Paraffin-embedded Breast Tumor Research Articles

CAR expression in invasive breast carcinoma and its effect on adenovirus transduction efficiency

BackgroundBreast cancer is the second leading cause of death in women, with invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC) as the two most common forms of invasive breast cancer. While estrogen receptor positive (ER+) IDC and ILC are treated similarly, the multifocality of ILC presents challenges in detection and treatment, worsening long-term clinical outcomes in patients. With increasing documentation of chemoresistance in ILC, additional treatment options are needed. Oncolytic adenoviral therapy may be a promising option, but cancer cells must express the coxsackievirus & adenovirus receptor (CAR) for adenoviral therapy to be effective. The present study aims to evaluate the extent to which CAR expression is observed in ILC in comparison to IDC, and how the levels of CAR expression correlate with adenovirus transduction efficiency. The effect of liposome encapsulation on transduction efficiency is also assessed.MethodsTo characterize CAR expression in invasive breast carcinoma, 36 formalin-fixed paraffin-embedded (FFPE) human breast tumor samples were assayed by CAR immunohistochemistry (IHC). Localization of CAR in comparison to other junctional proteins was performed using a multiplex immunofluorescence panel consisting of CAR, p120-catenin, and E-cadherin. ILC and IDC primary tumors and cell lines were transduced with E1- and E3-deleted adenovirus type 5 inserted with a GFP transgene (Ad-GFP) and DOTAP liposome encapsulated Ad-GFP (DfAd-GFP) at various multiplicities of infection (MOIs). Transduction efficiency was measured using a fluorescence plate reader. CAR expression in the human primary breast carcinomas and cell lines was also evaluated by IHC.ResultsWe observed membranous CAR, p120-catenin and E-cadherin expression in IDC. In ILC, we observed cytoplasmic expression of CAR and p120-catenin, with absent E-cadherin. Adenovirus effectively transduced high-CAR IDC cell lines, at MOIs as low as 12.5. Ad-GFP showed similar transduction as DfAd-GFP in high-CAR IDC cell lines. Conversely, Ad-GFP transduction of ILC cell lines was observed only at MOIs of 50 and 100. Furthermore, Ad-GFP did not transduce CAR-negative IDC cell lines even at MOIs greater than 100. Liposome encapsulation (DfAd-GFP) improved transduction efficiency 4-fold in ILC and 17-fold in CAR-negative IDC cell lines.ConclusionThe present study demonstrates that oncolytic adenoviral therapy is less effective in ILC than IDC due to differences in spatial CAR expression. Liposome-enhanced delivery may be beneficial for patients with ILC and tumors with low or negative CAR expression to improve adenoviral therapeutic effectiveness.

Prevalence of somatic PIK3CA mutations in hormone receptor-positive metastatic breast cancer: The Indian experience.

e13047 Background: The phosphatidylinositol 3-kinase (PI3K) inhibitors have been found to improve survival outcomes in hormone receptor-positive metastatic breast cancer (MBC) and are FDA-approved in this setting after progression on hormonal therapy. However, real-world data from India on the prevalence of PIK3CA mutations is scarce. Methods: This is a retrospective study including patients with hormone receptor-positive breast cancer registered at our center between January 2017- and December 2023 who developed metastatic disease on follow-up after getting appropriate treatment (adjuvant /neoadjuvant) as per institutional protocol. The PIK3CA mutation testing was done on the biopsy specimen. FDA-approved therascreen PIL3CA RGQ PCR kit from Qiagen was used and the assay is a real-time qualitative PCR test for the detection of 11 mutations phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) gene (Exon 7: C420R; Exon 9: E542K, E545A, E545D [1635G>T only], E545G, E545K, Q546E, Q546R; and Exon 20: H1047L, H1047R, H1047Y) using genomic DNA (gDNA) extracted from formalin-fixed, paraffin-embedded (FFPE) breast tumor tissue. The clinical demographics of patients with PIK3CA mutations were extracted from the hospital database and represented descriptively. Results: A total of 80 consecutive patients with hormone-positive breast cancer at relapse were screened and 26 were found to have PIK3CA mutations (32.5%) of which 24 were female (92.3%). The median age of patients with mutated PIK3CA cases was 50 years (range: 31 to 72 years). The clinical stage of disease at presentation was: stage II in 11.5%, IIIA in 23.1%, and IIIB in 65.4% of cases. The median time of relapse was 21 months from diagnosis (range: 10 to 42 months). The disease sites at relapse were multiple in 23 patients (88.46%) [including central nervous system (CNS) relapse in 3 patients) and CNS (brain) only in 3 patients (11.54%). The mutations detected included H1047R (3140A>G at exon 20) in 8 cases, E545K (1633 G>A at exon 9) in 5 cases, Q546R (1637 A>G at exon 9) in 4 cases, E542K (1624 G>A at exon7) in 3 cases and synchronous mutations (E542K/E545K at Exon 7) in 3 cases and (H1047R/H1047L at exon 20 ) in 3 cases. Only eight patients received alpelisib with fulvestrant due to financial constraints. Conclusions: This is the first Indian study that describes the prevalence of PIK3CA mutations in hormone-positive breast cancer at relapse. Around one-third of patients harbor this mutation and 23.1% of cases have CNS involvement at relapse. The most common PIK3CA mutation detected was H1047R (exon 20) followed by E545K (exon 9).

Abstract PO5-01-14: Genomic risk analyses in patients with clinical low risk ER-positive HER2-negative early breast cancer developing an early metastatic event

Abstract Background: Over the past decade, there have been notable changes in the definition of risk estimation in patients with early-stage breast cancer (EBC). Specifically, within the timeframe of 2000 to 2017, following Estrogen Receptor (ER) positive and HER2-negative EBC cases were classified as low risk according to the University Hospitals of Leuven (UHL) criteria of that time: if ER H-score was >200: all patients with tumors N0 and Grade (G) 1, ≤pT2; or G2-3 pT1a-b; in patients ≥40 years with tumors N0 and G2 pT1c-2 or G3 pT1c; in patients ≥ 55 years with tumors N0 G3 pT2 or N1 any Grade, pT≤2. For lower H-scores, other criteria applied. However, despite undergoing endocrine therapy, a subset of these patients with clinically low-risk tumors unexpectedly experienced metastasis within five years of initial diagnosis. This unforeseen outcome may indicate potential undertreatment, as these patients did not receive adjuvant chemotherapy. We have conducted a case-cohort study of those patients with early relapse and performed the current clinical risk assessment methods and genomic risk analyses. Methods: In this study, we included patients with ER-pos, HER2-neg early-stage breast cancer diagnosed between 1-1-2000 and 31-12-2017, who were classified as low-risk tumors by UHL guidelines at that time, who were older than 35 but younger than 71 years, had an unilateral, unifocal breast tumor with tumor size up to 5 cm. These patients did not receive adjuvant chemotherapy. We selected the patients that experienced early distant recurrence, defined as distant recurrence (DR) within 5 years after the initial diagnosis. To ensure comparability for the analyses they were matched to controls for grade, menopausal status, tumor size, lymphovascular invasion, progesterone receptor status, and the year of diagnosis. For each patient, we have calculated the clinical risk score according to the criteria outlined by Mymammaprint.com, based on the modified Adjuvant Online tool used in the MINDACT trial. For the analysis, formalin-fixed paraffin-embedded (FFPE) breast tumor tissue samples were subjected to in-house validated targeted RNA-based Mammaprint(MP) analysis by NGS (MP Agendia). Results: Of the 3190 patients with EBC at UHL during the study period, 2476 patients were considered for this analysis and fitted the clinical low-risk criteria defined low-risk at UHL. Among those, only 42 or 1.7% of the eligible cohort had developed metastasis within 5 years. Tissue for MP analysis was available for 35 of these cases and for their matched control. Due to technical failure, the 1:1 matching was lost and MP results were known for 28 cases and 27 controls. The patient and tumor characteristics of both groups were comparable. In this UHL clinical low-risk group, 21/28 (75%) cases would be identified as clinical high-risk by MyMammaprint.com and 18/27 (67%) controls. We identified 13 patients (46.43%) with high genomic risk within the group with DR and 9 (33.33%) within the control group. There was no statistically significant difference (p=0.412) in the proportion of genomically high-risk patients within the group with DR compared to the control group. Conclusions: In a database of patients with EBC of whom 98% did not develop metastasis within 5 years, we observed in a small series of chemotherapy naïve patients with early DR and UHL-defined clinical low-risk tumors, that nearly half had a genomically high risk of distant metastasis. Albeit numerically higher than the matched-control group, this observation was not significant and would warrant a larger sample size and power to confirm these results. Table. Genomic versus Clinical Risk by MyMammaprint.com Citation Format: Josephine Van Cauwenberge, Hava Izci, Hans Wildiers, Sileny Han, Christine Desmedt, Giuseppe Floris, Sara Vander Borght, Ann Smeets, Ines Nevelsteen, Isabelle Vanden Bempt, Patrick Neven. Genomic risk analyses in patients with clinical low risk ER-positive HER2-negative early breast cancer developing an early metastatic event [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-01-14.

Overexpression of WT1 in all molecular subtypes of breast cancer and its impact on survival: exploring oncogenic and tumor suppressor roles of distinct WT1 isoforms.

Breast cancer is a highly heterogeneous solid tumor, posing challenges in developing targeted therapies effective for all mammary carcinoma subtypes. WT1 emerges as a promising target for breast cancer therapy due to its potential oncogenic role in various cancer types. Previous works have yielded inconsistent results. Therefore, further studies are needed to clarify the behavior of this complex gene in breast cancer. In this study, we examined WT1 expression in both Formalin Fixed Paraffin Embedded breast tumors (n = 41) and healthy adjacent tissues (n = 41) samples from newly diagnosed cases of ductal invasive breast cancer. The fold change in gene expression between the tumor and healthy tissue was determined by calculating 2-∆∆Ct. Disease-free survival analysis was computed using the Kaplan-Meier method. To identify the expression levels of different WT1 isoforms, we explored the ISOexpresso database. Relative quantification of the WT1 gene revealed an overexpression of WT1 in most cases. The percentage of patients surviving free of disease at 8years of follow-up was lower in the group overexpressing WT1 compared to the group with down-regulated WT1. Interestingly, this overexpression was observed in all molecular subtypes of invasive breast cancer, underscoring the significance of WT1 as a potential target in all these subtypes. The observed WT1 down-expression in a few cases of invasive breast cancer, associated with better survival outcomes, may correspond to the down-regulation of a particular WT1-KTS (-) isoform: the WT1 A isoform (EX5-/KTS-). The co-expression of this WT1 oncogenic isoform with a regulated WT1- tumor suppressor isoform, such as the major WT1 F isoform (EX5-/KTS +), could also explain such survival outcomes. Due to its capacity to adopt dual roles, it becomes imperative to conduct individual molecular expression profiling of the WT1 gene. Such an approach holds great promise in the development of personalized treatment strategies for breast cancer.

A STUDY OF MMP7 EXPRESSION IN TRIPLE NEGATIVE BREAST CANCER PATIENTS

MMP7 has a part in preventing apoptosis and promoting angiogenesis. According to both in vivo and in vitro evidence, MMP7 is mostly seen during tumour invasion and metastasis. Controlling MMP7 expression may be a method for treating this cancer because its overexpression encourages the occurrence, progression, and invasiveness of breast cancer. The aim is to study the expression of MMP7 in triple negative breast cancer patients. Formalin fixed paraffin embedded breast tumor tissues from 50 triple negative breast cancer patients were collected for this study. immunohistochemistry method was used to study MMP7 expression and correlated with clinical and pathological parameters as well as disease status. MMP7 positivity was observed in 48% triple negative breast cancer patients. No significant correlation of MMP7 expression with age, menopausal status, tumour size, histopathology, stage and lymph node status were observed. A significant higher incidence of MMP7 expression was noted in patients with high grade tumor (63%) as compared to patients with low grade tumor (00%). Further, a significant higher incidence of MMP7 was observed in patients with high BR score (65%) as compared to the patients with low BR score (00%). Also, a significant higher incidence was observed in patients with relapse (63%) than patients in remission (35%). A significant higher incidence of MMP7 expression was observed with respect to disease free survival in univariate survival analysis. In multivariate survival analysis, for disease free survival MMP7 entered at step 1 and lymph node status and MMP7 entered at step 2. The findings imply that MMP7 is important for tumour invasion and metastasis, and that elevated MMP7 expression is associated with tumour development and recurrence.

Analytical validation of the 7-gene biosignature for prediction of recurrence risk and radiation therapy benefit for breast ductal carcinoma in situ.

Ductal carcinoma in situ (DCIS), is a noninvasive breast cancer, representing 20-25% of breast cancer diagnoses in the USA. Current treatment options for DCIS include mastectomy or breast-conserving surgery (BCS) with or without radiation therapy (RT), but optimal risk-adjusted treatment selection remains a challenge. Findings from past and recent clinical trials have failed to identify a 'low risk' group of patients who do not benefit significantly from RT after BCS. To address this unmet need, a DCIS biosignature, DCISionRT (PreludeDx, Laguna Hills, CA), was developed and validated in multiple cohorts. DCISionRT is a molecular assay with an algorithm reporting a recurrence risk score for patients diagnosed with DCIS intended to guide DCIS treatment. In this study, we present results from analytical validity, performance assessment, and clinical performance validation and clinical utility for the DCISionRT test comprised of multianalyte assays with algorithmic analysis. The analytical validation of each molecular assay was performed based on the Clinical and Laboratory Standards Institute (CLSI) guidelines Quality Assurance for Design Control and Implementation of Immunohistochemistry Assays and the College of American Pathologists/American Society of Clinical Oncology (CAP/ASCO) recommendations for analytic validation of immunohistochemical assays. The analytic validation showed that the molecular assays that are part of DCISionRT test have high sensitivity, specificity, and accuracy/reproducibility (≥95%). The analytic precision of the molecular assays under controlled non-standard conditions had a total standard deviation of 6.6 (100-point scale), where the analytic variables (Lot, Machine, Run) each contributed <1% of the total variance. Additionally, the precision in the DCISionRT test result (DS) had a 95%CI ≤0.4 DS units under controlled non-standard conditions (Day, Lot, and Machine) for molecular assays over a wide range of clinicopathologic factor values. Clinical validation showed that the test identified 37% of patients in a low-risk group with a 10-year invasive IBR rate of ~3% and an absolute risk reduction (ARR) from RT of 1% (number needed to treat, NNT=100), while remaining patients with higher DS scores (elevated-risk) had an ARR for RT of 9% (NNT=11) and 96% clinical sensitivity for RT benefit. The analytical performance of the PreludeDx DCISionRT molecular assays was high in representative formalin-fixed, paraffin-embedded breast tumor specimens. The DCISionRT test has been analytically validated and has been clinically validated in multiple peer-reviewed published studies.

Reckoning the Status of HER2/neu Protein and Oncogene in Breast Cancer Specimens: Comparison of IHC Assay with FISH Assay at a Tertiary Care Centre, West India

Introduction: Breast carcinoma is the most prevalent malignant neoplasm among women with more than one million new cases per year. The benefit of humanised anti-Human Epidermal growth factor Receptor 2 (HER2)/neu monoclonal antibody trastuzumab (Herceptin) in HER2/neu-positive breast cancers has been well documented. Immunohistochemistry (IHC) for protein overexpression and Fluorescence In Situ Hybridisation (FISH) for detecting HER2/neu gene amplification is widely used. Some laboratories use IHC as primary test with FISH for subset of cases while using FISH as primary investigation followed by IHC if needed is done by others. Aim: To evaluate IHC assay in breast carcinoma cases for HER2/neu as screening test before FISH. Materials and Methods: A cross-sectional descriptive type of observational study was conducted in the Department of Pathology and Multi-Disciplinary Research Unit, SMS Medical College and attached hospitals Jaipur, India, between April 2020 and December 2021. A total 122 samples of invasive breast carcinoma were included in study for IHC and FISH analysis. On paraffin embedded breast tumour tissue sections, IHC was performed using mouse monoclonal antibody targeting the intracellular domain of HER2/neu protein and FISH was implemented by dual colour probes targeting the HER2/neu gene on chromosome 17. Cases were classified according to HER2/neu status on IHC interpretation and FISH interpretation as per American Society of Clinical Oncology/ the College of American Pathologists (ASCO/CAP) HER2/neu-2018 testing guidelines. Chi-square test, kappa coefficient and Z-test were applied for statistical analysis. The p-value&lt;0.05 was considered significant. Results: Of the 122 patients of Invasive Duct Carcinoma (IDC), the mean age was 50.3±12.7 years with a age range of 25-70 years. HER2/neu IHC score 3+ (over expression) was seen in 19 (15.6%) cases while 2+ (equivocal) result was obtained in 84 (68.8%) cases and 19 (15.6%) cases showed negative (1+/0) HER2/neu expression. Out of 19 IHC positive cases, all cases were amplified by FISH and all 19 IHC negative cases, were non amplified by FISH. There were 84 IHC equivocal cases of which 37 (44%) cases were amplified, 45 (53.6%) non amplified and 2 (2.4%) cases were equivocal when analysed by FISH. Also, 11 (9%) cases were found Centromere Enumeration Probe (CEP) amplified in the study, they were negative or equivocal on IHC and none was IHC positive. Conclusion: The study concluded that combined FISH and IHC methodologies could optimise information on HER2/neu status in breast cancer patients. Also, testing algorithm is emphasised where laboratories may use IHC as a screening method and FISH can be used as accurate and specific method in IHC (2+) equivocal cases. Thus, patients with HER2/neu status positive of IHC (3+) or FISH (gene amplified) can be proposed to be treated with herceptin (trastuzumab).

Survivin expression as a prognostic marker for breast cancer.

Due to the speed of development observed in breast cancer, several studies aimed at discovering new biomarkers have been carried out in order to arrive at an early diagnosis. As survivin plays a fundamental role in the evasion of apoptosis in tumor cells, the aim of this study was to verify the expression profile of the survivin gene in paraffin-embedded breast tumor samples and associate it with the clinical characteristics of the patients. This is a cross-sectional study, for which 100 tumor samples were obtained from cancer patients treated throughout the year 2019 at Instituto de Mama do Cariri (Juazeiro do Norte, in the state of Ceará). This study included women over 30 years old who had confirmed breast cancer through anatomopathological examination but excluded those with non-neoplastic breast comorbidities, other neoplasms, or chronic diseases. Survivin gene expression was assessed by quantitative polymerase chain reaction. The expression of survivin is associated with the lack of expression of estrogen (p=0.027) and progesterone (p>0.0005) receptors. It means that survivin expression is higher in patients in which labeling was absent for estrogen receptor and progesterone receptor. Our data reinforce that survivin expression is higher in estrogen receptor-patients, thus representing an additional prognostic tool.

A STUDY OF OCT3/4 EXPRESSION IN HER2/NEU POSITIVE BREAST CANCER PATIENTS

Oct3/4 is found in germ cells is crucial to control pluripotency during normal development. There is mounting evidence for an association between Oct3/4 and carcinogenesis. Thus Oct3/4 immunohistochemistry has been found in studies to be a useful diagnostic technique for pluripotent germ-cell cancers, providing fresh insights into the cancer's histological heterogeneity. The aim is to study role of Oct3/4 expression in HER2/neu positive breast cancer patients. Paraffin embedded breast tumour tissues from 50 HER2/neu positive female breast cancer patients were enrolled in the study. Oct3/4 expression was studied by immunohistochemistry method and correlated with clinical and pathological parameters as well as disease status. Oct3/4 positivity was seen in 16% of HER2/neu positive breast cancer patients. No significant correlation of Oct3/4 expression with age, menopausal status, tumour size, histopathology, grade and BR score was observed. A trend of higher incidence of Oct3/4 expression was noted with lymph node positive (30%) than lymph node negative patients (07%). A significant higher incidence of Oct3/4 expression was noted in patients with advanced stage of disease (32%) as compared to patients with early stage of disease (04%) and in patients with relapse (44%) than patients in remission (10%). A significant higher incidence Oct3/4 expression was observed with respect to disease free survival in univariate survival analysis. In multivariate survival analysis, for disease free survival metastasis entered at step 1 and grade entered at step 2. For overall survival, metastasis is entered at step 1 as significant factor. The Oct3/4 gene expression may be implicated in self-renewal and tumorigenesis via activating its downstream target genes. It might serve as an important biomarker for contemplating the carcinogenesis, progression, metastasis, or invasive potential and prognosis of breast carcinoma. A larger prospective follow-up studies are recommended to further explore Oct3/4 significance.

Results and lessons from dual extraction of DNA and RNA from formalin-fixed paraffin-embedded breast tumor tissues for a large Cancer epidemiologic study

BackgroundThe use of archived formalin-fixed paraffin-embedded (FFPE) tumor tissues has become a common practice in clinical and epidemiologic genetic research. Simultaneous extraction of DNA and RNA from FFPE tissues is appealing but can be practically challenging. Here we report our results and lessons learned from processing FFPE breast tumor tissues for a large epidemiologic study.MethodsQiagen AllPrep DNA/RNA FFPE kit was adapted for dual extraction using tissue punches or sections from breast tumor tissues. The yield was quantified using Qubit and fragmentation analysis by Agilent Bioanalyzer. A subset of the DNA samples were used for genome-wide DNA methylation assays and RNA samples for sequencing. The QC metrices and performance of the assays were analyzed with pre-analytical variables.ResultsA total of 1859 FFPE breast tumor tissues were processed. We found it critical to adjust proteinase K digestion time based on tissue volume to achieve balanced yields of DNA and RNA. Tissue punches taken from tumor-enriched regions provided the most reliable output. A median of 1475 ng DNA and 1786 ng RNA per sample was generated. The median DNA integrity number (DIN) was 3.8 and median DV200 for RNA was 33.2. Of 1294 DNA samples used in DNA methylation assays, 97% passed quality check by qPCR and 92% generated data deemed high quality. Of the 130 RNA samples with DV200 ≥ 20% used in RNA-sequencing, all but 5 generated usable transcriptomic data with a mapping rate ≥ 60%.ConclusionsDual DNA/RNA purification using Qiagen AllPrep FFPE extraction protocol is feasible for clinical and epidemiologic studies. We recommend tissue punches as a reliable source material and fine tuning of proteinase K digestion time based on tissue volume.ImpactOur protocol and recommendations may be adapted by future studies for successful extraction of archived tumor tissues.

Analytical Validation of a Novel 6-Gene Signature for Prediction of Distant Recurrence in Estrogen Receptor-Positive, HER2-Negative, Early-Stage Breast Cancer

OncoMasTR is a recently developed multigene prognostic test for early-stage breast cancer. The test has been developed in a kit-based format for decentralized deployment in molecular pathology laboratories. The analytical performance characteristics of the OncoMasTR test are described in this study. Expression levels of 6 genes were measured by 1-step reverse transcription-quantitative PCR on RNA samples prepared from formalin-fixed, paraffin-embedded (FFPE) breast tumor specimens. Assay precision, reproducibility, input range, and interference were determined using FFPE-derived RNA samples representative of low and high prognostic risk scores. A pooled RNA sample derived from 6 FFPE breast tumor specimens was used to establish the linear range, limit of detection, and amplification efficiency of the individual gene expression assays. The overall precision of the OncoMasTR test was high with an SD of 0.16, which represents less than 2% of the 10-unit risk score range. Test results were reproducible across 4 testing sites, with correlation coefficients of 0.94 to 0.96 for the continuous risk score and concordance of 86% to 96% in low-/high-risk sample classification. Consistent risk scores were obtained across a > 100-fold RNA input range. Individual gene expression assays were linear up to quantification cycle values of 36.0 to 36.9, with amplification efficiencies of 80% to 102%. Test results were not influenced by agents used during RNA isolation, by low levels of copurified genomic DNA, or by moderate levels of copurified adjacent nontumor tissue. The OncoMasTR prognostic test displays robust analytical performance that is suitable for deployment by local pathology laboratories for decentralized use.

Identification of Novel BRCA1 Germline Deleterious Variant Among a Tunisian Family

Inherited predisposition to breast and ovarian cancer are most frequently due to germline mutations in the main genes BRCA1 (OMIM# 113705) and BRCA2 (OMIM# 600185). These inactivating mutations, essentially frameshift and nonsense variation, occurs mainly across conserved regions. The aim of the present study is to report a novel germline BRCA1 mutation identified in a Tunisian family case with early onset of breast and ovarian cancer and to evaluate the genotype phenotype correlation. The proband had high-grade tumors, invasive unilateral ductal carcinoma developed at the age of 38 and a serous ovarian adenocarcinoma after a gap of twelve years. The molecular analysis revealed a novel heterozygous nonsense BRCA1 mutation NM_007294.4: c.915T&gt;A p.(C305*) in the proband and her daughter. This mutation leads to a truncated protein which pathogenicity was validated by bioinformatics tools. This variant is subject to nonsense-mediated mRNA decay. We also underlined the immunohistochemistry usefulness by lack of expression of BRCA1 protein in paraffin embedded breast tumor contrasting with normal tissue. Clinical and pathological data tend to be homogeneous and led to the conclusion that there is a genotype phenotype correlation in BRCA1, an element that must be taken into account in genetic counselling. Conclusively, we are the first to report this novel BRCA1 germline likely deleterious variant extending the molecular and clinical spectrum of BRCA1 pathogenic point mutations. Further in vitro functional experiments needs to be established. High-risk individuals carrying this BRCA1 mutation benefit from preventive measures to reduce morbidity.

Abstract P3-10-04: Efficacy of RIDRPI103, a reactive oxygen species (ROS) activated prodrug in treatment of breast cancer

Abstract The Phosphatidylinositol-3 kinases (PI3Ks) is a family of lipid kinases encoded by PIK3C isoform genes. PI3K phosphorylates phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-triphosphate (PIP3), leading to AKT phosphorylation along with other proteins containing a PH domain. Phosphorylation of AKT stimulates protein synthesis and cancer cell growth. PI3K pathway is hyper-activated in &amp;gt;60% of clinical breast cancer patients due to aberrations in the genes encoding HER2, PTEN, PIK3CA, or AKT1-3 leading to de novo and acquired treatment resistance. There has been intense interest in developing drugs that target PI3K. However, drugs targeting PI3K activity are toxic, due to the physiological roles of PI3Ks in basic cellular processes. We have developed a novel technology, called RIDR (ROS-Induced Drug Release) which is a self-cyclizing reagent linked to a PI3K inhibitor (PI-103) to eject PI-103 in a controlled manner under oxidative stress in highly aggressive breast cancers including triple negative breast cancer cell lines (TNBCs) and other ER+, HER2+ breast cancer cell lines with activating PIK3CA mutations. We evaluated the efficacy of RIDR-PI-103 (5-100 µM) or PI-103 (0-5 µM) in normal fibroblasts, T47D, MCF-7, MDA-MB-361, BT474, MDA-MB-453 and MDA-MB-231 breast cancer cell lines. We noted that IC50 of PI-103 is 3.34 µM whereas IC50 of RIDR-PI-103 is &amp;gt;100 µM in normal fibroblasts, indicating the toxicity of the PI-103 alone but not the RIDR-PI-103 in normal fibroblasts. Our data indicated that ~30-40 µM RIDR-PI-103 significantly inhibited T47D, MDA-MB-231, MDA-MB-361 and MDA-MB-453 cell proliferation whereas higher concentrations of the drug were effective in BT474 and MCF-7 breast cancer cell lines. Our quantitative PCR data showed that the antioxidant catalase mRNA levels are statistically lower compared to normal fibroblasts in these cancer cell lines indicating that the efficacy of RIDR-PI-103 could be correlated to catalase expression. Doxorubicin is a clinically relevant chemotherapy known to induce reactive oxygen species (ROS) in breast cancer cell lines. We are currently assessing the levels of biomarkers for ROS in formalin-fixed paraffin embedded breast tumors treated without or with chemotherapy including 8-hydroxy-2'-deoxyguanosine (8-oxo-dG) and 4-hydroxy-2-noneal (4HNE). Our data indicated that doxorubicin significantly sensitized MDA-MB-453, MDA-MB-361 and MDA-MB-231 cells to RIDR-PI-103 indicated by significant lower IC50 values of the combined drug treatment versus the single agent. Doxorubicin and RIDR-PI-103 showed a synergistic effect in MDA-MB-361 and MDA-MB-231 cells to inhibit cancer cell proliferation. Doxorubicin was more effective than docetaxel, another chemotherapeutic drug, in sensitizing these cells to growth inhibition. Thus, this novel combination of the ROS-activatable PI3K inhibitor prodrug and chemotherapy provides strong justification for its continued development and future clinical trials for patients stricken with PI3K driven tumors. In other experiments, we show that a recent FDA-approved CDK4/6 inhibitor, palbociclib, sensitized ER+ breast cancer cell lines (T47D and MDA-MB-361) to RIDR-PI103 using cell viability assays. Experiments are ongoing to determine the mechanism of action of these novel drug combinations that could be potentially translated to clinic for treatment of breast cancer patients. Citation Format: Rosalin Mishra, Hima Patel, Haizhou Zhu, Aaron White, Long Yuan, Samar Alanazi, Kurt Hodges, Edward J Merino, Joan T Garrett. Efficacy of RIDRPI103, a reactive oxygen species (ROS) activated prodrug in treatment of breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-10-04.

Abstract 2896: Impact of intratumoral heterogeneity on gene expression profiles and known biomarkers in FFPE tumor biopsies from early stage breast cancer patients

Abstract Background: Cancer is a dynamic disease that becomes more heterogeneous during its course, with cellular subpopulations with distinct molecular profiles across different regions. Because this intratumoral heterogeneity may result in varying levels of gene expression patterns and sensitivity to treatment across different regions of the tumor, it is important to understand patterns of heterogeneity to develop effective biomarkers. We conducted multiregion sampling within single breast tumor biopsies to evaluate the effect of intratumoral heterogeneity on expression of known and novel biomarkers. Methods: We sampled three sequential regions of breast tumor biopsies to compare gene expression patterns determined by a novel biomarker panel (BA355), 5 known biomarkers (ERS1, ERBB2, PGR, AR, and MKi67) and a novel predictive classifier (BA100). RNA expression levels were measured using a proprietary custom designed nanoString nCounter assay. We investigated formalin-fixed paraffin-embedded (FFPE) breast tumor biopsies from a cohort of 40 early stage breast cancer patients (T1 N0 M0) collected prospectively. RNA was extracted from upper, middle, and lower regions of each biopsy, resulting in a total of 120 samples quantified and QC using standard procedures. Each layer consisted of 40 micrometers, with an interval of 50 micrometers of tissue. BA100 linear regression models were used to generate prediction scores. Analyses were done in R. Results: Eighteen of the 120 samples had poor data quality (sample median = 0 or inter quantile range &amp;gt; mean + 2*sd). Further analyses only included 102 good quality samples corresponding to 34 FFPE biopsies. Among 33 of 34 samples, gene expression patterns between the three regions of the biopsies were highly correlated, with correlation coefficients ranging from 0.90-0.99 (p-values ranged from 2.07e-19 to 8.45e-319). Among 31 of 34 samples, the three regions were clustered together in cluster analysis. The coefficient of variation (CV) for ESR1 ranged from 0.37%-15.5%, with a majority (32 of 34) of the samples having CV &amp;lt;10%. While the CVs of ERBB2, PGR, and AR were within the same range as ESR1, MKI67 had much larger variation (CV ranged from 0.99%-173%). BA100 scores yielded consistent predictions for the three regions in 33 of 34 samples. Conclusion: Examination of gene expression patterns across multiple regions of breast tumor biopsies did not reveal a significant impact of intratumoral heterogeneity on expression patterns using a novel gene signature panel. However, at the individual gene level, while a number of genes including ESR1, ERBB2, PGR, and ARdisplayed consistent gene expression, genes such as MKI67 showed significant variation, indicating that tumor heterogeneity may impact individual gene biomarkers. Further studies are ongoing to evaluate intratumoral heterogeneity. Citation Format: Lucas Delmonico, Joan Chen, Trushna Desai, Lisa Barnett, Aladdin Shadyab, John Obenauer, Gilda Alves Brown, Mauricio Magalhaes Costa, Marcia Fournier. Impact of intratumoral heterogeneity on gene expression profiles and known biomarkers in FFPE tumor biopsies from early stage breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2896.

Androgen Receptor Expression in Triple-Negative Breast Cancer

Background: Triple-negative breast cancer (TNBC) accounts for 15 to 20% of all breast cancers. These patients do not benefit from hormone therapy and other targeted treatments of breast cancer. Recently, researchers proposed the use of androgen receptor (AR)-targeted therapies in this subset of patients. The rate of AR expression in TNBC patients varies from 0 to 53%. AR positivity is associated with a better outcome for breast cancer patients. The purpose of this study was to evaluate AR status in TNBC patients and its association with other demographic and pathologic features.Methods: This cross-sectional study was conducted in the Cancer Institute of Iran, affiliated with Tehran University of Medical Sciences, in 2015. Archived formalin-fixed, paraffin-embedded breast tumor blocks were evaluated to determine the AR status of the tumors. Demographic and pathologic characteristics of the patients were retrieved from the department of pathology database. Data were analyzed with SPSS 18.0.Results: Seventy-seven TNBC patients with the mean age of 45.3 ± 11.5 were assessed. Twenty-six patients (34%) showed AR expression, and 51 patients (56%) did not have AR expression. There was no significant correlation between AR status and age, tumor size, histopathologic type of tumor, or lymph node involvement. However, AR positivity had a statistically significant association with a lower tumor grade and lymphovascular invasion (P = 0.029 and P = 0.01, respectively).Conclusion: TNBC patients with AR expression tend to have lower tumor grades and higher rates of lymphovascular invasion.

Expression and clinicopathological significance of DNA methyltransferase 1, 3A and 3B in tamoxifen-treated breast cancer patients

Progression of tamoxifen resistance remained as a crucial obstacle to treatment of estrogen receptor positive breast carcinoma patients. Recent studies demonstrated the importance of DNA methylation pattern on tamoxifen refractory. This study aimed to investigate the protein expression pattern and clinicopathological significance of DNA methyltransferase 1, 3A and 3B, as leading factors in regulation of DNA methylation process, in breast carcinoma patients with adjuvant tamoxifen therapy. Seventy two Formalin-Fixed Paraffin-Embedded (FFPE) breast tumor tissues of tamoxifen sensitive (TAMS) and tamoxifen resistance (TAM-R) patients were recruited for immunohistochemical experiments. DNMT1, DNMT3a, and DNMT3b expressions were observed in 86, 72.2 and 100% of tamoxifen resistance patients, respectively. Data analysis indicated that DNMTs were overexpressed in TAM-R tumors (P < 0.05). In TAM-S subgroup, DNMT1, DNMT3A and DNMT3B expression was associated with high histologic grade (P = 0.049, P = 0.01 and P = 0.02, respectively). DNMT3B expression was also correlated with lymphatic invasion (P = 0.034). In TAM-R subgroup, DNMT1 expression associated with extracapsular nodal extension (P = 0.019). DNMT3A and DNMT3B expression showed a significant association with high histologic grade (P = 0.001) and DNMT3A expression was also associated with HER-2 status (P = 0.027). Cox proportional hazard model demonstrated that overexpression of DNMT3B remained as an independent and unfavorable prognostic factor for disease free survival (P < 0.001). Taken together, these results suggest that DNMTs could be an effective factor in development of tamoxifen resistance in breast tumors.

Predicting efficacy of epirubicin by a multigene assay in advanced breast cancer within a Danish Breast Cancer Cooperative Group (DBCG) cohort: a retrospective-prospective blinded study

PurposeAnthracyclines remain a cornerstone in the treatment of primary and advanced breast cancer (BC). This study has evaluated the predictive value of a multigene mRNA-based drug response predictor (DRP) in the treatment of advanced BC with epirubicin. The DRP is a mathematical method combining in vitro sensitivity and gene expression with clinical genetic information from > 3000 clinical tumor samples.MethodsFrom a DBCG cohort, 140 consecutive patients were treated with epirubicin between May 1997 and November 2016. After patient informed consent, mRNA was isolated from archival formalin-fixed paraffin-embedded primary breast tumor tissue and analyzed using Affymetrix arrays. Using time to progression (TTP) as primary endpoint, the efficacy of epirubicin was analyzed according to DRP combined with clinicopathological data collected retrospectively from patients’ medical records. Statistical analysis was done using Cox proportional hazards model stratified by treatment line.ResultsMedian TTP was 9.3 months. The DRP was significantly associated to TTP (P = 0.03). The hazard ratio for DRP scores differing by 50 percentage points was 0.55 (95% CI –0.93, one-sided). A 75% DRP was associated with a median TTP of 13 months compared to 7 months following a 25% DRP. Multivariate analysis showed that DRP was independent of age and number of metastases.ConclusionThe current study prospectively validates the predictive capability of DRP regarding epirubicin previously shown retrospectively allowing the patients predicted to be poor responders to choose more effective alternatives. Randomized prospective studies are needed to demonstrate if such an approach will lead to increased overall survival.

PAX2 expression is correlated with better survival in tamoxifen-treated breast carcinoma patients.

PAX2 (paired box gene 2) is a transcription factor, which is involved in both cell proliferation and carcinogenesis. This study aimed to investigate PAX2 expression in tamoxifen resistant (TAM-R) and tamoxifen sensitive (TAM-S) breast carcinoma patients and analyze its correlation with clinicopathological characteristics and survival. Immunohistochemical analysis was performed to evaluate PAX2 protein expression in 36 TAM-R and 36 TAM-S formalin-fixed paraffin-embedded (FFPE) breast tumor tissues. Data analysis indicated that PAX2 expression was significantly higher in TAM-S group in comparison to TAM-R (P = 0.014). Overexpression of PAX2 was significantly correlated with perineural invasion (PNI) (P = 0.025). Kaplan-Meier survival analysis showed significant association between high expression of PAX2 and better disease-free survival (P < 0.001) and also overall survival (P = 0.031). Multivariate cox regression analysis demonstrated that patients with increased expression of PAX2 have a trend toward improved disease free survival (OR = 0.065, 95% CI: 0.009-0.476; P = 0.007) and overall survival (OR = 0.147, 95% CI: 0.020-1.105; P = 0.062). Our data suggested that high expression of PAX2 could be associated with better survival in estrogen receptor positive tamoxifen-treated breast carcinoma patients.

Clinical significance of CD73 in triple-negative breast cancer: multiplex analysis of a phase III clinical trial

BackgroundCD73 is an ecto-enzyme that promotes tumor immune escape through the production of immunosuppressive extracellular adenosine in the tumor microenvironment. Several CD73 inhibitors and adenosine receptor antagonists are being evaluated in phase I clinical trials. Patients and methodsFull-face sections from formalin-fixed paraffin-embedded primary breast tumors from 122 samples of triple-negative breast cancer (TNBC) from the BIG 02-98 adjuvant phase III clinical trial were included in our analysis. Using multiplex immunofluorescence and image analysis, we assessed CD73 protein expression on tumor cells, tumor-infiltrating leukocytes and stromal cells. We investigated the associations between CD73 protein expression with disease-free survival (DFS), overall survival (OS) and the extent of tumor immune infiltration. ResultsOur results demonstrated that high levels of CD73 expression on epithelial tumor cells were significantly associated with reduced DFS, OS and negatively correlated with tumor immune infiltration (Spearman’s R=−0.50, P<0.0001). Patients with high levels of CD73 and low levels of tumor-infiltrating leukocytes had the worse clinical outcome. ConclusionsTaken together, our study provides further support that CD73 expression is associated with a poor prognosis and reduced anti-tumor immunity in human TNBC and that targeting CD73 could be a promising strategy to reprogram the tumor microenvironment in this BC subtype.

Abstract PD6-07: Clinical significance of CD73 expression in triple-negative breast cancer from the BIG 02-98 adjuvant phase III clinical trial

Abstract Background: CD73 is an ecto-enzyme that promotes tumor immune escape through the production of immunosuppressive extracellular adenosine in the tumor microenvironment. Several CD73 inhibitors and adenosine receptor antagonists are being evaluated in phase I clinical trials. Objective: To investigate the prognosis significance of CD73 in human triple-negative breast cancer. Design and setting: This is a prospective-retrospective biomarker analysis. Using multiplex immunofluorescence and image analysis, we assessed CD73 protein expression on tumor cells, tumor-infiltrating leukocytes and stromal cells on full-face sections from formalin-fixed paraffin-embedded primary breast tumors. Participants: 122 samples of triple-negative breast cancer from the BIG 02-98 adjuvant phase III clinical trial were included in our analysis. This trial compared the addition of taxanes to anthracyclines-based chemotherapy in node-positive breast cancer. Results: Our results demonstrated that high levels of CD73 expression on epithelial tumor cells were significantly associated with reduced disease-free survival (DFS) and overall survival (OS) in patients with triple-negative breast cancer. Using the median as a threshold between low and high levels of CD73 on epithelial cells, hazard ratios (HR) adjusted for grade, number of positive lymph nodes and tumor size, were of 2.21 (95% confidence interval (CI): 1.15-4.25); p=0.02 for DFS and of 2.47 (95%CI: 1.21-5.07); p=0.01 for OS. CD73 expression negatively correlated with tumor immune infiltration (Spearman's R= -0.50, p&amp;lt;0.0001). Patients with high levels of CD73 and low levels of tumor-infiltrating leukocytes had the worse clinical outcome (HR: 4.24 (1.90-9.45), p&amp;lt;0.001 for DFS, HR: 3.91 (1.65-9.31), p=0.002 for OS) compared to patients with low CD73 and high tumor-immune infiltration. Flow cytometric analysis of tumor-infiltrating leukocytes revealed a high frequency of CD73-expressing B cells and higher CD73 expression on tumor-infiltrating myeloid cells and natural killer cells compared to peripheral blood. Conclusion and relevance: Taken together, our study provides further support that CD73 expression is associated with a poor prognosis and reduced anti-tumor immunity in human triple-negative breast cancer and that targeting CD73 could be a promising strategy to reprogram the tumor microenvironment in this breast cancer subtype. Citation Format: Buisseret L, Pommey S, Allard B, Garaud S, Bergeron MA, Cousineau I, Ameye L, Paesmans M, Crown JPA, Di Leo A, Piccart-Gebhart M, Willard-Gallo K, Sotiriou C, Stagg J. Clinical significance of CD73 expression in triple-negative breast cancer from the BIG 02-98 adjuvant phase III clinical trial [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD6-07.