Mutations In Breast Cancer Patients Research Articles

Benign and conserved DNA variants m.8860A>G and m.8701A>G indicating mitochondrial genetic drift in Pakistani population

BackgroundMitochondria are vital subcellular organelles that orchestrate the intricate process of oxidative phosphorylation (OXPHOS), generating adenosine triphosphate (ATP) as the primary energy molecule fueling cellular activities. During our research on mitochondrial mutations in breast cancer patients, we identified two notable single nucleotide polymorphisms (SNPs) present in both cancer patients and control individuals from the Pakistani population. Materials and methodsDNA was extracted from the blood samples of 30 individuals, and MT-ATP8 and MT-ATP6 were amplified using PCR with specific primers. Purified PCR products were sequenced and analyzed for mutations using SnapGene and BioEdit. Bioinformatics tools, Consurf and PolyPhen-2, were used to analyze the genetic variants and their impact on protein function and stability. ResultsThe analysis revealed two significant mutations in MT-ATP6 gene i.e., m.8860A>G (found in all 30 out of 30 samples) which results in the variant p.(Thr112Ala) and m.8701A>G (found in 13 out of 30 samples) which results in the variant p.(Thr59Ala). PolyPhen-2 analysis reveals the benign nature of both mutations, suggesting that the sequence variants are unlikely to cause any adverse effects on protein structure and function.

Analysis of Genetic Alterations in TP53 Gene in Breast Cancer – A Secondary Publication

Tumor protein p53 (TP53) mediates DNA repair and cell proliferation in growing cells. The TP53 gene is a tumor suppressor that regulates the expression of target genes in response to multiple cellular stress factors. Key target genes are involved in crucial cellular events such as DNA repair, cell cycle regulation, apoptosis, metabolism, and senescence. TP53 genetic variants and the activity of the wild-type p53 protein (WT-p53) have been linked to a wide range of tumorigenesis. Various genetic and epigenetic alterations, including germline and somatic mutations, loss of heterozygosity, and DNA methylation, can alter TP53 activity, potentially resulting in cancer initiation and progression. This study was designed to screen three reported mutations in the DNA-binding domain of the p53 protein in breast cancer, to evaluate the relative susceptibility and risk associated with breast cancer in the local population. Genomic DNA was isolated from 30 breast tumor tissues along with controls. Tetra and Tri ARMS PCR were performed to detect mutations in the TP53 coding region. For SNPs c.637C>T and c.733C>T, all analyzed cases were homozygous for the wild-type allele ‘C,’ while for SNP c.745A>G, all cases were homozygous for the wild-type allele ‘A.’ These results indicate no relevance of these three SNPs to cancer progression in our study cohort. Additionally, the findings from whole exon sequencing will help to predict more precise outcomes and assess the importance of TP53 gene mutations in breast cancer patients.

The Prevalence of BRCA1 and BRCA2 Mutations in Breast Cancer Patients

Aim: Genetic mutations in BRCA1 and BRCA2 are crucial for breast cancer risk assessment and treatment planning. Methods: This prospective observational study at Jinnah Postgraduate Medical Centre, Pakistan, involved female breast cancer patients aged 18 or older. The sample size calculation was based on a 3.4% prevalence rate of BRCA mutations, aiming for a 95% confidence level. Data on demographic, clinical characteristics, and genetic testing for BRCA mutations were collected and analyzed using SPSS version 23. Results: Among the study participants, 10% exhibited BRCA1 or BRCA2 mutations. The majority were diagnosed at stage 3 tumor development, with invasive ductal carcinoma being the predominant histological type. No significant familial predisposition to breast cancer was noted among the majority. Educational status and ethnicity showed varying associations with BRCA mutation presence. Conclusion: The study highlights a modest incidence of BRCA mutations among Pakistani breast cancer patients, underscoring the importance of genetic testing for risk assessment and targeted treatment. The findings support the need for comprehensive genetic screening programs in Pakistan, considering the diverse demographic and clinical characteristics of the population. Keywords: BRCA mutations, breast cancer, genetic testing, Pakistan, risk assessment, targeted treatment.

Abstract PO3-14-11: Association between BRCA alterations detected by circulating tumor DNA and germline mutations in breast cancer patients: a retrospective mono-institutional analysis

Abstract Background: PARP inhibitors (PARPi) are a standard of care for breast cancer (BC) patients (pts) with germline BRCA (gBRCA) mutation, with benefit demonstrated also in patients with somatic (s) BRCA1/2 mutations. The use of circulating tumor (ct) DNA testing allows to detect resistant and actionable alterations in BC, but it could be associated with the incidental identification of germinal mutations. The aim of this study was to describe the incidence of both, somatic and germinal BRCA1/2 alterations, and the impact on the therapeutic outcomes in a retrospective BC cohort with clinical ctDNA testing for their BC. Materials and methods: A retrospective cohort of 245 BC pts that underwent ctDNA analysis (Guardant 360) between August 2014 and May 2023 at Weill Cornell Medical College was identified. Pts with at least a BRCA1/2 alteration, including variant of uncertain significant (VUS) and synonymous, were enrolled in the study. A descriptive analysis was performed. Results: Among 35 patients included, 34 had metastatic disease, the median age was 61 years old (IQR 40-67), and 65.7% were post-menopausal at diagnosis. 57.1% of pts had hormone receptor positive/HER2 negative (HR+/HER2-) BC, 20% had HER2 positive disease and 22.9% were triple-negative (TN) BC. Ten (28.6%) and 20 (57.1%) pts had a sBRCA1 or a sBRCA2 alteration on ctDNA, respectively; 5 pts (14.3%) had a coexisting sBRCA1/2 mutation. The median variant allele frequency (VAF) was 1.2% for BRCA1 and 2.7% for BRCA2 alterations. Furthermore, 53.3% and 6.7% of sBRCA1 and 40% and 8% of sBRCA2 mutations were identified as VUS and synonymous, respectively. The most common associated genomic alterations included TP53 (62.9%), ESR1 (20%) and PI3KCA (52%) with a median number of 4 variants (IQR 2-7). Next generation sequencing (NGS) testing on tissue was available for 3 patients: two demonstrated concordance with BRCA alterations detected by ctDNA, whereas no alteration in BRCA1/2 genes was found for the third patient despite the tissue analysis was performed at the same time of the ctDNA. Germline testing was available for 24 (69%) pts; a corresponding gBRCA1 mutation was found for 3 pts (VAF 48.6%-84.3%), and a gBRCA2 for 8 pts (VAF 26.7%-49.5%). Due to the small sample size, a univocal VAF cut-off to detect gBRCA1/2 mutations could not be calculated. Twenty-nine pts had a sBRCA1/2 mutation detected by ctDNA test before starting a new therapy; of these 55.2% had HR+/HER2- disease, while 17.2% and 27.6% were HER2 positive and TN BC, respectively. The median progression free survival (mPFS) was 10.3 (3.2-17.4) months (ms) and the 1-year and 2-year overall survival (OS) rates were 82.3% and 70.5%, respectively. The mPFS was 10.3 ms (0-25.0) for patients (n=16) that underwent chemotherapy, 5.6 ms (0-15.1) for those who had received endocrine therapy (n=9), and not reached for patients treated with PARPi (n=4). Conclusions: ctDNA analysis allows the detection of sBRCA1/2 mutations that could be missed by tissue-based testing. The identification of BRCA alterations on ctDNA could guide the use of germline testing even when these are present at a lower VAF than expected, with relevant impact on the therapeutic choices and family screening. Further evaluation to establish the impact of sBRCA1/2 detected by ctDNA on the therapeutic algorithm decision are needed. Citation Format: Letizia Pontolillo, Eleonora Nicolò, Laura Munoz Arcos, Carolina Reduzzi, Mara Serena Serafini, Amanda Kaylan Strickland, Nadia Bayou, Jeannine Donahue, Elisabetta Molteni, Lorenzo Gerratana, Diana Giannarelli, Eleni Andreopoulou, Emilio Bria, Massimo Cristofanilli. Association between BRCA alterations detected by circulating tumor DNA and germline mutations in breast cancer patients: a retrospective mono-institutional analysis [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 PO3-14-11.

Abstract PO3-15-10: Enhanced Cancer Cell Proliferation and Aggressive Phenotype Counterbalance in Breast Cancer with High BRCA1 Gene Expression

Abstract Introduction: It is estimated that 5–10% of all breast cancer cases have a hereditary component with BRCA1 mutation being one of the most frequently observed gene mutations in breast cancer patients. BRCA1 plays a crucial role in DNA repair, and its mutation has been extensively studied. However, the clinical significance of BRCA1 gene expression remains largely unexplored. Given that heightened DNA repair mechanisms can potentially increase cancer cell proliferation, we hypothesized that breast cancer with high BRCA1 gene expression might be associated with aggressive tumor biology and worse survival. Methods: The clinical, pathological and gene expression levels of a total of 6,245 breast cancer patients were analyzed from three large independent cohorts: METABRIC (n = 1,903), GSE96058 (n = 3,273), and The Cancer Genome Atlas (TCGA, n = 1,069). The data was retrieved through cBioPortal. The xCell algorithm was used to correlate the BRCA1 gene expression with the stromal and immune cell fractions infiltrating the tumor microenvironment (TME). Results: BRCA1 gene expression was higher in breast cancer patients without BRCA1 mutation, and it correlated with the DNA repair activity (p = 0.026 and p < 0.01 respectively), however, it did not correlate with BRCA2 gene expression (p = 0.38). Breast cancer with high BRCA1 gene expression was associated with cancer cell proliferation as evidenced by higher Nottingham histological grade (p < 0.001), Ki67 expression (p < 0.001), significant enrichment of cell proliferation-related gene sets such as E2F Targets, G2M Checkpoint, MYC Targets V1 and V2, Mitotic Spindle (all FDR < 0.25), and significantly less infiltration of stromal cells such as adipocytes, fibroblasts, microvascular and lymphatic endothelial cells, and pericytes (all p < 0.05). High BRCA1 gene expression was associated with higher homologous recombination deficiency, intratumor genomic heterogeneity, and fraction altered (all p < 0.001); however, there was no correlation observed with mutation rates or neoantigens. It was observed that breast cancer with high BRCA1 gene expression had less infiltration of CD8 T cells, dendritic cells, regulatory T cells, and B cells and had higher infiltration of Th1 cells (all p < 0.05). To our surprise, there was no significant difference in overall survival regardless of the subtypes (except for ER+/HER2- subtype in METABRIC cohort, p = 0.03). Breast cancer with low BRCA1 gene expression enriched cancer aggravating pathway gene sets such as Cancer Stem Cell-related signaling pathways (NOTCH and HEDGEHOG), Angiogenesis, Epithelial Mesenchymal Transition, Inflammatory Response, and TGF-beta signaling (all FDR < 0.25). Conclusion: Contrary to our initial hypothesis, we did not find any significant association between BRCA1 gene expression and survival outcomes in breast cancer patients. Based on the results of our study, we speculate that the interplay between enhanced cancer cell proliferation and aggressive tumor characteristics may counterbalance the effect of high BRCA1 expression in breast cancer. Citation Format: Kohei Chida, Masanori Oshi, Arya Mariam Roy, Itaru Endo, Kazuaki Takabe. Enhanced Cancer Cell Proliferation and Aggressive Phenotype Counterbalance in Breast Cancer with High BRCA1 Gene Expression [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 PO3-15-10.

TP53-specific mutations serve as a potential biomarker for homologous recombination deficiency in breast cancer: a clinical next-generation sequencing study.

TP53 mutations and homologous recombination deficiency (HRD) occur frequently in breast cancer. However, the characteristics of TP53 pathogenic mutations in breast cancer patients with/without HRD are not clear. Clinical next-generation sequencing (NGS) of both tumor and paired blood DNA from 119 breast cancer patients (BRCA-119 cohort) was performed with a 520-gene panel. Mutations, tumor mutation burden (TMB), and genomic HRD scores were assessed from NGS data. NGS data from 47 breast cancer patients in the HRD test cohort were analyzed for further verification. All TP53 pathogenic mutations in patients had somatic origin, which was associated with the protein expression of estrogen receptor and progestogen receptor. Compared to patients without TP53 pathologic mutations, patients with TP53 pathologic mutations had higher levels of HRD scores and different genomic alterations. The frequency of TP53 pathologic mutation was higher in the HRD-high group (HRD score≥42) relative to that in the HRD-low group (HRD score<42). TP53 has different mutational characteristics between the HRD-low and HRD-high groups. TP53-specific mutation subgroups had diverse genomic features and TMB. Notably, TP53 pathogenic mutations predicted the HRD status of breast cancer patients with an area under the curve (AUC) of 0.61. TP53-specific mutations, namely HRD-low mutation, HRD-high mutation, and HRD common mutation, predicted the HRD status of breast cancer patients with AUC values of 0.32, 0.72, and 0.58, respectively. Interestingly, TP53 HRD-high mutation and HRD common mutation combinations showed the highest AUC values (0.80) in predicting HRD status. TP53-specific mutation combinations predict the HRD status of patients, indicating that TP53 pathogenic mutations could serve as a potential biomarker for poly-ADP-ribose polymerase (PARP) inhibitors in breast cancer patients .

Abstract A022: Assessment of clonal and subclonal PIK3CA mutations in patients with breast cancer

Abstract Background: PIK3CA missense mutations are commonly present in HR+HER2- breast cancer (BC) tumors, and these mutations vary in frequency. When common, mutations arise early in tumor progression and are deemed to be clonal events. In contrast, rarer subclonal mutations are present in a small subset of tumor cells. BC tumors with two or more PIK3CA mutations appear to respond better to PI3K inhibitors, though PI3K pathway activation may also confer resistance to HER2-targeted therapy in HER2+ BC patients. This study investigates the clonality of PIK3CA double mutations in BC patients to better understand tumor heterogeneity for this important therapy target. Methods: BC samples were analyzed using the OncoExTraTM assay, which identifies somatic mutations and gene fusions through whole-exome, whole-transcriptome sequencing with tumor-normal pairing. PIK3CA clonality was determined by calculating mutation frequency divided by tumor purity for samples with ≥20% tumor purity. Mutations with an adjusted mutation frequency ≥0.25 were classified as clonal, and those &amp;lt;0.25 as subclonal. We classified double PIK3CA mutations into three subgroups: both clonal (CC), both subclonal (SS) or one clonal and one subclonal (CS), and used Fisher’s Exact Test (p≤0.05) to test associations with other biomarkers. Results: Among 2161 BC samples, 739 (34.2%) had a single PIK3CA mutation and 87 (4.0%) had two or more PIK3CA mutations, of which 81 had tumor purity &amp;gt;20%. Among these 81 patient samples, 32 (39.5%) were CC, 20 (24.7%) were SS, and 29 (35.8.%) were CS. Most (72, 88.8%) samples were from primary tumors. The frequency of multiple PIK3CA mutations was 5.4% in HR+HER2- BC, 2.0% in HER2+ BC, and 0.3% in TNBC. The most common double PIK3CA mutations were in exon 20 and exon 9 (n=25, 30.9%), and in exon 20 and exon 1 (n=11, 13.6%). Examination of other biomarkers revealed that none were significantly associated with any of the three subgroups. We also investigated co-occurring alterations in the PI3K/PTEN/AKT/mTOR pathway. Among the 81 patient samples, three had a PTEN and one had an AKT1 mutation. Two of the three PTEN and the single AKT1 mutations were subclonal. Conclusion: Two or more PIK3CA mutations were present in 4.0% of BC patient samples, and in 39.5% of these both mutations were clonal, suggesting these tumors may be particularly sensitive to PI3K inhibitors. In addition, in this cohort 2% of HER2+ BC samples carried two or more PIK3CA mutations, suggesting strong PI3K pathway activation, and possible concomitant resistance to HER2-targeted treatments. Citation Format: Sameer S Udhane, Fadel Alyaqoub, Pawan Noel, Ariane Kemkes, Cynthia A Flannery, Paige E Innis, David W Hall, Snehal G Thakkar, Gargi Basu, Joyce O'Shaughnessy. Assessment of clonal and subclonal PIK3CA mutations in patients with breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A022.

Increased RUNX1 mutations in breast cancer disease progression

Despite advances in screening, therapy and surveillance, breast cancer remains threatening to women. Worst, patients suffer from side effects of treatments and cancer cells become resistant. The emergence of RUNX1 in breast cancer has put it in a spotlight due to its roles in the disease progression. It also plays important roles in normal mammary glands such as for cell growth, proliferation, migration and stemness. However, mutations in the RUNX1 gene have changed the regulation of these phenotypes and the full spectrum of its implications in breast cancer patients is unknown. In this study therefore, the pattern of RUNX1 mutations in breast cancer patients was examined to understand its fundamental impacts on the disease. The perturbation of RUNX1 and its mutations in breast cancer was elucidated through different studies reported in cBioPortal in the past ten years. From our analyses, the majority of RUNX1 mutations were found in the primary breast cancer, with women constituted most of the mutations, especially on the left side of the breast. Similarly, increased number of mutations was observed in ER-positive breast cancer patients and this was also the case at the early stage of the disease development. The level of RUNX1 mutations also increased gradually as patients got older and the peak was highest in the patients of 60–70 years old. Altogether, these data indicated that the mutated RUNX1 gene contributed to the progression of breast cancer and understanding of its regulatory mechanisms is crucial to therapeutically target this gene in the future.

A novel PIK3CA hot-spot mutation in breast cancer patients detected by HRM-COLD-PCR analysis.

The PI3K protein is involved in the PI3K/AKT/mTOR pathway. Deregulation of this pathway through PIK3CA mutation is common in various tumors. The aim of this work is to identify hotspot mutation at exons 9 and 20 in Tunisian patients with sporadic or hereditary breast cancer. Hotspot mutations in exon 9 and exon 20 of the PIK3CA gene were identified by QPCR-High Resolution Melting followed by COLD-PCR and sequencing in 63 (42 sporadic cases and 21 hereditary cases) tumor tissues collected from Tunisian patient with breast cancer. MCF7, and BT20 breast cancer cell lines harboring the PIK3CA hotspot mutations E545K and H1047R in exon 9 and exon 20 respectively, were used as controls in HRM experiments. PIK3CA hotspot mutations were detected in 66.7% (28 out of 42) of sporadic BC cases, and in 14.3% (3 out of 21) of hereditary BC. The E545K and the H1048Y were the most prevalent mutations identified in patients with sporadic and hereditary BC, whereas the H1047R hotspot mutation was not found in our patients. Statistical analysis showed that PIK3CA mutation associated with an aggressive behavior in patients with sporadic BC, while it's correlated with age, tumor stage and tumor size in the group patients with hereditary breast cancer. Our results showed a novel PIK3CA hotspot mutation in Tunisian breast cancer patients detected by HRM-COLD-PCR. Moreover, the absence of PIK3CA hotspot mutation associated with good prognosis.

BRCA mutation status and pathological characterization of breast cancer in Zhoushan Islands, China.

To investigate BRCA1/2 gene mutations and their relationship with clinicopathological features in patients with breast cancer in Zhoushan Islands. High-throughput whole-exome gene sequencing was used to detect BRCA1/2 mutations in 776 breast cancer patients in Zhoushan Islands. The BRCA1/2 mutation rate of breast cancer patients in Zhoushan Islands was 4.38% (34/776). BRCA1 mutations were significantly correlated with age, molecular type, and family history of breast and ovarian cancers. BRCA2 mutations were most commonly found in invasive lobular carcinoma. Moreover, the BRCA2 mutation rate of cancers with molecular type luminal B (receptor protein-tyrosine kinase [HER2]-negative) was also relatively high. The rate of BRCA1/2 mutations in breast cancer patients from Zhoushan Islands is approximately 4.38%, and BRCA1 mutation is related to age, molecular type, and family history of breast and ovarian cancers.

Detection of TP53 gene mutation in blood of breast cancer patients based on circulating tumor DNA and its application in prognosis.

This experiment was carried out to explore the application value of high throughput gene sequencing technology in detecting TP53 gene mutations in the blood of patients with breast cancer by detecting ctDNA gene mutations, and exploring the relationship between TP53 mutations and clinicopathological characteristics and prognosis of patients. The gene mutation of peripheral blood ctDNA and tissue paraffin DNA (tDNA) of 50 patients was detected by high-throughput sequencing technology. The basic data of 50 cases of Medium to high-risk breast cancer diagnosed and were retrospectively collected, and the clinicopathological characteristics and survival results of TP53 mutant and wild-type patients were compared and analyzed according to the ctDNA detection results and relevant follow-up data. Analyze the impact of TP53 mutations on overall survival and progression-free survival using univariate and multivariate Cox regression models. Among 50 patients, there were 29 cases of 7 kinds of gene mutations detected by ctDNA, and 37 cases of 9 kinds of gene mutations detected by tDNA. Using the gene mutation results detected by tDNA as the gold standard, the sensitivity and specificity of peripheral blood ctDNA in diagnosing TP53 gene mutations are 75% to 100%, 92.31% to 100%, and the overall coincidence rate with tDNA results was 83.33% to 100%. Exon 5 was the most prone to mutation, with a frequency of 24.14% (7/29). The most common type of mutation was the missense mutation of 37.93% (11/29). There was no significant correlation between TP53 mutation and PFS (HR=0.67, 95% CI: 0.41-1.08, P=0.102), while TP53 mutation was a protective factor for OS (HR=0.49, 95% CI: 0.27-0.90, P=0.022). The detection of ctDNA in peripheral blood of breast cancer patients by high-throughput gene sequencing technology can replace tumor tissue sections to understand gene mutation. The TP53 mutation in breast cancer patients is related to tumor size, lymph node metastasis and vascular tumor thrombus, but the prognosis of TP53 mutant patients is similar to that of wild-type patients.

Next-Generation Sequencing-Based Analysis of Clinical and Pathological Features of PIK3CA-Mutated Breast Cancer.

Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) is a well-known oncogene with a high prevalence of mutation in breast cancer patients. The effect of the mutation is a deregulation in phosphatidylinositol 3-kinase-related pathways, and, consequently, in unrestricted cell growth and differentiation. With the advent of precision oncology, PIK3CA has emerged as a pivotal treatment target, culminating in the recent approval of alpelisib. Despite years of research on this genetic alteration, certain aspects of its influence on the prognosis of breast cancer remain ambiguous. The purpose of this analysis is to characterize the clinical picture of breast cancer patients with PIK3CA mutation in comparison to the PIK3CA-wild-type group. We examined 103 tumor samples from 100 breast cancer patients using a next-generation sequencing panel. Presence of the mutation was linked to an older age at diagnosis, a lower expression of Ki67 protein, a greater percentage of tumors expressing progesterone receptors, and a notably higher incidence of metastatic disease at presentation. No significant differences were identified in overall and progression-free survival between the two groups. Our findings enhance the understanding of how PIK3CA mutations shape the clinical and prognostic landscape for breast cancer patients.

Landscape of germline BRCA1/BRCA2 variants in breast and ovarian cancer in Peru.

There is an increasing amount of data from Latin America on the characterization of BRCA variants; however, there is limited information from Peru. We conducted a retrospective study to describe germline pathogenic/likely pathogenic(P/LP) variants and variants of uncertain/unknown significance (VUS) in the BRCA1 and BRCA2 genes in Peru, in patients with breast and ovarian cancer, candidates for treatment with poly (adenosine diphosphate-ribose) polymerase (PARP) inhibitors. The patients were evaluated during the period 2019-2021. Genomic DNA was isolated from peripheral blood samples and targeted sequencing was performed using the Ampliseq BRCA panel. Genetic variant interpretation was carried out in accordance with the recommendations of the American College of Medical Genetics and ClinVar. During this period, 525 patients (143 with breast cancer and 382 with ovarian cancer) were studied. We found that 14.7% (21/143) of breast cancer patients and 20.7% (79/382) of ovarian cancer patients were carriers of P/LP variants in BRCA1/2. The most frequent pathogenic variants detected in BRCA1 were c.2105dupT (BIC: 2224insT, n=12, 18.75%), c.68_69delAG (BIC: 185delAG, n=6, 9.38%), c.140G>T and c.815_824dupAGCCATGTGG (n=5, 7.81%), while in BRCA2 were c.8023A>G (n=6, 16.67%), c.6024dupG (BIC: 6252insG, n=4, 11.11%), and c.9235delG (BIC: 9463delG, n=3, 8.33%). Regarding VUS, we found that 6.99% (10/143) of breast cancer patients and 7.33% (28/382) of ovarian cancer patients were carriers of a VUS in BRCA1/2. For BRCA1, the most frequent VUS was c.93C>G (n=2), and for BRCA2, c.5465A>T (n=4), c.3101T>C (n=3), c.205C>A and c.437T>C (n=2). We found a frequency of 14.7% germline mutations in breast cancer patients and 20.7% in ovarian cancer patients. The most recurrent mutations were BRCA1 c.2105dupT and BRCA2 c.8023A>G. We found that BRCA2 c.8023A>G, c.6024dupG, and c.9235delG were not previously reported in Peruvian patients. BRCA1 c.2344dupA is a novel mutation that has not been previously reported in any database. The frequency of VUS in our cohort was 7.2%.

Evaluation of PIK3CA Gene Mutations in Breast Cancer Patients Treated by Trastuzumab

Background: The phosphatidylinositol 3-kinases are known as a family of lipid kinases, playing a role in various cellular processes. A member of this family is PIK3CA which is frequently mutated in breast cancer. In this study, the association between H1047R, E542K and E545K mutations, and therapy resistance was investigated in Iranian breast cancer patients treated by Trastuzumab. Methods: Resistance and sensitive groups were chosen from Iranian patients treated by Trastuzumab. A PCR-RFLP approach was designed for detecting the H1047R mutation. Mutations in positions of codons E542K and E545K were detected using PCR-based DNA Sanger sequencing assay. The overall survival and disease-free survival were assessed. Results: A significant relationship was observed between the presence and absence of H1047R mutation and the overall survival (P = 0.025). In addition, there was a significant relationship between the response to Trastuzumab and some clinicopathological features, including the age and the status of ER/PR receptors (P-value&lt;0.05). E542K and E545K mutations were not observed in the patients. Conclusion: It can be said that probably H1047R mutation is a prognostic marker in the Trastuzumab-based therapy resistant breast cancer. Further studies can be performed to evaluate this mutation before using Trastuzumab to predict the effectiveness of this treatment.

Impact of early testing and analysis of germline genetic mutation patterns in patients with breast cancer: A single institution experience.

10613 Background: Approximately 3% of patients with breast cancer have a pathogenic or likely pathogenic variant (mutation) in a high-risk breast cancer gene, such as BRCA1, BRCA2and PALB2, which increases breast cancer risk more than fourfold. Approximately 3% have a germline mutation in a moderate-penetrance breast cancer gene, such as ATM or CHEK2, which confers a two- to threefold increased risk of breast cancer. Methods: In 2020 we started offering germline genetic testing to all newly diagnosed breast cancer patients at the time of diagnosis irrespective of their family history. Our objectives included the following: Prevalence of mutations in breast cancer patients treated at our institution. Germline mutations in patients with Ductal carcinoma in situ (DCIS). To identify if early universal germline testing affected the surgical modality. Results: A total of 525 patients were seen in the oncology clinic from January 2020 to December 2022 with a breast cancer diagnosis. The median age of diagnosis is 66 (32-91) years. Invasive ductal cancer (IDC 66 %) was the most common subtype, followed by DCIS (18%) (Table 1). Most patients had estrogen-positive cancer (82% vs. 18%), and approximately 13% had HER2-positive disease (61% HER2 low). Mutation in CHEK2 (9 patients) was our cohort's most common pathogenic mutation, followed by BRCA1 (6 patients) and MUYTH (6 patients) genes. Predominant germline mutations were identified in patients with IDC, followed by DCIS. One patient with a pure mucinous subtype had a germline pathogenic mutation (RECQL4 gene). We identified a higher percentage of pathogenic breast cancer-related mutations in patients above 60 years (63% vs. 37%) and all CHEK2 mutations were in patients above 60. Out of 41 patients with pathogenic mutations, 17 underwent a partial mastectomy, 12 had a bilateral mastectomy, and two had a unilateral mastectomy. All patients with BRCA1/2 mutation chose bilateral mastectomy, and all patients with a CHEK2 mutation underwent a partial mastectomy. Several variants of unknown significance (VUS) mutations were identified, and they followed a similar pattern, being more common in patients with IDC. VUS mutations in the ATM gene were the most reported abnormality. Conclusions: Pathogenic mutation in the CHEK2 gene is more prevalent, and we hypothesize that this finding could be related to the demographic population treated in our institution. Also, CHEK2 mutation was only identified in patients older than 60, which argues for more testing in patients in this age group. Approximately 9% of all IDC patients had a germline mutation compared to 4.2 % in patients with DCIS. Our review identifies that patients with BRCA1/2 mutations tend to undergo prophylactic mastectomy, and early testing likely impacted this decision. From our study, we believe that germline genetic testing should be offered to all breast cancer patients.

Survival outcomes of BRCA1/BRCA2 mutated breast cancers: Study from a tertiary care cancer centre in India.

e22546 Background: There exists a discrepancy amongst various studies over the years on survival outcomes of BRCA (Breast cancer susceptibility gene) mutated breast cancers. Prevalence of BRCA mutation varies from 10 to 18% among Indian population and there is a paucity of data on the survival outcomes of this population. Methods: This ambispective observational study assessed the survival outcomes of 71 BRCA mutated breast cancer patients, treated with uniform protocol at AIIMS New Delhi between January 2014 to December 2022, using survival analysis and cox regression methods. Results: Amongst the 71 patients, BRCA1 and BRCA2 mutated cases were 52 (73.2%) and 19 (26.8%) respectively. The median age was 39 years (24-63 years) in both the groups. Positive family history for malignancy (BRCA1- 69.2% vs BRCA2- 36.8%) and incidence of Triple negative histology (BRCA1-82.7% VS 36.8%) were more commonly associated with BRCA1 positivity. Eight patients (11.3%) had synchronous cancer, most common being a contralateral breast primary. 4 patients (5.6%) developed metachronous cancer. Eighty percent of patients were premenopausal at presentation. There was no difference in stage at presentation, incidence of synchronous and metachronous cancers across both the groups. Of 71 patients, 18 (25%) and 17 (24%) patients underwent risk reducing mastectomy and risk reducing oophorectomy respectively. Thirty five (49.3%) presented with stage I &amp; II disease, while 28 (34.4%) and 7 (9.9%) presented with stage III and metastatic disease respectively. Forty seven patients (67.1%) received neoadjuvant chemotherapy with pathologic complete response rate of 42.5% which was uniform among both the groups. Over the study period, 10 (14.1%) patients had disease relapse with 7 (70%) being metastatic and 3 patients died of the disease. With a median follow up of 36.9 months, 3-year disease free survival (DFS) was 84.2% (BRCA1: 81.1% vs BRCA2: 93.1%) and overall survival (OS) was 94.7% for both groups. Median DFS and OS were not achieved. BRCA type, receptor status, age and menopausal status were not predictors of survival in our study. Conclusions: In our study population of BRCA1/BRCA2 mutated breast cancer, survival outcomes were comparable to historical cohorts of Indian breast cancer patients. Thus, BRCA mutation positivity doesn’t seem to have any prognostic significance in breast cancer outcome.

Frequency and mutational spectrum of PIK3CA gene mutations in breast cancer patients: Largest and first report from Lebanon

The PIK3CA pathway is one of the most frequently altered pathways in human cancers, especially in breast cancer with approximately 40% of HR+/HER2– advanced breast cancer cases exhibiting mutations in the PIK3CA gene. While the mutations can occur across the entire gene, the most common are observed in exon 9 corresponding to the helical domain, and in exon 20 encompassing the kinase domain. This study constitutes the first attempt at determining the frequency and mutational spectrum in Lebanese breast cancer patients. For this purpose, DNA samples from 280 breast cancer patients from across Lebanon were screened for PIK3CA mutations using the Therascreen® PIK3CA RGQ Real-time PCR assay. In line with previous reports, 38.57% of cases were positive for at least one PIK3CA mutation, among which approximately 59% were in exon 9 and 37% in exon 20. However, PIK3CA mutations are breast cancer are heterogeneous whereby 20% of known PIK3CA mutants might not be detected by compact PCR based assays. Thus, the adoption of comprehensive Next Generation Sequencing based panels to decipher the complete clinical, molecular and immunohistochemical profile of breast cancer tumor requires further investigation.

Abstract P5-12-06: Comprehensive analysis of DNA damage repair gene germline mutations in Chinese breast cancer patients

Abstract Background: Germline DNA damage repair (DDR) mutations has been associated with increased cancer risk, PARP inhibitor therapeutic opportunity for breast cancer (BC) patients. However, the profile of germline mutations in BC covering comprehensive DDR genes remains unclear. Methods: A total of 341 women with breast cancer who tested 102 germline related genes (including 50 DDR genes) between April 2021 to May 2022 in Guangdong Provincial People’s Hospital were identified. Variants were classified into pathogenic, likely pathogenic, variant of uncertain significance (VUS), likely benign and benign groups according to the ACMG/AMP Standards and Guidelines. We defined pathogenic and likely pathogenic variants as deleterious mutations. Results: The median age of 341 breast cancer patients was 48 (range, 20-89) at the first diagnosis of BC. A total of 47 patients (13.78%) carried 53 deleterious germline variants in 21 cancer predisposition genes, 16 of which were DDR genes. DDR deleterious mutations were detected in genes including BRCA2 (n=18), BRCA1(n=7), FANCA (n=4), PMS2 (n=4), PALB2(n=2), RECQL4(n=2), PALB2 (n=2), etc. The younger age at diagnosis (less than 40-year-old) were significantly associated with deleterious mutations in DDR pathway(P=0.02). At least one VUS was identified in 238 (69.79%) patients. The top 5 DDR VUS genes were FANCM (n=21), ATM (n=20), RAD54L (n=17), FANCD2 (n=15) and ATR (n=14). Breast or ovarian cancer family history were significantly correlated with VUS germline mutations in DDR pathway(P=0.039). Interesting, we found that patients with pCR efficacy of neoadjuvant therapy were more likely to have VUS mutations in DDR pathway (table 1). Conclusion: We provided a comprehensive view of germline DDR gene mutations in BC patients and also analyzed the association between clinical characteristics and germline DDR mutation status. DDR mutations are prevalent in Chinese BC patients. Patients with younger and breast or ovarian cancer family history were more likely to carry DDR alterations. Moreover, patients with higher frequency of DDR VUS mutations may benefit from neoadjuvant therapy. Table 1. Clinicopathological characteristics between germline mutation carriers and non-carriers Citation Format: Ning Liao, Li Cao, Guochun Zhang, Junyun Wang, Airong Yang, Yulei Wang, Kai Li, Lingzhu Wen, Chongyang Ren, Minghan Jia, Cheukfai Li, Hsiaopei Mok, Bo Chen, Jianguo Lai, Weikai Xiao. Comprehensive analysis of DNA damage repair gene germline mutations in Chinese breast cancer patients [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 P5-12-06.

Abstract P2-26-09: Synergistic activity of PI3K inhibitor in combination with AZD6738, ATR inhibitor in breast cancer preclinical model via DNA damage response pathway

Abstract Background: The PI3K inhibition has been an appealing approach for anticancer therapy due to its crucial role in cell growth, proliferation, and survival in various cancers. Emerging data suggest the PI3K pathway is also involved in DNA replication and genome stability, making DNA damage response (DDR) inhibitors as an attractive combination treatment for PI3K pathway blockades. Here, to enhance the efficacy of PI3K inhibitors, we investigated the novel approach combining PI3K inhibitors (alpelisib, AZD8835, AZD8186, NVP-BKM120) with DDR blockade using ATR inhibitor (AZD6738) in breast cancer preclinical models. Methods: Hormone receptor-positive breast cancer cells, MCF7 (PIK3CA E545K mutation) and T47D (PIK3CA H1047R mutation) were used. First, cell viability and the synergistic effect of combination drug was carried out by MTT assy. Next, western blot, cell cycle analysis, apoptosis assay, and immunocytochemistry were conducted to validate the hypothesis. Finally, tumor xenograft experiments were conducted using BALB/c nude mice to validate the in vitro data. Results: A synergistic antiproliferative effect was observed over a wide range of combination (PI3K inhibitor + ATR inhibitor) concentrations (even in a lower concentration as 1/10 x IC50) in vitro, suggesting that when alpelisib is combined with an ATR inhibitor, there may be possibility of lowering the effective dosage of alpelisib. After confirmation of the strong synergistic effect in sub-G1/G0-G1 arrest-mediated apoptosis by the combination treatment, we demonstrated that expressions of phospho ATR, ATM, and CHK1 were suppressed but -H2AX markedly increased by combination treatment as compared to PI3K inhibitors and ATR inhibitor alone. This suggests that combination treatment activates apoptotic pathway through enhanced DNA double strand breaks. Finally, the in vivo study showed that the combination treatment group tended to show a greater tumor growth inhibition compared with PI3K inhibitor or ATR inhibitor alone. Conclusion: The combined inhibition of PI3K and ATR showed a synergistic anticancer effect in vitro and in vivo. ATR inhibitor in combination with PI3K inhibitor merits further clinical investigation to enhance the activity of PI3K inhibitor for the treatment of PIK3CA mutated breast cancer patients. Citation Format: Yong Wha Moon, Mithun Gosh, Nahee Park, Kamal Pandey, Nar Bahadur Katwal, Sa Deok Hong. Synergistic activity of PI3K inhibitor in combination with AZD6738, ATR inhibitor in breast cancer preclinical model via DNA damage response pathway [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 P2-26-09.

BRCA1 and BRCA2 mutations and their clinical relevance in selected women diagnosed with triple-negative breast cancer in Kenya: a descriptive cross-sectional study.

triple-negative breast cancer (TNBC) is a heterogeneous breast cancer type with a poor prognosis. About 25% of TNBC patients carry breast cancer susceptibility genes 1 and 2 (BRCA1 and BRCA2) mutations. Screening for BRCA mutations would facilitate early detection and initiation of personalized therapy, thus improving prognosis. However, this has not been explored in our population. We aimed at identifying BRCA1 and BRCA2 gene mutations and their clinical relevance among selected women with TNBC in Kenya. six participants enrolled in a larger descriptive cross-sectional study who met the inclusion criteria were selected. Structured questionnaires were used to obtain qualitative data. Deoxyribonucleic acid (DNA) was extracted from saliva. Whole exome sequencing of BRCA1 and BRCA2 genes using a next-generation sequencer was done. overall, 83.3% of BRCA1 and BRCA2 gene mutations with clinical relevance were detected. Most of the variants (63%) were found in BRCA1 whereas 37% were found in BRCA2. Pathogenic mutations in BRCA1 gene included c.5513T>A, c.5291T>C, c.5297T>G, c.110C>A, c.5212G>C, c.122A>C, c.5117G>A, c.5095C>T, c.5054C>T, c.5053A>G, c.115T>A, c.5143A>G, and c.130T>G. Those in BRCA2 gene were c.7878G>A, c.9154C>T, c.8243G>A, c.7976G>A, c.8165C>G, c.8167G>C, and c.8168A>T. One variant (c.5352delG: p. Leu1785Terfs) not matching any in the BRCA Exchange and ClinVar databases was detected. our study revealed BRCA mutations that could be common among our population. Further, it has shown that BRCA1 and BRCA2 genetic mutations identified are of clinical relevance and there is a need to screen for these mutations in breast cancer patients to understand their implication in patient management outcomes.