PIK3CA Gene Research Articles

Gastric cancer actionable genomic alterations across diverse populations worldwide and pharmacogenomics strategies based on precision oncology.

Introduction: Gastric cancer is one of the most prevalent types of cancer worldwide. The World Health Organization (WHO), the International Agency for Research on Cancer (IARC), and the Global Cancer Statistics (GLOBOCAN) reported an age standardized global incidence rate of 9.2 per 100,000 individuals for gastric cancer in 2022, with a mortality rate of 6.1. Despite considerable progress in precision oncology through the efforts of international consortia, understanding the genomic features and their influence on the effectiveness of anti-cancer treatments across diverse ethnic groups remains essential. Methods: Our study aimed to address this need by conducting integrated in silico analyses to identify actionable genomic alterations in gastric cancer driver genes, assess their impact using deleteriousness scores, and determine allele frequencies across nine global populations: European Finnish, European non-Finnish, Latino, East Asian, South Asian, African, Middle Eastern, Ashkenazi Jewish, and Amish. Furthermore, our goal was to prioritize targeted therapeutic strategies based on pharmacogenomics clinical guidelines, in silico drug prescriptions, and clinical trial data. Results: Our comprehensive analysis examined 275,634 variants within 60 gastric cancer driver genes from 730,947 exome sequences and 76,215 whole-genome sequences from unrelated individuals, identifying 13,542 annotated and predicted oncogenic variants. We prioritized the most prevalent and deleterious oncogenic variants for subsequent pharmacogenomics testing. Additionally, we discovered actionable genomic alterations in the ARID1A, ATM, BCOR, ERBB2, ERBB3, CDKN2A, KIT, PIK3CA, PTEN, NTRK3, TP53, and CDKN2A genes that could enhance the efficacy of anti-cancer therapies, as suggested by in silico drug prescription analyses, reviews of current pharmacogenomics clinical guidelines, and evaluations of phase III and IV clinical trials targeting gastric cancer driver proteins. Discussion: These findings underline the urgency of consolidating efforts to devise effective prevention measures, invest in genomic profiling for underrepresented populations, and ensure the inclusion of ethnic minorities in future clinical trials and cancer research in developed countries.

Abstract PO4-13-12: PIK3CA mutational status in tissue & plasma as a prognostic tool in HR+/HER2- breast cancer (BC)

Abstract Background: Activating mutations of PIK3CA gene are described in about 30-40% of BC. They confer overall worse prognosis and resistance to endocrine and chemotherapeutic therapy. Concordance between testing methods (tissue & plasma) are not widely studied. We aim to correlate tissue & plasma assays and to analyze the discordant cases and prognostic value of PIK3CA mutations (PIK3CAm) in HR+/HER2- BC. Methods: We performed a retrospective and unicentric analysis of PIK3CA mutational status in tissue & plasma samples in patients (pts) with HR+/HER2- BC from February/21 to April/23. PIK3CA test: Cobas®PIK3CA Mutation Kit. We correlated both diagnostic methods. Kaplan-Meier and Cox models were used to analyze progression-free survival (PFS) and comparison outcomes in PIK3CAm vs wild-type (wt). Results: We analyzed 225 samples from 161pts with HR+/HER2- BC (149 in tissue & 76 in plasma). PIK3CA mutations were detected in 62pts (38.5%), of which 39.6% (59pts) were detected in tissue and 11.8% (9pts) in plasma. Hotspot mutations: H1047X (45.7%), E545X (20%) and E542K (12.8%). In advanced disease, metastatic BC (mBC), tissue & plasma concordance was conducted in 64 cases, with overall correlation rate of 70.3%. We found PIK3CAm in 28pts: by both methods in 9pts (32.1%) and exclusively tissue detection in 19 cases (67.8%). Plasma detection was correlated with the presence of ≥3 metastatic sites (66.7% vs 31.6%; p=0.08) and with collection of samples during disease progression (66.7% vs 47.4%; p=0.43). 80pts received treatment with CDK4/6 inhibitors + endocrine therapy. PFS was slightly shorter in PIK3CAm vs wt (24m vs 30m; HR=1.39 [95%CI,0.7-2.4], p=0.26). A sub-analysis was performed based on exons 9 & 20, showing a significantly lower PFS in PIK3CAm exon 9 vs 20 population (9.7m vs 30.3m; HR=2.84 [95%CI,1.1-7.4], p=0.02). In addition, plasma detection of PIK3CAm was associated with worse PFS compared with PIK3CAm detected only in tissue (12.4m vs 29.3; HR=2.4 [95%CI,0.8-6.5], p=0.08). Although we observed a trend towards a poorer PFS in pts with visceral involvement in PIK3CAm (21.9m vs 30.1m; HR=2.82 [95%CI,0.6-12.3], p=0.14), in a multivariate analysis, mutations in exon 9 were an independent poor prognostic factor regardless visceral involvement and detection in plasma; p=0.05. Conclusions: Our results support the PIK3CA determination in tissue as the diagnostic method of choice, although further studies could better define the role of liquid biopsy in the detection of PIK3CAm. In our population, the presence of PIK3CAm confers poorer prognosis in luminal BC, being significantly worse in mutation carriers in exon 9 regardless visceral involvement and plasma mutation detection. Table. Tissue & plasma correlation in patients with PIK3CA mutational status tested in our population (mBC). Citation Format: Eduardo Terán Brage, Rebeca Lozano Mejorada, Aline Rodrigues Françoso, José Antonio Muñóz Leon, Álvaro López Gutiérrez, Luis Figuero-Pérez, Daniel Morchón Araujo, María Garijo Martínez, Jonnathan Roldán Ruiz, María Mar Abad Hernández, Magdalena Sancho de Salas, Emilio Fonseca Sánchez, César Augusto Rodríguez Sánchez. PIK3CA mutational status in tissue & plasma as a prognostic tool in HR+/HER2- breast cancer (BC) [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 PO4-13-12.

Abstract PO4-16-05: Identification of functional HLA-A*11:01-restricted driver gene PIK3CA mutation specific T-cell receptors

Abstract The adoptive transfer of genetically engineered T-cell receptors (TCR-T) has emerged as a promising therapeutic strategy for the treatment of solid tumors. Recent clinical trials have demonstrated the safety and efficacy of TCR-T cell therapies against certain types of metastatic solid cancers. In our study, we aimed to investigate the potential of TCR-T cell therapies targeting neoantigens, which are exclusively expressed in cancer cells, with a focus on metastatic breast cancer (MBC). Specifically, we identified four driver missense mutations (PIK3CA E542K, E545K, H1047L, and H1047R) frequently observed in MBC patients and utilized computational tools to predict the generation of neoantigens. Utilizing NetMHCpan V.4.1, we performed screening of peptides spanning the mutation region and predicted a neoantigen epitope, PIK3CA_H1047L, to bind with HLA-A*11:01. To validate the presentation and stability of the neoantigen epitope, we employed TAP1-deficient K562 cells and observed stable expression of the mutant peptide-major histocompatibility complex (MHC) complex on the cell membrane. Subsequently, we isolated T cells from healthy donors possessing the HLA-A11:01 genotype, and subjected them to successive stimulations with neopeptide-pulsed dendritic cells. T cells stimulated with neopeptide were examined for neoantigen specificity using an IFN-γ ELISpot assay. Employing pMHC tetramer staining and single-cell sorting via flow cytometry, we successfully isolated five HLA-A11:01-restricted PIK3CA_H1047L-specific TCRs. Through lentivirus transduction of these TCRs into T cells, we achieved the generation of TCR-T cells that exhibited potent and specific activity exclusively against the PIK3CA_H1047L neoantigen. The functionality of the TCR-T cells was confirmed through assessments of CD137 expression, IFN-γ secretion, and cytotoxicity assays. In conclusion, our study demonstrates T cell responses towards neoantigens derived from PIK3CA, a commonly observed driver mutation in MBC. Furthermore, we successfully isolated five distinct neoantigen-specific TCRs that specifically recognized the PIK3CA_H1047L mutation and were restricted to the prevalent HLA-A*11:01 allele. The transfer of these TCRs into T cells resulted in the generation of TCR-T products exhibiting remarkable activity against the PIK3CA_H1047L neoantigen. These findings strongly encourage further investigation into TCRs targeting driver mutations in MBC, with the aim of advancing neoantigen-targeted TCR-T therapies into clinical trials. Additionally, the PIK3CA_H1047L neoantigen holds great promise as a potential candidate for the development of effective cancer vaccines. Citation Format: Meiying Shen, Xiaojian Han, Siyin Chen, Aishun Jin, Shengchun Liu. Identification of functional HLA-A*11:01-restricted driver gene PIK3CA mutation specific T-cell receptors [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 PO4-16-05.

In Silico and In Vitro Study of Isoquercitrin against Kidney Cancer and Inflammation by Triggering Potential Gene Targets.

Kidney cancer has emerged as a major medical problem in recent times. Multiple compounds are used to treat kidney cancer by triggering cancer-causing gene targets. For instance, isoquercitrin (quercetin-3-O-β-d-glucopyranoside) is frequently present in fruits, vegetables, medicinal herbs, and foods and drinks made from plants. Our previous study predicted using protein-protein interaction (PPI) and molecular docking analysis that the isoquercitrin compound can control kidney cancer and inflammation by triggering potential gene targets of IGF1R, PIK3CA, IL6, and PTGS2. So, the present study is about further in silico and in vitro validation. We performed molecular dynamic (MD) simulation, gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, cytotoxicity assay, and RT-PCR and qRT-PCR validation. According to the MD simulation (250 ns), we found that IGF1R, PIK3CA, and PTGS2, except for IL6 gene targets, show stable binding energy with a stable complex with isoquercitrin. We also performed gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of the final targets to determine their regulatory functions and signaling pathways. Furthermore, we checked the cytotoxicity effect of isoquercitrin (IQ) and found that 5 μg/mL and 10 μg/mL doses showed higher cell viability in a normal kidney cell line (HEK 293) and also inversely showed an inhibition of cell growth at 35% and 45%, respectively, in the kidney cancer cell line (A498). Lastly, the RT-PCR and qRT-PCR findings showed a significant decrease in PTGS2, PIK3CA, and IGF1R gene expression, except for IL6 expression, following dose-dependent treatments with IQ. Thus, we can conclude that isoquercitrin inhibits the expression of PTGS2, PIK3CA, and IGF1R gene targets, which in turn controls kidney cancer and inflammation.

Prognostic and predictive biomarkers with therapeutic targets in nonsmall-cell lung cancer: A 2023 update on current development, evidence, and recommendation.

Since the publication of the original work in 2014, significant progress has been made in the characterization of genomic alterations that drive oncogenic addiction of nonsmall cell lung cancer (NSCLC) and how the immune system can leverage non-oncogenic pathways to modulate therapeutic outcomes. This update evaluates and validates the recent and emerging data for prognostic and predictive biomarkers with therapeutic targets in NSCLC. We performed a literature search from January 2015 to October 2023 using the keywords non-small cell lung cancer, clinical practice guidelines, gene mutations, genomic assay, immune cancer therapy, circulating tumor DNA, predictive and prognostic biomarkers, and targeted therapies. We identified, reviewed, and evaluated relevant clinical trials, meta-analyses, seminal articles, and published clinical practice guidelines in the English language. Regulatory-approved targeted therapies include those somatic gene alterations of EGFR ("classic" mutations, exon 20 insertion, and rare EGFR mutations), ALK, ROS1, BRAF V600, RET, MET, NTRK, HER2, and KRAS G12C. Data for immunotherapy and circulating tumor DNA in next-generation sequencing are considered emerging, whereas the predictive role for PIK3CA gene mutation is insufficient. Advances in sequencing and other genomic technologies have led to identifying novel oncogenic drivers, novel resistance mechanisms, and co-occurring mutations that characterize NSCLC, creating further therapeutic opportunities. The benefits associated with immunotherapy in the perioperative setting hold initial promise, with their long-term results awaiting.

Pathologic features of brain hemorrhage after radiation treatment: case series with somatic mutation analysis

BackgroundRadiation treatment for diseases of the brain can result in hemorrhagic adverse radiation effects. The underlying pathologic substrate of brain bleeding after irradiation has not been elucidated, nor potential associations with induced somatic mutations. MethodsWe retrospectively reviewed our department's pathology database over 5 years and identified 5 biopsy specimens (4 patients) for hemorrhagic lesions after brain irradiation. Tissues with active malignancy were excluded. Samples were characterized using H&E, Perl's Prussian Blue, and Masson's Trichrome; immunostaining for B-cells (anti-CD20), T-cells (anti-CD3), endothelium (anti-CD31), macrophages (anti-CD163), α-smooth muscle actin, and TUNEL. DNA analysis was done by two panels of next-generation sequencing for somatic mutations associated with known cerebrovascular anomalies. ResultsOne lesion involved hemorrhagic expansion among multifocal microbleeds that had developed after craniospinal irradiation for distant medulloblastoma treatment. Three bleeds arose in the bed of focally irradiated arteriovenous malformations (AVM) after confirmed obliteration. A fifth specimen involved the radiation field distinct from an irradiated AVM bed. From these, 2 patterns of hemorrhagic vascular pathology were identified: encapsulated hematomas and cavernous-like malformations. All lesions included telangiectasias with dysmorphic endothelium, consistent with primordial cavernous malformations with an associated inflammatory response. DNA analysis demonstrated genetic variants in PIK3CA and/or PTEN genes but excluded mutations in CCM genes. ConclusionsDespite pathologic heterogeneity, brain bleeding after irradiation is uniformly associated with primordial cavernous-like telangiectasias and disruption of genes implicated in dysangiogenesis but not genes implicated as causative of cerebral cavernous malformations. This may implicate a novel signaling axis as an area for future study.

Comprehensive Analysis of Clinically Relevant Copy Number Alterations (CNAs) Using a 523-Gene Next-Generation Sequencing Panel and NxClinical Software in Solid Tumors.

Copy number alterations (CNAs) are significant in tumor initiation and progression. Identifying these aberrations is crucial for targeted therapies and personalized cancer diagnostics. Next-generation sequencing (NGS) methods present advantages in scalability and cost-effectiveness, surpassing limitations associated with reference assemblies and probe capacities in traditional laboratory approaches. This retrospective study evaluated CNAs in 50 FFPE tumor samples (breast cancer, ovarian carcinoma, pancreatic cancer, melanoma, and prostate carcinoma) using Illumina's TruSight Oncology 500 (TSO500) and the Affymetrix Oncoscan Molecular Inversion Probe (OS-MIP) (ThermoFisher Scientific, Waltham, MA, USA). NGS analysis with the NxClinical 6.2 software demonstrated a high sensitivity and specificity (100%) for CNA detection, with a complete concordance rate as compared to the OS-MIP. All 54 known CNAs were identified by NGS, with gains being the most prevalent (63%). Notable CNAs were observed in MYC (18%), TP53 (12%), BRAF (8%), PIK3CA, EGFR, and FGFR1 (6%) genes. The diagnostic parameters exhibited high accuracy, including a positive predictive value, negative predictive value, and overall diagnostic accuracy. This study underscores NxClinical as a reliable software for identifying clinically relevant gene alterations using NGS TSO500, offering valuable insights for personalized cancer treatment strategies based on CNA analysis.

Abstract 1752: Comprehensive genomic profiling of advanced breast cancer subtypes: Insights from liquid biopsy analysis and implications for personalized therapies

Abstract Background: Breast cancer stands as the most prevalent malignancy affecting women’s health. The disease consists of three subtypes, including hormone receptor (HR) positive, HER2-positive, and triple-negative breast cancer (TNBC). In recent years, the advent of targeted therapies such as PI3K inhibitors has significantly improved the prognosis of breast cancer patients. However, the detection of relevant molecular markers, such as PIK3CA mutations, relies on tissue samples, which imposes limitations on the clinical application of these therapies. Consequently, this study presents a comprehensive analysis of genomic profiling that compares breast cancer patients across distinct pathological classifications, utilizing a liquid biopsy approach. Methods: In this retrospective study, 214 patients with advanced breast cancer were recruited. Plasma samples collected prior to first line treatment were analyzed using PredicineCARE, a targeted next-generation sequencing (NGS) liquid biopsy assay, to detect somatic alterations in ctDNA of blood, including single nucleotide variations (SNVs), gene fusions, and copy number variations (CNVs). Results: Based on the IHC classifications of tumor tissue, this cohort comprised 119 HR-positive, 61 HER2-positive, and 34 TNBC patients. The assay identified a total of 1456 mutations and 2357 gene copy number variants in plasma samples. Among these, the most frequently mutated genes and those with copy number variations (top 5) in HR-positive patients were TP53 (40%), PIK3CA (39%), ATM (22%), ERBB2 (19%), and FGFR1 (19%). In HER2-positive patients, they were TP53 (39%), PIK3CA (31%), ERBB2 (30%), NTRK1 (20%), and RAD50 (20%). For TNBC patients, the top genes were TP53 (59%), ERBB2 (44%), PIK3CA (35%), NTRK1 (35%), and BRCA2 (26%). Regarding the PIK3CA gene, the most prevalent mutation site was H1047, occurring in 12.61% of HR-positive patients, 14.75% of HER2-positive patients, and 11.76% of TNBC patients. Similarly, both E542 (4.20% in HR-positive, 3.28% in HER2-positive, and 2.94% in TNBC patients) and E545 (3.36% in HR-positive, 3.28% in HER2-positive, and 2.94% in TNBC patients) showed equivalent prevalence across the three classifications. These findings suggest the potential efficacy of PI3K inhibitors in various pathological types of breast cancer. Conclusions: This study offers insights into the genomic landscape of advanced breast cancer subtypes through liquid biopsy. The observed prevalence of PIK3CA mutations supports the potential efficacy of PI3K inhibitors across these diverse types of breast cancer, laying the groundwork for personalized therapeutic strategies and biomarker identification for prognosis and targeted therapies. Citation Format: Xiaoran Liu, Yaxin Liu, Cancan Jia, Yue Zhang, Feng Xie, Haoran Tang, Shidong Jia, Huiping Li. Comprehensive genomic profiling of advanced breast cancer subtypes: Insights from liquid biopsy analysis and implications for personalized therapies [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 1752.

Abstract 6448: Prevalence and spectrum of PIK3CA mutations in Indian breast cancer patients

Abstract Introduction: Breast carcinomas are the most commonly encountered cancers in women globally. Due to advancements in cancer research the death rate in breast cancer has reduced by 41% since 1989; however, the downward trend has recently slowed emphasizing on new breast carcinomas treatment discovery. Breast carcinomas are molecularly categorized by the expression of specific hormone receptors, as well as the overexpression of human epidermal growth factor receptor 2 (HER2). Approximately 80% of breast cancers have been termed HER2-negative, but researchers now recognize that 55% of all breast cancers may actually contain low levels of HER2, levels that are still targetable by HER2-directed therapies such as Trastuzumab Deruxtecan. The phosphoinositide 3-kinase (PI3K) pathway is the most frequently altered pathway in HR+/HER2- breast carcinomas and is associated with tumor development, disease progression, and endocrine resistance. Methodology: The study entailed histological verification of breast cancer, and thence the extraction of DNA from formalin fixed, paraffin embedded (FFPE) tissue samples. A tumor content of 20% was the cut-off for performing the PIK3CA mutation analysis. The DNA was subjected to mutation analysis using a FDA-approved PIK3CA RGQ Real Time PCR based assay that has ability to detect 11 mutations in the PIK3CA gene encompasses exons 7, 9, and 20. Results: A total of 696 breast cancer patient samples (with more than 20% neoplastic tissue content) were screened for PIK3CA mutations. Of these, 275 (39.5%) (Male: Female ratio = 1:26.5; Age range = 30-80 years) harbored a mutation of the PIK3CA gene. Females were commonly affected; however, the males were also found to have PIK3CA mutation. The most common affected age group is from 41 to50 years. The positivity rate exon wise were as follows: Exon 7 (1.45%), Exon 9 (39.27%), and Exon 20 (44.27%). There was presence of co-mutations amongst different exons, which comprised of 4%. HR+/HER2- (50%) followed by HR-/Her2- (3.27%) and HR+/HER2+ (1.82%). The HER2- cases (Score 1 and Score 2) by IHC were considered as low positive cases (14%). Conclusion: This is one of the largest study to report frequency of PIK3CA mutations in the Indian population. PIK3CA mutations are commonly encountered in hormone receptor positive and HER2 Negative metastatic breast cancer and have shown promising clinical outcome leading to better prognosis. It provides insights into genotyping each breast cancer and exploits the usage of PI3K/AKT inhibitors together with other combination of drugs for effective target treatment strategies. Keywords: Breast carcinomas, PIK3CA, HR/HER2, Targeted Therapeutic Citation Format: Rahul Katara, Tina Bhardwaj, Monika Rawat, Deepak K. Sharma, Vipin Kumar, Shivani Sharma, Vaishali Dagar, Pawan Kumar, Sambit K. Mohanty. Prevalence and spectrum of PIK3CA mutations in Indian breast cancer patients [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 6448.

Abstract 6861: PIK3CA activating mutation promotes an immune suppressive microenvironment in squamous cell carcinoma

Abstract Head and neck squamous cell carcinoma (HNSCC) is a leader in cancer incidence worldwide. Recently, immunotherapies such as immune checkpoint blockade (ICB) have offered great promise in treating many solid tumors, including HNSCCs, that are refractory to chemotherapy and radiation. Challengingly, HNSCC patients often relapse following ICB treatment due to the lack of a thorough understanding of the mechanisms underlying cancer immune evasion. These disparities in patient outcomes strongly suggest the existence of genetic variations that underlie their distinct responses to ICB. Although the specific oncogenic mutations responsible for driving relapse in SCC patients remain unclear, it is imperative to grasp the mechanisms of cancer immune evasion in individuals with specific genetic profiles to enhance the precision of immunotherapy. Cancer immune evasion has emerged as a hallmark of cancer and is facilitated by a highly complex tumor microenvironment (TME). We aim to understand the genetic basis shaping the immune suppressive TME and hypothesize that oncogenic driver mutations play a dominant role in preventing the immune clearance of transformed cells by reprogramming the immune landscape in the tumors. To determine the critical genetic signatures enriched in SCC patients that can impact anti-tumor immunity, we analyzed TCGA data which revealed a strong negative correlation between PIK3CA level and CD8 T cell signatures. We have identified that activating mutations in the PIK3CA gene, found in 20% of HNSCCs, promote rapid tumor relapse after initial response to anti-PD-L1 and anti-CTLA-4 ICB treatments. Utilizing single-cell analysis, quantitative immune profiling, and multiplexed imaging, our lab showed that tumor-initiating cells (TICs) in SCCs can have an intricate dialogue with myeloid-derived suppressor cells (MDSCs) where TICs secrete factors to enhance MDSC recruitment and suppressive function on cytotoxic T cells. Additionally, our data supports that the tumor interstitial fluid (TIF) of PIK3CA mutant SCC tumors can enhance the suppressive activity of MDSCs on cytotoxic T cells. These results suggest that the acquisition of PIK3CA mutation may trigger TICs to release additional factors to further modify MDSC activities. A critical method by which MDSCs suppress T cells is through the production and respiratory burst of reactive oxygen species (ROS). RNA sequencing revealed that PIK3CA mutant SCCs have multiple mechanisms of increasing ROS in the TME and specifically in MDSCs, while simultaneously upregulating lipid metabolism genes, such as SCD1, to protect itself from MDSC-derived ROS. This study reveals a potential strategy for targeting PIK3CA mutant SCCs as a promising precision immunotherapy to enhance the efficacy of ICB treatments. Citation Format: Benjamin T. Nicholson, Weijie Guo, Yuxuan Miao. PIK3CA activating mutation promotes an immune suppressive microenvironment in squamous cell carcinoma [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 6861.

Abstract 5696: Exploring the association between miR-9-5p levels and the mTORC1/mTORC2 pathway in laryngeal cancer cell lines

Abstract Background: We previously demonstrated that differing levels of miR-9-5p could predict chemosensitivity to cisplatin, and that miR-9-5p is a regulator of potential chemotherapeutic target MAP1B. As the mTOR/PIK3CA pathway has been implicated in the pathogenesis of laryngeal squamous cell carcinoma (LSCC), we sought to determine an association between differing miR-9-5p levels, mTOR pathway mediators including MAP1B, and methylation. Methods: UM-SCC-98, UM-SCC-12, UM-SCC-11A, UM-SCC-10A (obtained from the University of Michigan) laryngeal cancer cell lines were used in this study. We previously showed UM-SCC-98 and UM-SCC-12 to have significantly lower miR-9-5p levels as compared to UM-SCC-11A and UM-SCC-10A (Northern blot analysis). The IC50 of an mTORC1 inhibitor (Rapamycin) and an mTORC1/C2 inhibitor (AZD-8055) were evaluated using the Cell Titer Blue Viability assay. Cells were treated with the de-methylating agent, 5-Azacytidine (1μM). Gene expression of mTOR pathway mediators- miR-9-5p, PTEN, EGFR, PIK3CA, and MAP1B- were determined in each cell line pre-and post-treatment via qPCR. Results: All cell lines were resistant to treatment with Rapamycin (IC50 > 60μM) with the UM-SCC-12 cell line yielding the highest IC50 (> 150μM). Conversely, decreased cell proliferation was detected with AZD-8055. This was significantly associated with their miR-9-5p levels– a higher AZD-8055 IC50 was associated with cell lines UM-SCC-98 (~40μM) and UM-SCC-12 (~23μM) compared to UM-SCC-10A (~16μM) and UM-SCC-11A (~8μM). Following demethylation treatment, miR-9-5p levels were significantly increased in UM-SCC-12 only (2-fold), and this corresponded to a significant decrease in MAP1B gene expression (2-fold) but not with EGFR, PIK3CA, and PTEN gene expression. Conclusion: In conclusion, our study suggests that miR-9-5p may regulate MAP1B gene expression via the mTORC2 pathway. In addition, methylation may play a role in the regulation of the miR-9-5p gene and its downstream targets in laryngeal cancer tumorigenesis. Future studies will involve exploring epigenetic mechanisms of miR-9-5p inhibition and defining the role of key mediators of the mTOR pathway in LSCC. Citation Format: Christina Gobin, Matthew Chang, Chayil C. Lattimore, Kristianna M. Fredenburg. Exploring the association between miR-9-5p levels and the mTORC1/mTORC2 pathway in laryngeal cancer cell lines [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 5696.

Abstract 2425: Pairwise analysis of plasma cell-free DNA before and after paclitaxel plus ramucirumab treatment in patients with metastatic gastric cancer

Abstract Background: Plasma cell-free DNA (cfDNA) analysis has emerged as an appealing alternative to detect somatic mutations. This study compared cfDNA at baseline (baseline-cfDNA) and progressive disease (PD-cfDNA) and tumor tissue DNA (ttDNA) in patients with metastatic gastric cancer who underwent palliative second-line paclitaxel+ramucirumab treatment. Methods: We conducted targeted sequencing of 106 cancer-related genes using germline DNA, baseline-cfDNA, and PD-cfDNA samples. The results were then compared with conventional cancer panel results using ttDNA. Results: Of 76 consecutively enrolled patients who received paclitaxel+ramucirumab treatment, 46 patients (27 males; median age 57.5 [range, 32-73] years) with access to all three samples were analyzed along with their ttDNA data. A total of 138, 145, and 80 mutations were detected in baseline-cfDNA, PD-cfDNA, and ttDNA respectively. Combined analysis of baseline-cfDNA and ttDNA revealed that TP53 (56.5%) was the most frequently mutated gene, followed by CDH1 (26.1%), KRAS (21.7%), and APC (13.5%). For the top four genes, the sensitivity and positive predictive value of baseline-cfDNA compared with ttDNA were 71.8% and 51.9%, respectively. Compared with ttDNA alone, 32 patients (70.0%) benefited from baseline-cfDNA in detecting novel mutations. When combined with baseline-cfDNA and PD-cfDNA, novel mutations were discovered in 34 patients (73.9%). Of note, PD-cfDNA analysis detected 9 novel pathogenic mutations in TP53, APC, PIK3CA, CDH1, RNF43, CTNNB1, and BRCA2 genes in 8 patients (17.4%) after treatment. In baseline-cfDNA, patients with a circulating ttDNA fraction concentration at 110-160 bp of >4.3777 ng/µL had significantly shorter progression-free survival (PFS) (median 3.5 vs. 5.3 months, P=0.016). In addition, maximal variant allele frequency (VAF) values of >0.1045 (median 3.4 vs. 5.2 months, P=0.022) and the sum of VAF values of >0.2071 (median 3.4 vs. 5.2 months, P=0.028) were significantly associated with shorter PFS. In patients with TP53 mutations, those with TP53 VAF values of >0.1014 significantly had worse PFS (median 4.6 vs. 6.4 months, P=0.022). Conclusions: Although cfDNA could not entirely take over the role of ttDNA, cfDNA analysis identified additional somatic mutations that were otherwise missed by ttDNA alone. Moreover, PD-cfDNA analysis detected novel pathogenic mutations that developed during treatment, implicating the clonal evolution of cancer. In addition, baseline cfDNA predicted PFS of patients receiving paclitaxel+ramucirumab therapy. Citation Format: Ji-Won Kim, Dong Soo Kyung, Won Yeong Ko, Hwang-Phill Kim, Sung-Hyun Hwang, Kui-Jin Kim, Ju Hyun Lee, Jeongmin Seo, Minsu Kang, Eun Hee Jung, Koung Jin Suh, Se Hyun Kim, Jin Won Kim, Yu Jung Kim, Jee Hyun Kim, Keun-Wook Lee. Pairwise analysis of plasma cell-free DNA before and after paclitaxel plus ramucirumab treatment in patients with metastatic gastric 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 2425.

Abstract 2449: Case report: DETECT-A participants with pre-malignant conditions

Abstract Background: Blood based tests for multi-cancer early detection (MCED) are being developed to facilitate the earlier detection of various cancer types. The Detecting cancers Earlier Through Elective mutation-based blood Collection and Testing (DETECT-A) study evaluated the CancerSEEK MCED test, an early version of the Exact Sciences MCED test in development, in 9,911 women, age 65-75, without previous history of cancer (Lennon et al., Science 2020). The degree to which MCED testing will facilitate detection of precancerous conditions or incidental findings is unclear. Here we focus on DETECT-A participants who had pre-cancerous conditions diagnosed during a diagnostic work-up for a positive CancerSEEK MCED test. Methods: In a post-hoc analysis, we report on the detection of precancerous conditions consequent to CancerSEEK MCED testing and follow-up. Electronic health records were reviewed for diagnostic procedures performed, details of premalignant and cancer diagnoses, and vital status. Results: In three individuals, mutations in PIK3CA, TP53, or KRAS genes led to a positive CancerSEEK MCED test result. The prescribed imaging protocol using 2-deoxy-2[fluorine-18] fluoro-D-glucose positron emission tomography-computed tomography (18-FDG PET-CT), revealed a 10.3 × 9.8 × 7.8(cm) ovarian mucinous cystadenoma, a 0.8 cm appendiceal mucinous neoplasm, and 3 colonic polyps displaying high-grade dysplasia. All three participants were diagnosed with clinically significant pre-cancerous lesions, subsequently underwent surgical treatment, and remain alive and cancer-free as of February 2023 (Table 1). Conclusion: The diagnostic evaluation of a positive MCED test may occasionally reveal clinically significant pre-cancerous conditions amenable to interventions. The frequency of such findings and their clinical impact warrants further study. Citation Format: Omair A. Choudhry, Angana Kharge, Seema P. Rego, Paul Z. Elias, Adam H. Buchanan, Anne Marie Lennon, Nickolas Papadopoulos, Frank Diehl, Tomasz M. Beer. Case report: DETECT-A participants with pre-malignant conditions [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 2449.

Abstract 1955: Novel long-acting covalent PI3Kα inhibitors boost potential action in cancer

Abstract Phosphoinositide 3-kinase (PI3K) is a key regulator of cell proliferation, survival, and metabolism. Hotspot mutations in the PIK3CA gene constitutively activate the catalytic subunit of the PI3Kα isoform and are found in 10-30% of solid tumors. The development of PI3K pathway inhibitors has been hampered by poor drug tolerance and release of negative feedback loops, leading to rapid reactivation of signaling in response to reversible inhibitors. To remedy these challenges, we developed highly selective covalent PI3Kα inhibitors that irreversibly bind to a non-conserved cysteine (C862) located 11 Å outside the ATP-binding site. Here we present the properties of an optimized drug-like covalent lead, dubbed compound 9, and describe the generation of a biotinylated probe developed to be used in conjunction with compound 9 to monitor PI3Kα target occupancy in relation to downstream PI3K signaling outputs. Biochemical investigations of compound 9 using TR-FRET assays showed high affinity binding (Ki), high reaction rates (kinact) for covalent bond formation with PI3Kα, and negligible off-target reactivity. Cellular NanoBRET assays revealed that compound 9 diffuses three times more rapidly into cells as compared to BYL719 (alpelisib), and leads to rapid and potent on-target engagement. Covalent bond formation to residue C862 of PI3Kα was further confirmed by X-ray crystallography. In PIK3CA mutant cancer cell lines, covalent binding of PI3Kα occurred at low nanomolar concentrations and led to persistent inhibition of Akt/PKB phosphorylation for more than 72 hours after drug removal. Notably, investigation of PI3Kα re-synthesis after labeling with compound 9 showed a negligible reappearance in cancer cell lines, which indicates that persistent suppression of PI3Kα signaling by compound 9 results in a much longer half-life for PI3Kα than previously reported. The irreversible inactivation of PI3Kα by compound 9 provides a marked gain in growth inhibition potency (10-600-fold) in PIK3CA mutant cancer cell lines in comparison to clinical reversible PI3Kα inhibitors and allows for intermittent dosing without compromising efficacy. These data collectively demonstrate that compound 9 enables precise and selective targeting of PI3Kα with high potency and long-lasting efficacy in cancer models. The new class of irreversible PI3Kα inhibitors have a unique pharmacology among PI3K inhibitors characterized by strong decoupling of drug exposure from efficacy which may provide opportunities to lower treatment burden in patients. Citation Format: Theodora A. Constantin, Lukas Bissegger, Erhan Keleş, Luka Raguž, Clara Orbegozo, Thorsten Schaefer, Isobel Barlow-Busch, John E. Burke, Chiara Borsari, Matthias P. Wymann. Novel long-acting covalent PI3Kα inhibitors boost potential action in 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 1955.

Abstract 4125: Exploring the efficacy of alpelisib and combined PI3Kα-S6K inhibition in LIN28B-mediated colorectal cancer metastasis

Abstract Introduction: Colorectal cancer (CRC) is the third most diagnosed cancer worldwide. LIN28B, an RNA-binding protein that regulates mRNA translation, has garnered significant interest in recent cancer research. Our lab has established that LIN28B is an oncogene and promotes CRC metastasis. However, the exact mechanisms through which LIN28B exerts these effects remain elusive. The phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway is altered in 50-70% of CRCs and is characterized by PIK3CA gene mutations in 20-30% of CRC cases. PIK3CA encodes the catalytic subunit p110α of the PI3Kα enzyme, which is critical for PI3K/AKT signaling. Activation of this pathway is associated with worse CRC prognosis. Among the components of this signaling cascade, the ribosomal protein S6 kinase (S6K) is a crucial downstream effector, playing a pivotal role in protein synthesis and cell growth. Despite the frequent occurrence of PIK3CA mutations in CRC, targeted therapies for PIK3CA-mutant CRC are lacking. Alpelisib, a PI3Kα-specific inhibitor approved by the FDA for a subset of PIK3CA-mutant breast cancers, remains largely unassessed in CRC. We hypothesize that LIN28B-mediated CRC metastasis is facilitated by the activation of the PI3K/AKT pathway, representing a potential target for Alpelisib or other related inhibitors. Results: Our data indicate that LIN28B expression in CRC cells promotes liver metastasis in mouse models of metastatic CRC (mCRC). Furthermore, LIN28B expression activates the PI3K/AKT pathway, as measured by an AKT phosphorylation array. Treatment of CRC cells with SC79, a pan-AKT activator, enhanced cell migration and invasiveness. Similarly, VillinCre;R26Pik3ca transgenic mice, expressing a constantly active mutant form of PI3Kα in the intestinal epithelium, demonstrated crypt hyperplasia and an amplified propensity for tumorigenesis, mirroring the effects of LIN28B expression in CRC. Importantly, Alpelisib effectively hinders LIN28B-driven CRC progression in cell lines, transgenic mice-derived organoids, and mCRC mouse models, as well as the growth rate of CRC patient-derived organoids (PDOs). The effects observed with Alpelisib treatment were replicated with inhibition of S6K using either LY25284702 or PF4708671. Moreover, a combination of low-dose Alpelisib with low-dose LY25284702 or PF4708671 synergistically restricted the growth of PDOs. Conclusion: Our data provide compelling evidence that the PI3K/AKT pathway regulates LIN28B-mediated CRC metastasis, and its inhibition by Alpelisib is feasible. This underscores the potential for precision medicine in CRC, offering targeted therapies to PIK3CA-mutant CRC patients who lack specific treatment options. Furthermore, our study highlights the clinical advantages of combined inhibition strategies for improved anti-metastatic outcomes, a field in dire need of interventions in mCRC. Citation Format: Alice E. Shin, Kensuke Sugiura, Secunda W. Kariuki, David A. Cohen, Christopher J. Lengner, Peter A. Sims, Joel T. Gabre, Anil K. Rustgi. Exploring the efficacy of alpelisib and combined PI3Kα-S6K inhibition in LIN28B-mediated colorectal cancer metastasis [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 4125.

Association of preoperative and postoperative circulating tumour DNA (ctDNA) with PIK3CA gene mutation with risk of recurrence in patients with non-metastatic breast cancer

BackgroundCirculating tumour DNA (ctDNA), contains tumour-specific gene mutation in blood circulation and could aid in postoperative risk stratification of non-metastatic breast cancer. In this study, we investigated the feasibility of detecting PIK3CA gene mutations in ctDNA in the preoperative (preop) and postoperative period (postop), and its prognostic significance in patients with breast cancer. MethodsA cohort of patients with breast cancer undergoing curative surgery with available blood samples preoperatively and postoperatively (Post op) at either Post op time period; week 1–2, week 3–4 or weeks 5–12 were enrolled. PIK3CA gene mutations at exons 9 and 20 were detected in ctDNA with High resolution melting (HRM) PCR and Allele specific fluorescence probe-based PCR. ResultsA total of 62 patients (age, median (IQR), 51.50 (45.0–65.0) years), with a median follow-up of 90 months (interquartile range (IQR),60–120 months) were enrolled. In total, 25 (40.3%) and 22 (35%) patients with breast cancer had detectable PIK3CA gene mutations in ctDNA in preoperative and postoperative period, respectively. PIK3CA gene mutations in ctDNA in postoperative period (hazard ratio (H.R: 18.05, p = 0.001) were a negative prognostic factor for recurrencefree survival (RFS) and overall survival (OS) (H.R: 11.9, p = 0.01) in patients with breast cancer. Subgroup analysis of ctDNA indicate that positive ctDNA in both preoperative/postoperative period and post op period only were found to have prognostic effect on RFS and OS (RFS; p < 0.0001, O·S; p = 0.0007). Moreover, ctDNA-based detection preceded clinical detection of recurrence in patients with an average lead time of 12 months (IQR:20–28.5 months) across all the breast cancer subtypes. ConclusionWe highlighted the prognostic ability of ctDNA in patients with breast cancer in perioperative period. However, future prospective studies are needed to assess the utility of ctDNA in clinical practice

Cold Tumour Phenotype Explained Through Whole Genome Sequencing in Clinical Nasopharyngeal Cancer: A Preliminary Study.

Nasopharyngeal cancer (NPC) is a complex cancer due to its unique genomic features and association with the Epstein-Barr virus (EBV). Despite therapeutic advancements, NPC prognosis remains poor, necessitating a deeper understanding of its genomics. Here, we present a comprehensive whole genome sequencing (WGS) view of NPC genomics and its correlation with the phenotype. This study involved WGS of a clinical NPC biopsy specimen. Sequencing was carried out using a long read sequencer from Oxford Nanopore. Analysis of the variants involved correlation with the phenotype of NPC. A loss of genes within chromosome 6 from copy number variation (CNV) was found. The lost genes included HLA-A, HLA-B, and HLA-C, which work in the antigen presentation process. This loss of the major histocompatibility complex (MHC) apparatus resulted in the tumour's ability to evade immune recognition. The tumour exhibited an immunologically "cold" phenotype, with mild tumour-infiltrating lymphocytes, supporting the possible etiology of loss of antigen presentation capability. Furthermore, the driver mutation PIK3CA gene was identified along with various other gene variants affecting numerous signaling pathways. Comprehensive WGS was able to detect various mutations and genomic losses, which could explain tumour progression and immune evasion ability. Furthermore, the study identified the loss of other genes related to cancer and immune pathways, emphasizing the complexity of NPC genomics. In conclusion, this study underscores the significance of MHC class I gene loss and its probable correlation with the cold tumour phenotype observed in NPC.

Combined PIK3CA and SOX2 Gene Amplification Predicts Laryngeal Cancer Risk beyond Histopathological Grading.

The PIK3CA and SOX2 genes map at 3q26, a chromosomal region frequently amplified in head and neck cancers, which is associated with poor prognosis. This study explores the clinical significance of PIK3CA and SOX2 gene amplification in early tumorigenesis. Gene copy number was analyzed by real-time PCR in 62 laryngeal precancerous lesions and correlated with histopathological grading and laryngeal cancer risk. Amplification of the SOX2 and PIK3CA genes was frequently detected in 19 (31%) and 32 (52%) laryngeal dysplasias, respectively, and co-amplification in 18 (29%) cases. The PIK3CA and SOX2 amplifications were predominant in high-grade dysplasias and significantly associated with laryngeal cancer risk beyond histological criteria. Multivariable Cox analysis further revealed PIK3CA gene amplification as an independent predictor of laryngeal cancer development. Interestingly, combined PIK3CA and SOX2 amplification allowed us to distinguish three cancer risk subgroups, and PIK3CA and SOX2 co-amplification was found the strongest predictor by ROC analysis. Our data demonstrate the clinical relevance of PIK3CA and SOX2 amplification in early laryngeal tumorigenesis. Remarkably, PIK3CA amplification was found to be an independent cancer predictor. Furthermore, combined PIK3CA and SOX2 amplification is emerging as a valuable and easy-to-implement tool for cancer risk assessment in patients with laryngeal precancerous lesions beyond current WHO histological grading.

Comparison of RNA-Seq and microarray in the prediction of protein expression and survival prediction.

Gene expression profiling using RNA-sequencing (RNA-seq) and microarray technologies is widely used in cancer research to identify biomarkers for clinical endpoint prediction. We compared the performance of these two methods in predicting protein expression and clinical endpoints using The Cancer Genome Atlas (TCGA) datasets of lung cancer, colorectal cancer, renal cancer, breast cancer, endometrial cancer, and ovarian cancer. We calculated the correlation coefficients between gene expression measured by RNA-seq or microarray and protein expression measured by reverse phase protein array (RPPA). In addition, after selecting the top 103 survival-related genes, we compared the random forest survival prediction model performance across test platforms and cancer types. Both RNA-seq and microarray data were retrieved from TCGA dataset. Most genes showed similar correlation coefficients between RNA-seq and microarray, but 16 genes exhibited significant differences between the two methods. The BAX gene was recurrently found in colorectal cancer, renal cancer, and ovarian cancer, and the PIK3CA gene belonged to renal cancer and breast cancer. Furthermore, the survival prediction model using microarray was better than the RNA-seq model in colorectal cancer, renal cancer, and lung cancer, but the RNA-seq model was better in ovarian and endometrial cancer. Our results showed good correlation between mRNA levels and protein measured by RPPA. While RNA-seq and microarray performance were similar, some genes showed differences, and further clinical significance should be evaluated. Additionally, our survival prediction model results were controversial.

Real-World Data and Clinical Implications of Next-Generation Sequencing (NGS)-Based Analysis in Metastatic Breast Cancer Patients.

Over the last two decades, the use of Next-Generation Sequencing (NGS) in medical oncology has increased the likelihood of identifying druggable mutations that may be potentially susceptible to targeted treatments. The European Society for Medical Oncology (ESMO) currently does not recommend the use of the NGS test to determine the therapeutic course of patients with metastatic breast cancer (mBC) in daily clinical practice. However, the aim of this work is to evaluate the potential contribution of the NGS test in selecting targeted therapies for patients with mBC. Data were retrospectively collected from 101 patients diagnosed with metastatic breast cancer and treated at the Modena Cancer Center between January 2015 and April 2022. A NGS test was performed on the tumor tissue of each patient at the Laboratory of Molecular Pathology of the University Hospital of Modena. This study analyzed the clinical-pathological characteristics and mutational profile of the population using NGS tests, with a focus on actionable mutations that could be targeted in advanced stages of clinical development. The indicator of this study was to quantify the actionable mutations that resulted in a change of cancer treatment. In total, 101 patients with metastatic breast cancer were analyzed, including 86 with luminal phenotype, 10 who were HER2-positive and 5 who were triple-negative. Median age was 52 years. NGS analysis was conducted on 47 samples of primary breast cancer, 52 on metastatic sites of disease and 2 on liquid biopsies. A total of 85 gene mutations were found. The most common mutations were identified in the PIK3CA (47%), FGFR (19%) and ERBB2 genes (12%), and to a lesser extent in other genes. Of the 61 patients with pathogenic mutations, 46 (75%) had at least one actionable mutation. Of these, nine received treatment with a molecular target drug: eight patients with a mutation of the PIK3CA gene were treated with alpelisib and fulvestrant; one patient with FGFR1/2 amplifications received TAS120. Median PFS for these patients was 3.8 months. The study results show that using the NGS test on cancer tissue of metastatic breast cancer could influence the therapeutic choices, considering the small sample size and limited follow-up. About 9% of the study population had their therapy modified based on the results of NGS. The growing number of detectable mutations and increased accessibility of the test may lead to a greater number of potential therapeutic implications for the NGS assay. Perspectives suggest that NGS analysis can be implemented in daily clinical practice, particularly in contexts where a Molecular Tumor Board (MTB) is active.