Breast Cancer Panel Research Articles

Breast cancer genomic analyses reveal genes, mutations, and signaling networks.

Breast cancer (BC) is the most commonly diagnosed cancer and the predominant cause of death in women. BC is a complex disorder, and the exploration of several types of BC omic data, highlighting genes, perturbations, signaling and cellular mechanisms, is needed. We collected mutational data from 9,555 BC samples using cBioPortal. We classified 1174 BC genes (mutated ≥ 40 samples) into five tiers (BCtier_I-V) and subjected them to pathway and protein‒protein network analyses using EnrichR and STRING 11, respectively. BCtier_I possesses 12 BC genes with mutational frequencies > 5%, with only 5 genes possessing > 10% frequencies, namely, PIK3CA (35.7%), TP53 (34.3%), GATA3 (11.5%), CDH1 (11.4%) and MUC16 (11%), and the next seven BC genes are KMT2C (8.8%), TTN (8%), MAP3K1 (8%), SYNE1 (7.2%), AHNAK2 (7%), USH2A (5.5%), and RYR2 (5.4%). Our pathway analyses revealed that the five top BC pathways were the PI3K-AKT, TP53, NOTCH, HIPPO, and RAS pathways. We found that BC panels share only seven genes. These findings show that BC arises from genetic disruptions evident in BC signaling and protein networks.

Cancer risk assessment of premalignant breast tissues from patients with BRCA mutations by genome profiling

Patients with germline pathogenic variants of BRCA1/2 genes have a particular predisposition to develop breast cancer. No clinical test has been developed to accurately and quantitatively evaluate their risk of developing breast cancer. We hypothesized that aberrant cell clonal expansion may be initiated in normal breast tissues without manifesting pathologic changes. To assess the prevalence of clonal expansion in the normal breast, we collected normal breast tissue from 24 breast cancer patients who had undergone surgical resection and 5 carriers of pathogenic BRCA1/2 variant who had undergone prophylactic mastectomy. Whole-exome sequencing (WES) was conducted in 97 specimens from 14 individuals, and TOP panel, a gene panel targeting 464 genes, was conducted in 321 specimens from 26 individuals, including 8 individuals with germline pathogenic variants of BRCA1/2 genes. Recurrent oncogenic mutations within PIK3CA, ARHGAP35, HRAS, and NF1 were identified in normal breast tissue at considerable variant allelic frequencies (VAF), suggesting clonal expansion. In addition, 937 normal breast tissues were evaluated using the Breast Cancer Panel (BCP) targeting 25 genes to determine the exact prevalence and distribution of clonal expansion. To assess the clonal expansion, we developed the clonality score, which is the mean value of clonal cell fractions for samples obtained from a given breast. The average clonality score in macroscopically normal breast tissue was 0.95 (0–2.46), with a significant difference between cases with and without a history of breast cancer of stage 2 or more advanced stage (p = 0.01). Additional WES on 42 samples with relatively large clone size (VAF > 3%) confirmed that these cell clones harbored multiple mutations (10.7 mutations/sample), and the number of existing mutations was consistent with the clone size (R = 0.50). The results suggest that clonal changes occur in normal breast tissue of women at high risk for breast cancer even before cancer is detected pathologically and/or radiologically, and the clonality score shows the potential to be a valid method of evaluating clonal expansion for cancer-risk assessment that provides appropriate preventive options for patients at high risk for breast cancer.

Co-occurrence of germline pathogenic variants in a Brazilian cohort of patients with breast cancer.

e22532 Background: The estimated prevalence of germline pathogenic variants (PV) or likely pathogenic variants (LPV) in high and moderate penetrance genes is around 7-10% among women with breast cancer (BC). The emergence of next-generation sequencing (NGS) technology allowed multi-gene panel testing (MGPT) analysis at a lower cost. However, the detection of incidental findings with unknown significance (VUS) and challenges with variant interpretation increase substantially with MGPT. The effect of co-existing PV/LPV in two or more genes is still understudied. Here, we present the prevalence of concurrent germline LPV/PV in a cohort of Brazilian women with BC who underwent a MGPT in a single reference laboratory. Methods: Retrospective observational study using a laboratory cohort (Oncoclínicas Precision Medicine). This study includes women with BC who performed a germline MGPT (able to call SNVs, indels and CNVs) from 2019 to 2023. All patients were tested in a single reference laboratory (OC Precision Medicine) and originated from several Brazilian centers within the Oncoclínicas network. Two distinct germline NGS assays were used: breast and ovarian cancer (BOC) panel (36 genes) and a pan-cancer (PC) panel (105 genes). Results: In the overall cohort, 2,651 women with BC were included in this analysis. The median age at genetic testing was 51 years. 1,867 pts (70.4%) underwent a PC panel testing and the remaining 784 pts (29.6%) a BOC panel.Overall, 314 (12%) pts carrier of LP/PV in high- and/or moderate-penetrance genes. Two or more heterozygous LP/PV were found in 35 pts (1.32%), including 8 pts (1.02%) in the BOC panel and 27 pts (1.44%) in the PC panel subgroups. There were no differences in age of onset of BC in single vs co-occurring double mutants. The most frequent co-existing PV/LPV were found in BRCA2 (11) / BRCA1 (8) / PALB2 (4) / TP53 (3) and MUTYH (7) / CHEK2 (3) in the high and moderate penetrance genes, respectively. Six pts (0.23%) had a double heterozygous involving high and/or moderate penetrance genes. Interestingly, there was a case with four heterozygous variants in TP53, MUTYH, MUTYH and APC. Conclusions: In times of rapid incorporation of MGPT in the clinical practice, our real-world data showed that a small proportion of BC patients could be carriers of two or more germline heterozygous LP/PV in cancer genes. However, this represents a challenging scenario for patient and family management, especially in a co-occurrence of high and moderate penetrance genes.

Update Breast Cancer 2024 Part 1 - Expert Opinion on Advanced Breast Cancer.

Clinical evidence is interpreted based on clinical studies and personal experience which can lead to different interpretations of data. This makes the opinions issued by panels of experts such as the Advanced Breast Cancer Panel which convened in November 2023 for the seventh time (ABC7) particularly important. At the conference, current issues around advanced breast cancer were evaluated by an international team of experts. In 2023 the data on CDK4/6 inhibitors was so extensive that the answers to questions about the sequencing of therapy and the potential use of chemotherapy as an alternative therapy were relatively clear. Moreover, data on antibody drug conjugates which provides a good overview of their uses is available for all molecular subtypes. Some therapeutic settings, including patients with brain metastases or leptomeningeal disease, older patients, locally advanced breast cancer and visceral crises, continue to be particularly important and were discussed in structured sessions. The scientific context of some of the topics discussed at ABC7 is presented and assessed here.

PAK6 acts downstream of IQGAP3 to promote contractility in triple negative breast cancer cells

Breast cancer is a heterogeneous disease that remains the most common malignancy among women worldwide. During genomic analysis of breast tumours, mRNA levels of IQGAP3 were found to be upregulated in triple negative tumours. IQGAP3 was subsequently found to be expressed across a panel of triple negative breast cancer (TNBC) cell lines. Depleting expression levels of IQGAP3 delivered elongated cells, disrupted cell migration, and inhibited the ability of cells to form specialised invasive adhesion structures, termed invadopodia. The morphological changes induced by IQGAP3 depletion were found to be dependent on RhoA. Indeed, reduced expression of IQGAP3 disrupted RhoA activity and actomyosin contractility. Interestingly, IQGAP3 was also found to interact with p-21 activated kinase 6 (PAK6); a protein already associated with the regulation of cell morphology. Moreover, PAK6 depletion phenocopied IQGAP3 depletion in these cells. Whereas PAK6 overexpression rescued the IQGAP3 depletion phenotype. Our work points to an important PAK6-IQGAP3-RhoA pathway that drives the cellular contractility of breast cancer cells promoting both cell migration and adhesive invasion of these cells. As this phenotype is relevant to the process of metastasis and re-seeding of metastasis, the pharmacological targeting of PAK6 could lead to clinical benefit in TNBC patients.

Dynamic change of cancer genome profiling in metachronous bilateral breast cancer with BRCA pathogenic variant.

A 61-year-old woman with BRCA2 pathogenic variant had been treated for 20years and showed dynamic changes in the genomic profile of her metachronous bilateral breast cancer and metastases. She underwent right breast conservation surgery at age 42-Genome 1, lung metastasis and left axillary lymph node metastasis at age 51, partial excision under local anesthesia for left breast cancer at age 53-Genome 2, left axillary lymph node dissection was added 6month later-Genome 3. Then, olaparib was administered, and subsequently, left mastectomy was performed for the recurrence of left breast cancer at age 59-Genome 4. Genomic profile of the tumor was analyzed at four points (Genome 1-3 were analyzed by in house breast cancer panel, and Genome 4 was analyzed by Foundation One CDx). Two interesting findings emerged from these analyses. First, the genomic profile revealed that the left axillary lymph node metastasis, considered histologically from right breast cancer, was a metastasis from the left breast cancer. The second finding is that as the disease progressed, mutation profile became more diverse. The profile of the left breast cancer removed after olaparib and other treatments showed reversion mutation of BRCA2 and was diagnosed as tumor mutation burden high. Subsequent response to pembrolizumab was favorable.

Abstract PO2-08-01: Universal Multi-Gene Panel Testing of Newly Diagnosed Breast Cancer Patients in a Community Healthcare System

Abstract Introduction Multi-gene panel, germline genetic testing has emerged over the last decade as a useful tool in the assessment of hereditary cancer syndromes. As barriers to genetic testing are reduced, offering universal genetic testing to all cancer patients is a practice gaining traction. We present outcomes data specific to the offering of universal, multi-gene panel testing to newly diagnosed breast cancer patients in a community hospital setting. Methods A retrospective chart review was performed for all newly diagnosed breast cancer patients (n=1005) at a community hospital between 8/5/2019 and 8/5/2022. Uptake and outcomes of genetic counseling and genetic testing were assessed. All patients were offered genetic counseling, and those who consented to genetic testing were offered a multi-gene panel (37-93 genes). The National Comprehensive Cancer Network (NCCN) Genetic/Familial High-Risk Assessment: Breast, Ovarian, and Pancreatic (2020) guidelines were utilized to distinguish which patients met traditional genetic testing criteria. Results Of all newly diagnosed breast cancer patients, 915 (91.1%) patients underwent genetic counseling and 871 (86.7%) patients elected for genetic testing. Of those who declined genetic testing (n=134), 39 (29.1%) reported previous genetic testing. Of patients tested, 489 (56.1%) met NCCN criteria. Overall, 114 (13.1%) of tested patients received results with likely pathogenic/pathogenic (LP/P) variants. Of those, 86 (75.4%) had a clinically significant variant, a high-risk, moderate-risk or another dominantly inherited gene variant, while 28 (24.6%) were identified as genetic carriers; Table 1 summarizes genes with LP/P variants in this cohort, with 10 patients receiving results for 2 variants. Of the patients with clinically significant genetic results, 58 (67.4%) met NCCN criteria. Furthermore, 65 patients (75.6%) reported a family history of cancer relevant to the identified LP/P variant. The average age of the total population, and of those with a positive result, was 62 years. While nearly 19% of all patients tested were under age 50, patients under 50 comprised only 14.0% (n=12) of patients with a clinically significant result. Breast cancer management changes, including addition of a PARP inhibitor to chemotherapy regimen and/or elected bilateral mastectomies in the setting of unilateral disease, were noted in 71.4% (n=20) of patients with high-risk genetic findings and 20.6% (n=7) of patients with moderate-risk genetic findings. Conclusion The 86.7% uptake of genetic testing suggests implementation of universal testing in a community setting is feasible and favored by patients. Clinically significant genetic results would have gone undetected in 32.6% of patients if NCCN criteria was strictly utilized to offer testing. In addition to the 62 patients with high and moderate risk genetic results, utilization of expanded panel testing provided benefit for an additional 24 patients found to have an actionable variant in a gene that would not have been detected on traditional breast cancer panels. Our data supports the offering of genetic testing to all newly diagnosed breast cancer patients, regardless of age, family history, or other criterion, to inform precise breast cancer management and overall, lifetime cancer risks. Genetic Findings of Universal Multi-Gene Panels in a Community Setting Citation Format: Natalie Johns, Jody Wallace, Darbey Raible, Jordan Brady, Grace Miller, Brooke Phillips. Universal Multi-Gene Panel Testing of Newly Diagnosed Breast Cancer Patients in a Community Healthcare System [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-08-01.

Abstract 3297: Discovery and evaluation of GT19870, a GSPT1/Myc molecular glue drug (MGD) with oral bioavailability for targeting c-Myc and n-Myc-addictive blood cancer and solid tumors

Abstract c-Myc and n-Myc are oncogenic transcriptional factors driving tumor initiation, progression and poor prognosis in a variety of blood cancers and solid tumors, such as acute myeloid leukemia (AML), B-cell malignancies, castration resistance prostate cancer (mCRPC), small cell lung cancer and (SCLC) and neuroblastoma. Both c-Myc and n-Myc dysregulations have been directly linked to the poor clinical outcome in some of these cancers which are addictive to deregulated Myc proteins. Therefore, it is highly warranted to discover and develop novel therapeutical agents for targeting Myc-addictive cancers. GT19870 was discovered through a SAR effort for Myc degradation with improved bioavailability (F % 92.52) from GT19630, a GSPT1/Myc degrader series 1-4by using phenotypic screenings with c-Myc-addictive HL-60 AML cells and growth factor-dependent hematopoiesis progenitor TF-1 cells, followed by c-Myc ELISA and Western blot assays. GT19870 selectively inhibited the proliferation of HL60 cells with an IC50 of 2.6 nM as compared to an IC50 of 70.03 nM (26.5-fold selectivity) and effectively degraded c-Myc protein with an IC50 of 2.54 nM and IC90 <10 nM in HL-60 cells, as compared to an IC90>30 nM in CD3/CD28-stimulated PBMC assays. GT19870, as a MGD, was confirmed to selectively degrade GSPT1, a neo-substrate, by proteomic and Western Blot assays. Further, GT19870 was tested in SCLC, triple negative breast cancer (TNBC) and brain cancer cell panels, in which GT1987 was revealed potent anti-proliferation activities with an IC50<50 nM in 9 of 19 SCLC, 8 of 13 TNBC, and 7 of 24 brain cancer cell lines tested. Importantly GT19870 was also shown potent antitumor activities with IC50 of ≦5.4 nM in 8 of 8 SCLC patient derived organoids. Furthermore, GT19870 (salt form-GT19884) was demonstrated to induce target-engaged (c-Myc and n-Myc) tumor regression in AML, SCLC, mCRPC and neuroblastoma xenograft and PDX models with in a dose ranges of 3-12 mpk/qd/po. In brief, GT19870 is an oral MGD degrading GSPT1/c-Myc/n-Myc. This novel MGD has been demonstrated target-engaged antitumor activities in vitro and in vivo with selectivity of c-Myc degradation in c-Myc-addictive cancer cells as compared to growth factor-stimulated TF-1 cells and CD3/CD28 activated PBMC. GT19870 has been progressing IND enabling stage.

Digital RNA sequencing using unique molecular identifiers enables ultrasensitive RNA mutation analysis

Mutation analysis is typically performed at the DNA level since most technical approaches are developed for DNA analysis. However, some applications, like transcriptional mutagenesis, RNA editing and gene expression analysis, require RNA analysis. Here, we combine reverse transcription and digital DNA sequencing to enable low error digital RNA sequencing. We evaluate yield, reproducibility, dynamic range and error correction rate for seven different reverse transcription conditions using multiplexed assays. The yield, reproducibility and error rate vary substantially between the specific conditions, where the yield differs 9.9-fold between the best and worst performing condition. Next, we show that error rates similar to DNA sequencing can be achieved for RNA using appropriate reverse transcription conditions, enabling detection of mutant allele frequencies <0.1% at RNA level. We also detect mutations at both DNA and RNA levels in tumor tissue using a breast cancer panel. Finally, we demonstrate that digital RNA sequencing can be applied to liquid biopsies, analyzing cell-free gene transcripts. In conclusion, we demonstrate that digital RNA sequencing is suitable for ultrasensitive RNA mutation analysis, enabling several basic research and clinical applications.

LC-MS/MS platform-based serum untargeted screening reveals the diagnostic biomarker panel and molecular mechanism of breast cancer

BackgroundBreast cancer (BC), the most common form of malignant cancer affecting women worldwide, was characterized by heterogeneous metabolic disorder and lack of effective biomarkers for diagnosis. The purpose of this study is to search for reliable metabolite biomarkers of BC as well as triple-negative breast cancer (TNBC) using serum metabolomics approach. MethodsIn this study, an untargeted metabolomics technique based on ultra-high performance liquid chromatography combined with mass spectrometry (UHPLC-MS) was utilized to investigate the differences in serum metabolic profile between the BC group (n = 53) and non-BC group (n = 57), as well as between TNBC patients (n = 23) and non-TNBC subjects (n = 30). The multivariate data analysis, determination of the fold change and the Mann-Whitney U test were used to screen out the differential metabolites. Additionally, machine learning methods including receiver operating curve analysis and logistic regression analysis were conducted to establish diagnostic biomarker panels. ResultsThere were 36 metabolites found to be significantly different between BC and non-BC groups, and 12 metabolites discovered to be significantly different between TNBC and non-TNBC patients. Results also showed that four metabolites, including N-acetyl-D-tryptophan, 2-arachidonoylglycerol, pipecolic acid and oxoglutaric acid, were considered as vital biomarkers for the diagnosis of BC and non-BC with an area under the curve (AUC) of 0.995. Another two-metabolite panel of N-acetyl-D-tryptophan and 2-arachidonoylglycerol was discovered to discriminate TNBC from non-TNBC and produced an AUC of 0.965. ConclusionThis study demonstrated that serum metabolomics can be used to identify BC specifically and identified promising serum metabolic markers for TNBC diagnosis.

Gene expression alterations predict the pathological complete response in triple-negative breast cancer exploratory analysis of the NACATRINE trial

This exploratory analysis of the Neoadjuvant Carboplatin in Triple Negative Breast Cancer (NACATRINE) study aimed to identify the biomarkers of pathological complete response (pCR) in patients with triple-negative breast cancer (TNBC) treated with neoadjuvant chemotherapy (NAC) within the context of a clinical trial. The NACATRINE trial is a phase II, single-center, randomized, open-label clinical trial that investigated the addition of carboplatin to sequential anthracycline- and taxane-based NAC for TNBC. We evaluated the gene expression in untreated samples to investigate its association with pCR, overall survival (OS), and disease-free survival (DFS). RNA was extracted from the tissue biopsy, and the nCounter Breast Cancer panel was used to analyze gene expression. Of the 66 patients included in the gene expression profiling analysis, 24 (36.4%) achieved pCR and 42 (63.6%) had residual disease. In unsupervised hierarchical clustering analyses, differentially expressed genes between patients with and without pCR were identified irrespective of the treatment (24 genes), carboplatin (37 genes), and non-carboplatin (27 genes) arms. In receiver operating characteristic (ROC) curve analysis, 10 genes in the carboplatin arm (area under the ROC curve [AUC], 0.936) and three genes in the non-carboplatin arm (AUC, 0.939) were considered to be potential pCR-associated biomarkers. We identified genes that were associated with improvements in OS and DFS in addition to being related to pCR. We successfully identified gene expression signatures associated with pCR in pretreatment samples of patients with TNBC treated with NAC. Further investigation of these biomarkers is warranted.

Machine learning-based somatic variant calling in cell-free DNA of metastatic breast cancer patients using large NGS panels

Next generation sequencing of cell-free DNA (cfDNA) is a promising method for treatment monitoring and therapy selection in metastatic breast cancer (MBC). However, distinguishing tumor-specific variants from sequencing artefacts and germline variation with low false discovery rate is challenging when using large targeted sequencing panels covering many tumor suppressor genes. To address this, we built a machine learning model to remove false positive variant calls and augmented it with additional filters to ensure selection of tumor-derived variants. We used cfDNA of 70 MBC patients profiled with both the small targeted Oncomine breast panel (Thermofisher) and the much larger Qiaseq Human Breast Cancer Panel (Qiagen). The model was trained on the panels’ common regions using Oncomine hotspot mutations as ground truth. Applied to Qiaseq data, it achieved 35% sensitivity and 36% precision, outperforming basic filtering. For 20 patients we used germline DNA to filter for somatic variants and obtained 245 variants in total, while our model found seven variants, of which six were also detected using the germline strategy. In ten tumor-free individuals, our method detected in total one (potentially germline) variant, in contrast to 521 variants detected without our model. These results indicate that our model largely detects somatic variants.

Comprehensive analysis of patients with cancer with a positive CHEK2 test: Are we getting closer to a genotype/phenotype correlation?

10588 Background: The CHEK2 gene encodes a protein kinase that acts as a tumor suppressor with an important role in DNA damage repair. This gene is included in most hereditary breast, ovarian and prostate cancer panels, but the clinical actionability of CHEK2 pathogenic/likely pathogenic variants (PVs) may be challenging. Indeed, decisions concerning clinical surveillance and/or risk reducing procedures as well as screening for multiple associated cancer risks are not consensual. Our objective was to identify a potential genotype/phenotype association regarding CHEK2 PV identified in our population. Methods: Study group included all patients (pts) who tested positive for CHEK2 PVs between July 2000 and June 2022; control group included pts without PVs after multigene panel testing (wt) between January 2020 and June 2022. PVs were categorized as: truncating (includes nonsense, frameshift, splice site variants, gross deletions and duplications) and not truncating (missense and in-frame deletions). Clinical characteristics from both groups included gender, personal and family cancer history and tumor characteristics. Results: From 8604 pts tested, we identified 101 CHEK2 and 1506 wt pts. CHEK2 pts were mainly women (95; 92%) and Portuguese (94; 93.1%). All non-Portuguese pts had Eastern Europe ancestry. CHEK2 pts were either heterozygous (94;93%), homozygous (3; 3%) or had digenic inheritance (4; 6%: both a CHEK2 and a BRCA1/2 PV). Compared to wt pts, heterozygous CHEK2 pts were younger at 1st cancer diagnosis (44.5 yrs vs 47 yrs) and had more multiple primary cancers (OR: 1.8; p: 0.02). CHEK2 breast cancer was mostly but not exclusively estrogen receptor positive (69/78; 88,4%) and Her2 negative (54/69; 78%). Two BC cases had triple negative phenotype. The most frequent PVs detected were c.349A &gt; G (25 pts), c.1100del (24 pts) and c.319+2T &gt; A (20 pts). Considering heterozygous PVs, 57 were truncating and 37 non-truncating, with c. 1100del and c.349A &gt; G being the most frequent, in each group, respectively. For truncating versus non-truncating PVs there was no difference in phenotype regarding breast (OR: 0.69; p: 0.15), CRC (OR: 0.94; p: 0.91), gastric (OR: 0.76; p: 0.65), thyroid (OR: 1.3; p: 0,69) and kidney cancer (OR: 0.36; p: 0.27). Further stratification of PVs according to cancer type revealed a correlation of CRC with c.1100del (OR: 4.57; p: 0.0025) and c.349A &gt; G (OR: 5.49; p: 0.0003), while gastric, kidney and thyroid cancers correlated with c.349A &gt; G (OR: 5.8; p: 0.0069), c.1100del (OR: 26.86; p: 0.02) and c.319+2T &gt; A (OR: 3.31; p: 0.05) PVs, respectively. Conclusions: This analysis suggests a potential genotype-phenotype correlation regarding CHEK2 pathogenic variants, regardless of its truncating potential. The study of clinical outcomes will further contribute to determine how this information may influence the clinical and risk management of CHEK2 pts and their families.

Validation of a breast cancer polygenic risk score in 15,490 Brazilians using exome sequencing.

10631 Background: Brazil has a highly admixed population with significant Southern European, African and Native American genetic ancestries. Approximately 75% of the population has two or more continental ancestries. Therefore, breast cancer polygenic risk scores (PRS) developed in predominantly European populations requires validation in the Brazilian population. Compared with genotyping arrays and whole genome sequencing, affordable and scalable whole exome sequencing (WES) allows genotyping of both rare and common variants in the targeted regions and imputation of common variants not directly captured. In this study we validate a previously developed 3820-variant PRS using WES in 15,490 Brazilians. Methods: The cohort consisted of 6,362 women with a history of breast cancer (mean age 49.6, stddev 11.6) and 9,128 unphenotyped adults as controls (mean age 41.6, stddev 13.3). WES were performed on germline DNA and variants were called and curated. Imputation was done using the 1000 Genomes Phase 3 reference panel for variants falling outside the WES captured regions. After excluding subjects with known pathogenic or likely pathogenic variants in BRCA1, BRCA2, PALB2, PTEN, or TP53, and first or second-degree relatives of the probands, 14,577 individuals remained in the final analysis (5,730 cases and 8,847 controls). We restricted PRS calculation to 2,893 of the original 3,820 variants with an estimated global imputation accuracy of 96%. Odds ratios (OR) were adjusted using the top 10 principal components. Results: The breast cancer PRS showed a highly significant correlation with cancer risk (p-value = 7.1e-81, OR = 1.43, 95% confidence interval (CI) = 1.38-1.48 per PRS standard deviation). Compared with women in the middle deciles, those in the highest 10% had an OR of 1.93, while those in the lowest 10% had an OR of 0.52 (Table). Conclusions: In this study we show that WES can capture 76% of the variants included in a previously developed and validated breast cancer PRS, and the calculated PRS is correlated with breast cancer risk in the admixed Brazilian population. This opens the door for clinical studies into the application of PRS in Brazil, particularly in women without mutations in breast cancer high-penetrance genes. In addition, our WES-based PRS strategy can be quickly, affordably, and seamlessly adopted by labs that already offer Hereditary Breast Cancer panels that use WES as a backbone. [Table: see text]

Benzimidazole carbamate induces cytotoxicity in breast cancer cells via two distinct cell death mechanisms

Metastatic breast cancer (mBC) is responsible for >90% of breast cancer-related deaths. Microtubule-targeting agents (MTAs) are the front-line treatment for mBC. However, the effectiveness of MTAs is frequently limited by the primary or acquired resistance. Furthermore, recurrent mBC derived from cancer cells that survived MTA treatment are typically more chemoresistant. The overall response rates for the second- and third-line MTAs in mBC patients previously treated with MTAs are 12–35%. Thus, there is an ongoing search for novel MTAs with a distinct mode of action that can circumvent chemoresistance mechanisms. Our results show that methyl N-(6-benzoyl-1H-benzimidazol-2-yl)carbamate (BCar), a microtubule-disrupting anthelmintic that binds to the colchicine binding site separate from the binding sites of clinically used MTAs, has the potential to treat MTA-resistant mBC. We have comprehensively evaluated the cellular effects of BCar in a panel of human breast cancer (BC) cell lines and normal breast cells. BCar effects on the clonogenic survival, cell cycle, apoptosis, autophagy, senescence, and mitotic catastrophe were measured. Approximately 25% of BCs harbor mutant p53. For this reason, the p53 status was included as a variable. The results show that BC cells are >10x more sensitive to BCar than normal mammary epithelial cells (HME). p53-mutant BC cells are significantly more sensitive to BCar treatment than p53 wild-type BC cells. Furthermore, BCar appears to kill BC cells primarily via either p53-dependent apoptosis or p53-independent mitotic catastrophe. When compared to docetaxel and vincristine, two clinical MTAs, BCar is fairly innocuous in HME cells, providing a much wider therapeutic window than docetaxel and vincristine. Together, the results strongly support the notion that BCar-based therapeutics may serve as a new line of MTAs for mBC treatment.

Abstract 2175: Gene signature predicts pathological complete response in triple negative breast cancer patients: NACATRINE trial

Abstract In triple negative breast cancer (TNBC), achieving pathological complete response (pCR) is associated with improved survival outcomes. However, only one-third of patients receiving neoadjuvant chemotherapy (NAC) achieve pCR. Recognizing molecular markers that predict response to chemotherapy and selecting patients who can benefit from NAC in clinical practice has been a challenge in many studies. This exploratory analysis of the NACATRINE trial aimed to identify biomarkers that predict pCR TNBC patients receiving NAC. The NACATRINE trial is a Brazilian, phase II, single-center, randomized, open-label study that compared adding carboplatin to sequential neoadjuvant therapy based on anthracycline and taxane for TNBC. We evaluated gene expression in untreated samples to associate with the response to systemic therapy. RNA was extracted from the tissue biopsy, and the nCounter Breast Cancer panel analyzed gene expression. Gene counts were normalized by housekeeping. For statistical significance, the t-test was used with a p-value &amp;lt;0.05 and fold-change ≥2. Of the 66 patients included in the gene expression profile analysis, 24 (36.4%) had pCR, and 42 (63.6%) had residual disease. Thirty-seven genes were differentially expressed when associated with pCR in the carboplatin arm and twenty-seven in the non-carboplatin arm (p-value &amp;lt;0.001). We performed a linear regression model to evaluate gene performance, and the area under the curve (AUC) was calculated to assess the predictive value of differentially expressed genes. Eleven genes (KCNB1, FGF1, SNAI1, CXCL9, FGL2, HAS1, CXCL8, VEGFD, HEMK1, MS4A2, and NFKBIZ) in the carboplatin arm (AUC 0.938) and eight genes (ALDH1A1, FZD8, HIST1H3H, CXADR, WDR77, PRKACB, JUN, and CDK6) in the non-carboplatin arm (AUC 0.943) was associated with pCR. Overall survival (OS) was higher among patients who had downregulated KCNB1, FGF1, and SNAI1 (HR = 4.43, 95% CI 1.00 - 1.58; p=0.04; HR = 4.92, 95% CI 1.10 - 2.83; p= 0.03; HR = 4.86, 95% CI 1.38 - 1.11; p= 0.01) respectively. Similarly, upregulated of ALDH1A1, CXCL9 and FGL2 were associated with a better prognosis (HR = 0.17, 95% CI 0.05 - 0.50; p= 0.003; HR = 0.15, 95% CI 0.04 - 0.50; p = 0.004; HR = 0.21, 95% CI 0.07 - 0.60; p = 0.009), respectively. We identified potentially beneficial gene expression signatures (OS and pCR) for neoadjuvant chemotherapy, suggesting that these markers validated in a prospective study can be used in selecting patients for neoadjuvant chemotherapy in TNBC. Citation Format: Ana J. Freitas, Caroline R. Nunes, Rhafaela L. Causin, Iara V. Santana, Marco A. Oliveira, Stéphanie Calfa, Cristiano P. Souza, Márcia M. Marques. Gene signature predicts pathological complete response in triple negative breast cancer patients: NACATRINE trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2175.

Abstract P2-23-19: Interrogating multicellular signaling in breast cancer using a bio-orthogonal chemistry-based proteomics platform

Abstract Tumors are multicellular ecosystems that communicate through the exchange of extracellular signaling molecules. In breast cancer, this multicellular communication can enable tumor cells to cooperate in a range of contexts, including tumor invasion and the outgrowth of distal metastases. Illuminating the mechanisms by which tumor cells communicate may reveal new therapies. To this end, three-dimensional tumor organoid models have emerged as versatile platforms for modeling multicellular behavior ex vivo. However, organoid culture typically requires the use of poorly defined, animal-derived extracellular matrices, such as Matrigel. These exogenous matrices contain thousands of proteins that dominate over and conceal cell-secreted factors in conventional proteomics approaches. Revealing low abundance cell-secreted factors from this complex exogenous background presents a formidable challenge. To surmount this challenge, we develop a new method to isolate the pericellular proteome in 3D organotypic culture models. This technology harnesses biorthogonal click chemistry to bypass exogenous factors, infiltrate intercellular spaces, and retrieve the endogenous intercellular proteome. This approach requires no genetic manipulation, requires only 1 million cells, and can be adapted to diverse organotypic models. These capabilities enable isolation of intercellular signaling factors in models that yield precious amounts of material and that can sustain only limited manipulation ex vivo, which we demonstrate using organoids established from patient-derived xenografts. To establish the generalizability of our approach, we apply this technology to a panel of breast cancer and small-cell lung cancer organoid models. Furthermore, we demonstrate that this method can be readily adapted to different extracellular matrix environments that are widely employed in organoid research, including basement membrane matrices and collagen gels. Taken together, our results establish this technology as a scalable and generalizable platform that may open opportunities for researchers investigating diverse questions in organoid models of cancer. Moving forward, we are leveraging these capabilities to investigate changes in the intercellular proteome that emerge after the acquisition of therapy resistance. We hope to uncover new mechanisms of collective signaling that may be targeted to overcome therapy resistance. Citation Format: Brad A. Krajina, Ami Yamamoto, Kevin J. Cheung, Samuel Madasu. Interrogating multicellular signaling in breast cancer using a bio-orthogonal chemistry-based proteomics platform [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 P2-23-19.

Abstract P4-03-39: Breast density and Body Mass Index association with breast cancer molecular subtypes in early breast cancer Spanish population

Abstract Obesity is a major health problem and is closely related to the incidence and progression of breast cancer (BC). Likewise, mammogram breast density (MBD) is a risk factor for breast cancer. This study aims to analyze the potential association between BMI (Body Mass Index) and MBD with different molecular subtypes of BC. The interaction with cholesterol, vitamin D, insulin levels, and lifestyle, will also be evaluated. Materials and methods: This is a transversal, descriptive, multicenter study involving women with a recent diagnosis of early BC for one-year duration (November 2019 to October 2020). BMI was classified as obesity (BMI &amp;gt;=30 kg/m2), overweight (&amp;gt;=25 kg/m2), and normal weight (18-25 Kg/m2). American college of radiology was used to classify MBD into 4 categories: A, B, C, and D. Data of weight, height, MBD, molecular subtype (according to 13th St Gallen International Breast Cancer Panel), cholesterol, insulin, vitamin-D, hypertension, diabetes, exercise, and lifestyle were collected at the moment of diagnosis. Results: 162 women with a recent diagnosis of early BC have been successively evaluated in 3 Spanish hospitals. The median age was 52 years, and 49.1% were postmenopausal. Molecular subtypes were: 43% luminal-A, 25% luminal-B, 17% HER2+, and 15% triple-negative. 7% of patients were MBD type A, 17% type B, 55% type C, and 21% type D. 52% were normal weight, 32% overweight, and 16% obese. 48% of women had normal cholesterol levels and 60% normal vitamin D. There was not association between BMI and molecular subtype, however considering menopausal status, BMI was significantly higher in postmenopausal patients with Luminal A subtype (p= 0.011) and HER2+ subtype (p= 0.027). There was no association between MBD and molecular subtype. However, there were significant differences between BMI and MBD (p&amp;lt; 0.001) with lower BMI in patients who had MBD type C or D. Patients with higher BMI had lower HDL-Cholesterol (p&amp;lt; 0.001) and higher levels of insulin (p&amp;lt; 0.001). Conclusion: This study showed a higher BMI in Luminal A and HER2 positive postmenopausal BC patients. Higher BMI was shown in patients with low MBD independently of menopausal status. More studies are needed to understand the role of obesity and molecular subtype of breast cancer. Citation Format: Isabel Calvo, Marta González, Fernando Neria, Isabel Gallegos, Lourdes García-Sánchez, Silvia Pérez, Laura G. Estévez. Breast density and Body Mass Index association with breast cancer molecular subtypes in early breast cancer Spanish population. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-03-39.

Frequency of Pathogenic Germline Mutations in Early and Late Onset Familial Breast Cancer Patients Using Multi-Gene Panel Sequencing: An Egyptian Study.

Precision oncology has been increasingly used in clinical practice and rapidly evolving in the oncology field. Thus, this study was performed to assess the frequency of germline mutations in early and late onset familial breast cancer (BC) Egyptian patients using multi-gene panel sequencing to better understand the contribution of the inherited germline mutations in BC predisposition. Moreover, to determine the actionable deleterious mutations associated with familial BC that might be used as biomarker for early cancer detection. Whole blood samples were collected from 101 Egyptian patients selected for BC family history, in addition to 50 age-matched healthy controls. A QIAseq targeted DNA panel (human BC panel) was used to assess the frequency of germline mutations. A total of 58 patients (57.4%) out of 101 were found to have 27 deleterious germline mutations in 11 cancer susceptibility genes. Of them, 32 (31.6%) patients carried more than one pathogenic mutation and each one carried at least one pathogenic mutation. The major genes harboring the pathogenic mutations were: ATM, BRCA2, BRCA1, VHL, MSH6, APC, CHEK2, MSH2, MEN1, PALB2, and MUTYH. Thirty-one patients (30.6%) had BRCA2 mutations and twenty (19.8%) had BRCA1 mutations. Our results showed that exon 10 and exon 11 harbored 3 and 5 mutations, respectively, in BRCA1 and BRCA2 genes. Our analysis also revealed that the VHL gene significantly co-occurred with each of the BRCA2 gene (p = 0.003, event ratio 11/21), the MSH2 gene (p = 0.01, 4/10), the CHEK2 gene (p = 0.02, 4/11), and the MSH6 gene (p = 0.04, 4/12). In addition, the APC gene significantly co-occurred with the MSH2 gene (p = 0.01, 3/7). Furthermore, there was a significant mutually exclusive event between the APC gene and the ATM gene (p = 0.04, 1/36). Interestingly, we identified population specific germline mutations in genes showing potentials for targeted therapy to meet the need for incorporating precision oncology into clinical practice. For example, the mutations identified in the ATM, APC, and MSH2 genes. Multi-gene panel sequencing was used to detect the deleterious mutations associated with familial BC, which in turns mitigate the essential need for implementing next generation sequencing technologies in precision oncology to identify cancer predisposing genes. Moreover, identifying DNA repair gene mutations, with focus on non-BRCA genes, might serve as candidates for targeted therapy and will be increasingly used in precision oncology.

Abstract 3115: Establishment and characterization of patient-derived breast cancer models for cancer studies

Abstract BACKGROUND: Despite the progress and development of targeted agents and Immunotherapeutics, breast cancer is still the leading cause of death among women worldwide. At present, targeted therapy for breast cancers include endocrine blockage therapy for estrogen receptor positive (ER+) tumors and trastuzumab or lapatinib for HER2 positive (HER2+) tumors and CDK inhibitors for HER negative (HER-) tumors as well as PARP inhibitors for BRCA1/2 mutations. There is still a high unmet need for triple negative breast cancer (TNBC) and relapsed breast cancers with specific mutations. Breast cancer is a highly heterogeneous disease and reliable breast cancer models are needed to develop new clinical therapies and better reflect tumor biology. Patient-derived xenograft (PDX) models have been widely used for understanding tumor characteristics and represent human heterogeneity more faithfully and thus are suitable for mouse clinical studies for predicting drug efficacy in the clinic. Here we report the establishment and characterization of a panel of breast cancer (BC) PDX for preclinical research. METHODS: Tumor samples from patients’ breast tumor or metastastic lesion obtained via surgery, biopsy, or pleural effusions/ascites were engrafted subcutaneously in immunodeficient mice to establish PDX models. The models were classified and characterized by histopathology, immunohistochemistry (IHC), and breast cancer specific biomarkers such as estrogen receptor (ER), progesterone receptor (PR) and HER2. Genomic analysis of these models was performed by next generation sequencing (NGS). In these breast cancer models, the effects of estrogen on tumor growth and targeted endocrine therapy such as Fulvestrant were evaluated. The change of tumor volume over time was used to determine the growth rate and response to treatment in mice. RESULTS: A series of BC PDX models derived from primary patient breast tumor or metastatic lesions were successfully established and characterized. Key characteristics of parental tumors, such as histopathology, clinical markers, gene expression and copy number, as well as estrogen dependence was retained. The IHC data indicated various expression levels of ER, PR and HER2 across the panel, with TNBC being the dominant subtype. ESR1 mutation, which may play an important role in BC progression and endocrine therapy resistance, was also identified in an estrogen-independent model. In vivo, these models showed distinct tumor growth rates and different pharmacodynamic responses to standard of care agents. For example, ESR1 mutant model was responsive to Fulvestrant. CONCLUSIONS: The established BC PDX panel represent a range or ER/PR/HER2 expression status from primary and advanced cancer providing a series of preclinical models for breast cancer research and potential for mouse clinical trial enrollment. Citation Format: Leilei Chen, Xueying Yang, Likun Zhang, Xiaobo Chen, Henry Q.X. Li, Jingjing Wang. Establishment and characterization of patient-derived breast cancer models for cancer studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3115.