sara Tolaney, Laura MacConaill, Neal Lindeman, Bruce Johnson, Matthew Meyerson, Eric Winer, Deborah A. Dillon, Andrew Cherniack, Nancy U. Lin. Tumor Genomic Landscape in Older Women with Metastatic Breast Cancer (MBC) [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 P5-14-06.

Abstract P4-07-04: STX-478, a mutant-selective PI3Kα H1047X inhibitor clinical candidate with a best-in-class profile: Pharmacology and therapeutic activity as monotherapy and in combination in breast cancer xenograft models

Abstract PI3Kα is highly mutated in cancer resulting in hyperactivation of lipid kinase activity and downstream AKT signaling. H1047 is the most common site of oncogenic mutation and occurs in ~14% of all breast cancers. Initial therapeutic benefit of targeting PI3Kα was established with alpelisib, an alpha-selective PI3K inhibitor that is equipotent against wild-type and mutant forms. However, wild-type PI3Kα inhibition results in frequent dose-limiting toxicities including hyperglycemia, restricting the full potential of this drug. Selective targeting of H1047X-mutant PI3Kα is expected to both improve anti-tumor activity and reduce toxicity. STX-478 is an allosteric, CNS-penetrant, selective PI3Kα H1047X inhibitor, having excellent drug-like properties and exceptional kinome selectivity. STX-478 demonstrated minimal inhibition of CYP enzymes in vitro, supporting the potential for combinations with a wide range of therapeutics in breast cancer and a variety of other tumor types. STX-478 selectivity extended to the inhibition of other activating kinase domain mutations in biochemical assays. In a diverse panel of PI3Kα H1047X mutant cell lines, STX-478 selectively reduced the cellular levels of pAKT (S473) with a strong correlation between pAKT inhibition and cell viability (R = 0.8). In a high-throughput viability screen of 467 cancer cell lines, the presence of PIK3CA H1047X and other kinase domain mutations were the single strongest predictor of STX-478 sensitivity with potency superior to alpelisib. STX-478 also selectively inhibited the proliferation of cell lines with PI3Kα helical domain mutations, potentially due to the selective dependency of these cells on mutant PI3Kα. When combined with fulvestrant, lapatinib, or abemaciclib, STX-478 demonstrated synergistic anti-proliferative activity in cell lines with relevant ER/HER2 status. Unlike alpelisib, STX-478 did not impair glucose metabolism or cause insulin resistance at efficacious doses. In the T47D (PI3Kα H1047R) breast cancer model, STX-478 (100 mg/kg) monotherapy caused tumor regression whereas alpelisib caused only stasis. STX-478 combination with fulvestrant was well-tolerated, with more consistent and deeper tumor regression. Similar results were observed in a PI3Kα H1047R mutant ER+/HER2- PDX model, where fulvestrant monotherapy showed minimal activity, while combination with STX-478 yielded tumor regressions. In an ER+/HER2+ PDX model (PI3Kα H1047R/R108H), palbociclib and STX-478 (100 mg/kg) monotherapy resulted in similar efficacy while the combination was well tolerated and yielded tumor regression. Together these data indicate robust STX-478 monotherapy activity that was well tolerated and improved when dosed in combination with fulvestrant or CDK4/6 inhibitors. Finally, we investigated the effect of STX-478 treatment in an ER+ PDX model carrying a helical domain mutation. STX-478 treatment resulted in tumor growth inhibition at doses that did not result in metabolic dysfunction, suggesting that STX-478 may also be efficacious in treating PIK3CA mutant tumors with helical domain mutations. In summary, STX-478 efficacy was superior to alpelisib at a dose level that exceeds the clinically relevant exposure in mice without causing metabolic dysfunction. STX- 478 has a predicted low human dose, CNS exposure, low risk of DDI, and a predicted long half-life with minimal variation in peak-to-trough plasma concentrations which further supports a favorable therapeutic index. STX-478 has the potential to provide a best-in-class profile to improve outcomes in patients harboring tumors with prevalent PI3Kα H1047X mutations as well as other kinase and helical domain mutant tumors. The significant CNS exposure of STX-478 is expected to enable this treatment for patients with brain tumors and brain metastases not afforded by existing options. STX-478 is currently in IND enabling studies and is expected to enter human clinical trials in 2023. Citation Format: Leonard Buckbinder, David J. St. Jean, Brendon Ladd, Trang Tieu, Philip Jonsson, Jacob Alltucker, Samantha Manimala, Weixue Wang, Angel Guzman-Perez, Darrin D. Stuart, Gregory Dowdell. STX-478, a mutant-selective PI3Kα H1047X inhibitor clinical candidate with a best-in-class profile: Pharmacology and therapeutic activity as monotherapy and in combination in breast cancer xenograft models [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 P4-07-04.

Genetic Heterogeneity and Tissue-specific Patterns of Tumors with Multiple PIK3CA Mutations.

To comprehensively characterize tissue-specific and molecular subclasses of multiple PIK3CA (multi-PIK3CA) mutations and assess their impact on potential therapeutic outcomes. We profiled a pan-cancer cohort comprised of 352,392 samples across 66 tumor types using a targeted hybrid capture-based next-generation sequencing panel covering at least 324 cancer-related genes. Molecularly defined subgroups, allelic configuration, clonality, and mutational signatures were identified and tested for association with PI3K inhibitor therapeutic response. Multi-PIK3CA mutations are found in 11% of all PIK3CA-mutant tumors, including 9% of low tumor mutational burden (TMB) PIK3CA-mutant tumors, and are enriched in breast and gynecologic cancers. Multi-PIK3CA mutations are frequently clonal and in cis on the same allele and occur at characteristic positions across tumor types. These mutations tend to be mutually exclusive of mutations in other driver genes, and of genes in the PI3K pathway. Among PIK3CA-mutant tumors with a high TMB, 18% are multi-PIK3CA mutant and often harbor an apolipoprotein B mRNA-editing enzyme, catalytic polypeptide (APOBEC) mutational signature. Despite large differences in specific allele combinations comprising multi-PIK3CA mutant tumors, especially across cancer types, patients with different classes of multi-PIK3CA mutant estrogen receptor-positive, HER2-negative breast cancers respond similarly to PI3K inhibition. Our pan-tumor study provides biological insights into the genetic heterogeneity and tissue specificities of multi-PIK3CA mutations, with potential clinical utility to guide PI3K inhibition strategies.

Genetic and epigenetic characteristics of patients with colorectal cancer from Uruguay.

Colorectal cancer (CRC), the 3rd most frequent cancer worldwide, affects both men and women. This pathology arises from the progressive accumulation of genetic and epigenetic alterations. In this study, KRAS, NRAS, PIK3CA, and BRAF gene mutations, mismatch repair (MMR) genes methylation profile, microsatellite instability (MSI) and CpG Island Methylator Phenotype (CIMP) status were assessed. The associations of these molecular features with clinicopathological data were also investigated. A hundred and eight unselected CRC samples and their histological and clinical data, were gathered between 2017 and 2020. The prevalence of KRAS, NRAS and BRAF gene mutations was similar to that described in other populations. 28.7% of tumors were KRAS-mutated, mostly in men, distal location, with a CIMP-negative status. BRAFV600E frequency was 6.5% and associated with MSI (p=0.048), MLH1-methylated (p<0.001) and CIMP-High (p<0.001) status. We also confirmed that BRAFV600E tumors were more prevalent in older women and proximal location. A striking different result was the lack of most common variants in the PIK3CA gene. A complete absence of PIK3CA-mutated tumors in a population has not been previously reported. Among MMR genes, the only with an aberrant methylation pattern was MLH1 gene. Its frequency was 9.25%, lower than previously reported. Methylated tumors were most frequent in patients older than 70 years old and proximal tumor location. Finally, CIMP-High status was mainly observed in moderately differentiated tumors with a rate of 15.7%. Our findings were consistent with previous reports in other populations, but also showed some features unique to our cohort. This study is the first to report the analysis of a large number molecular biomarkers of CRC in Uruguay and one of the few performed in Latin-America.

Abstract 2682: Small molecule targeting the lipoic acid post-translational modification impacts proliferation of colorectal and PIK3CA-mutant cell lines

Abstract To identify novel therapeutic targets, we utilize the PRISM platform, a multiplexed cell line viability technology of 500 solid tumor cell lines and correlate responses to functional genomic and baseline genetic data. We describe ESD0140656, a small molecule with selective anti-proliferative effect on colorectal and PIK3CA-mutant cell lines. Response to ESD0140656 is correlated to sensitivity to CRISPR/Cas9 KO of components of the protein lipoylation pathway and OGDH complex members, which catalyze a step of the TCA cycle. Lipoylation is a rare post-translational modification attached to just four enzymes in humans, including the OGDH complex. Knockout of the protein that transfers lipoic acid to these four enzymes (LIPT1) sensitizes cells to ESD0140656, and ESD0140656 treatment leads to reduction of lipoic acid in cells. These results suggest ESD0140656 targets the lipoylation pathway and may represent a novel therapeutic angle for colorectal and PIK3CA-mutant tumors. Citation Format: Laura Doherty, Tenzin Sangpo, Peter Tsvetkov, John Davis, Navid Dianati, Wolfgang Schwede, Katja Zimmermann, Laura Evans, Aldo Amatucci, Henrik Seidel, Atanas Kamburov, Gizem Akcay, Todd Golub, Ashley Eheim, Nils Burkhardt, Knut Eis, Sven Christian, Matt Rees, Jennifer Roth. Small molecule targeting the lipoic acid post-translational modification impacts proliferation of colorectal and PIK3CA-mutant cell lines [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 2682.