ER-negative Breast Cancer Research Articles

Abstract 3011: Psychosocial stress, social support, and incident breast cancer by estrogen receptor status

Abstract Introduction: Chronic stress affects immune function and hormonal signaling and has been hypothesized to be associated with breast cancer, although results from the few prior studies are mixed and have not examined potential differences by estrogen receptor (ER) status. We investigated the relationship between stressful life events and incident breast cancer by ER status among postmenopausal women. We further evaluated whether this association is modified by social support. Methods: Our analysis included 76,951 postmenopausal women enrolled in the Women’s Health Initiative. Participants were followed for events for up to 26 years (median, 16.7). We generated Cox proportional hazards models adjusting for demographic, clinical, lifestyle/behavioral, and social factors to estimate hazard ratios (HR) and 95% confidence intervals (95%CI) for associations between baseline self-reported stressful life events and incident breast cancer by ER status. Results: The mean age was 63 (SD 7.3), and majority of participants were White race (83.5%) and married or in a marriage-like relationship (63.0%). In analyses stratified by ER status, there was no relationship between stressful life events and ER-positive breast cancer. In contrast, compared to women in the lowest quartile, those in higher quartiles had an increased risk of ER-negative breast cancer, where those in quartile 4 had the highest risk (Quartile 4 vs Quartile 1; HR=1.30; 95%CI 1.01-1.68; Ptrend=0.050). Additionally, associations were stronger for the highest versus lowest quartile of stressful life events among widowed women (HR=2.39; 95%CI 1.29-4.44; Pinteraction<0.001). Moreover, while the interaction was not statistically significant, we observed an elevated risk for ER-negative breast cancer among women in the highest quartile of stressful life events reporting lower social support. Conclusion: In this cohort of postmenopausal women, higher experiences of pre-diagnostic stressful life events were associated with increased risk of ER-negative breast cancer. Citation Format: Wayne R. Lawrence, Jasmine A. McDonald, Faustine Williams, Meredith S. Shiels, Neal D. Freedman, Ziqiang Lin, Jared W. Magnani. Psychosocial stress, social support, and incident breast cancer by estrogen receptor status [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 3011.

Abstract 1937: Racial differences of epigenome in normal breast tissue reveal biological pathways implicated in racial disparities in breast cancer

Abstract Racial disparity in breast cancer is well recognized. Compared to European American (EA) women, African American (AA) women are more often diagnosed with breast cancer at younger ages, have a more advanced or aggressive disease, and have poorer outcomes. Increasing evidence suggests that biological differences between EA and AA women, particularly differences in epigenetic changes of DNA methylation (DNAm), may contribute to the observed cancer disparity. We hypothesize that racial differences in DNAm in normal breast tissue may reflect distinct genomic susceptibility to breast cancer in different racial groups. Therefore, we investigated epigenome-wide differences of DNAm in normal breast tissue between 178 EA and 272 AA women using the Illumina TruSeq Methyl Capture EPIC library and NGS technology. We identified 17,340 differentially methylated CpG loci (DMLs) between EA and AA women, of which 7,744 were hypermethylated in AA women and 9,596 were hypermethylated in EA women. Pathway analyses of genes annotated to these racial DMLs suggested enrichment of biological processes including cell movement, cell motility, and cell migration, features that are likely linked to the aggressiveness and metastatic potentials of tumor cells. We further examined racial differences in DNAm of breast cancer-related candidate genes and GWAS-identified breast cancer risk loci. Approximately 30% of candidate genes were found to have racial differences in DNAm at more than three CpG sites and these candidate genes were enriched for pathways related to response to stimulus and anatomic structure development. Most interestingly, racial DMLs were found to be enriched for breast cancer risk loci that are associated with risk of estrogen-receptor negative (ER-) breast cancer, treatment response to aromatase inhibitor, and breast cancer-specific morality, phenotypes of which we observed differences between EA and AA breast cancer patients. Our results suggest that racial differences in DNAm in normal breast tissue may induce different biological processes that lead to distinct clinical features of breast tumors observed in EA and AA women and provide an epigenetic mechanism underlying the observed racial disparities. Further research is need to validate the findings of this study. Citation Format: Nan Lin, James Castle, Jinpeng Liu, Chunyan He. Racial differences of epigenome in normal breast tissue reveal biological pathways implicated in racial disparities in breast cancer [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 1937.

Abstract 2099: Predicting the metastatic potential in breast cancer patients in Bangladesh

Abstract Background and Aims: Breast cancer (BC) is the leading cause of mortality among all cancers in Bangladeshi women. The prognosis of metastatic breast cancer depends on certain factors, like surgery, radiotherapy, chemotherapy, hormonal receptor content of the tumor or associated illness such as diabetes mellitus and obesity. Among the associated illness, diabetes mellitus is one of the most common metabolic disease worldwide, which is linked to an increased risk of developing breast cancer and an even higher risk of recurrence and dying from this disease. In this context, we studied diabetic and metabolic disease related growth and metabolic factors in breast cancer patients for assessing the prognosis of BC. Methods: A total of 180 mastectomy patients were purposively taken in the present study. Data were collected from retrospective information. Anthropometric and biochemical parameters were taken by standard methods. Growth and metabolic factors were determined by enzyme-linked immunosorbent assay (ELISA) method. Association of diabetes treatment modalities with breast cancer metastasis was evaluated by correlation analysis among 80 diabetic patients. Results: Serum level of insulin-like growth factor 1 (IGF1) was found to be significantly higher (p=0.001) in ER and PR negative breast cancer than their positive counterparts. Patients who had lymph node positive and Her2 borderline showed a significant reduction in serum adiponectin level (p<0.05) and this association was retained after adjusting the confounding variables. Furthermore, delayed treatment, IGF-1, ALP, sleeping hours were significant predictors for metastasis of breast cancer patients in Bangladesh. The Diabetic BC patients treated with both insulin and oral drug was found to be negatively associated with breast cancer metastasis compared to Diabetic-BC patients treated with only oral drugs (p<0.05). However, BC metastasis did not any have an association with serum fasting insulin or insulin resistance. Obesity also did not show any relation with BC prognosis. Conclusions: Serum IGF-1 and adiponectin may have an association with invasiveness and metastasis of breast cancer. Diabetes treatment with combination therapy ameliorates breast cancer metastasis.Breast cancer prognosis would be independently predict by serum levels of IGF-1 as well as IGF1 could be used as a screening biomarker when validated in a larger sample size. Citation Format: Salima Akter, Muhammad Saiedullah, Mousumi Bhowmik, Hajara Akhter, Shahina Ansary, Waheed Akhtar, Sufi Hannan Zulfiqur Rahman, Tae Gyu Choi, Sung Soo Kim. Predicting the metastatic potential in breast cancer patients in Bangladesh [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 2099.

Relationship between tumor microbiota transcriptional activity and gene expression in breast cancer

BackgroundA few studies have reported the distribution of the microbiota in breast cancer tissues, but few reports have compared the microbiota in different subtypes of breast cancer tissue. Moreover, no study has reported on the relationship between the microbiota and gene expression in breast tumor.MethodsSections of formalin-fixed paraffin-embedded (FFPE) tissue were prepared from the breast tumors of 70 patients and were subjected to microarray analysis to identify gene expression profiles. The same total RNA samples were also used to analyze the microbiota activity in tumor tissues by performing 16 S rRNA sequencing and internal transcribed spacer (ITS) sequencing of reverse transcript cDNA with Illumina Miseq. Pearson’s correlation coefficient was used for calculating the correlation between microbial relative activity and gene expression.ResultsThe microbiota transcriptional activity of 70 FFPE samples mainly consisted of the phyla Bacteroidetes, Firmicutes and Proteobacteria. Prevotella_9, Bacteroides and Alloprevotella were the most active genera in ER+/HER2-, ER+/HER2 + and ER-/HER2 + tumors, while triple-negative samples exhibited a higher activity of Lactobacillus. In ER-negative samples (triple-negative and ER-/HER2+), 479 genes, including the breast carcinogenesis genes phospholipase A2, histone cluster 2, Crk-like, and cyclin D1, were significantly positive associated with the activity of Lactobacillus.ConclusionThis was the first study to clarify an association between the breast tumor microbiota transcriptional activity and the expression of carcinogenesis genes in ER-negative breast cancer. Changes in the microbiota of breast tissue induced by external factors might be one of the key causes of ER negative breast cancer.

Hyperinsulinemic and Pro-Inflammatory Dietary Patterns and Metabolomic Profiles Are Associated with Increased Risk of Total and Site-Specific Cancers among Postmenopausal Women.

We evaluated associations of the Empirical Dietary Index for Hyperinsulinemia (EDIH), Empirical Dietary Inflammatory Pattern (EDIP) and Healthy Eating Index (HEI2015) and their metabolomics profiles with the risk of total and site-specific cancers. We used baseline food frequency questionnaires to calculate dietary scores among 112,468 postmenopausal women in the Women's Health Initiative. We used multivariable-adjusted Cox regression to estimate hazard ratios (HR) and 95% confidence intervals for cancer risk estimation. Metabolomic profile scores were derived using elastic-net regression with leave-one-out cross validation. In over 17.8 years, 18,768 incident invasive cancers were adjudicated. Higher EDIH and EDIP scores were associated with greater total cancer risk, and higher HEI-2015 with lower risk: HRQ5vsQ1(95% CI): EDIH, 1.10 (1.04-1.15); EDIP, 1.08 (1.02-1.15); HEI-2015, 0.93 (0.89-0.98). The multivariable-adjusted incidence rate difference(Q5vsQ1) for total cancer was: +52 (EDIH), +41 (EDIP) and -49 (HEI-2015) per 100,000 person years. All three indices were associated with colorectal cancer, and EDIH and EDIP with endometrial and breast cancer risk. EDIH was further associated with luminal-B, ER-negative and triple negative breast cancer subtypes. Dietary patterns contributing to hyperinsulinemia and inflammation were associated with greater cancer risk, and higher overall dietary quality, with lower risk. The findings warrant the testing of these dietary patterns in clinical trials for cancer prevention among postmenopausal women.

Low-dose aspirin and risk of breast cancer: a Norwegian population-based cohort study of one million women

Several studies evaluated the association between aspirin use and risk of breast cancer (BC), with inconsistent results. We identified women aged ≥ 50 years residing in Norway between 2004 and 2018, and linked data from nationwide registries; including the Cancer Registry of Norway, the Norwegian Prescription Database, and national health surveys. We used Cox regression models to estimate the association between low-dose aspirin use and BC risk, overall and by BC characteristics, women’s age and body mass index (BMI), adjusting for sociodemographic factors and use of other medications. We included 1,083,629 women. During a median follow-up of 11.6 years, 257,442 (24%) women used aspirin, and 29,533 (3%) BCs occurred. For current use of aspirin, compared to never use, we found an indication of a reduced risk of oestrogen receptor-positive (ER +) BC (hazard ratio [HR] = 0.96, 95% confidence interval [CI]: 0.92–1.00), but not ER-negative BC (HR = 1.01, 95%CI: 0.90–1.13). The association with ER + BC was only found in women aged ≥ 65 years (HR = 0.95, 95%CI: 0.90–0.99), and became stronger as the duration of use increased (use of ≥ 4 years HR = 0.91, 95%CI: 0.85–0.98). BMI was available for 450,080 (42%) women. Current use of aspirin was associated with a reduced risk of ER + BC in women with BMI ≥ 25 (HR = 0.91, 95%CI: 0.83–0.99; HR = 0.86, 95%CI: 0.75–0.97 for use of ≥ 4 years), but not in women with BMI < 25.Use of low-dose aspirin was associated with reduced risk of ER + BC, in particular in women aged ≥ 65 years and overweight women.

Abstract P4-03-20: Association between reproductive factors (parity and age first full term birth) and the frequency of estrogen receptor negative breast cancer according to age at diagnosis

Abstract Background: The frequency of early estrogen receptor (ER)-negative breast cancers (BC) decreases with increasing age at diagnosis (Partridge P, JCO 2016). Giving birth is generally considered a protective factor for the occurrence of BC in a woman’s lifetime. However, for up to more than 20 years following childbirth, BC risk may be higher. This appears to be due to an increase in hormone receptor-positive BC, especially in women older at the time of 1st childbirth (Nichols HB, Ann Int Med 2019). This means that the ER-negative BC are relatively less frequent in these 2 decades after delivery. In this study, we aimed at retrospectively investigating the impact of parity and age at first full term pregnancy (FFTP) on the frequency of ER-negative BC according to the age at diagnosis in a large institutional cohort of patients diagnosed with BC. Patients and Methods: We considered all patients diagnosed and/or treated with early BC in UZ Leuven between January 2000 and November 2020. ER-negativity was defined as &amp;lt; 1% positive cells. Age at diagnosis was subdivided in categories of 5 years. Parous women could be having a low (1 or 2 children) or high parity (&amp;gt; 2 children) and age of FFTP was arbitrarily divided into &amp;lt; or ≥ 27 years of age. BMI was considered a possible confounder and was corrected for. A logistic regression model was used for data analysis with ER-negative status as binary outcome and FFTP class and multiparity as explanatory variables. To test whether the difference in ER-negative proportions between FFTP classes depends on multiparity, we modelled the interaction between multiparity and FFTP class. Results: We included 9955 consecutive female patients after excluding missing values. 8358 out of 9955 women had at least 1 child (84%). In our study population, parity as such was not an independent variable for BC subtype. Women with a FFTP ≥ 27y as compared to those with a FFTP &amp;lt; 27y were less likely to have an ER-negative BC. The p-value for the interaction term between high parity (&amp;gt;2 children) and FFTP class equals p= 0.0044. Hence, there is statistical evidence to suggest that the differences between FFTP categories with regards to ER-negative BC may depend on multiparity. Table 1 shows a lower incidence of ER-negative BC in the FFTP ≥ 27y group compared to the FFTP &amp;lt; 27y group in case of high parity. Table 2 shows the absolute proportion. These results seem to be independent of BMI. Conclusion: Women with a FFTP at 27 years of age or older and more than 2 children have proportionally less ER-negative type breast cancers as compared to women with a FFTP before 27 years of age. This difference between FFTP classes is not observed in absence of high parity. Table 1: effect of FFTP class by multiparity Table 2: proportion of ER-negative breast cancer subtype Citation Format: Maja Vangoitsenhoven, Evert Theys, Karen Van Baelen, Annouschka Laenen, Christine Desmedt, Giuseppe Floris, Hans Wildiers, Kevin Punie, Sileny Han, Thaïs Baert, Frédéric Amant, Ann Smeets, Ines Nevelsteen, Rani Vanhoudt, Patrick Neven. Association between reproductive factors (parity and age first full term birth) and the frequency of estrogen receptor negative breast cancer according to age at diagnosis [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 P4-03-20.

Abstract GS4-04: Population-based Estimates of contralateral Breast Cancer Risk among Carriers of Germline Pathogenic Variants in ATM, BRCA1, BRCA2, CHEK2, and PALB2

Abstract Purpose To estimate the risk of contralateral breast cancer (CBC) among women in the general population with germline pathogenic variants (PVs) in ATM, BRCA1, BRCA2, CHEK2, and PALB2. Methods Among 15,104 prospectively followed women within the CARRIERS study treated with ipsilateral surgery for invasive breast cancer, a subset of 14,237 women were identified from population-based studies. The risk of CBC was estimated for PV carriers in each gene compared to women without PVs in a multivariate proportional hazard regression analysis accounting for the competing risk of death and adjusting for patient and tumor characteristics. The primary analyses focused on the overall cohort and on women from the general population. Secondary analyses examined associations by race/ethnicity, age at primary breast cancer diagnosis, menopausal status, and tumor estrogen receptor status. Results Germline BRCA1, BRCA2, and CHEK2 PV carriers with breast cancer were at significantly elevated risk (Hazard ratio ≥ 1.9, p&amp;lt; 0.05) of CBC, whereas only the PALB2 PV carriers with ER-negative breast cancer had elevated risks. In contrast, ATM PV carriers did not have significantly increased CBC risks. African American PV carriers had similarly elevated risks of CBC as non-Hispanic White PV carriers. Among premenopausal women, the 15-year cumulative incidence of CBC was &amp;gt;20% for BRCA1, BRCA2 and CHEK2 PV carriers with breast cancer, and for PALB2 PV carriers with ER-negative breast cancer. The 15-year cumulative incidence of CBC among postmenopausal PV carriers was &amp;lt; 20% for PV carriers in any of the 5 genes. Conclusions Women diagnosed with breast cancer and known to carry germline PVs in BRCA1, BRCA2, CHEK2, or PALB2 are at substantially increased risk of CBC and may benefit from enhanced surveillance and risk-reduction strategies. Citation Format: Siddhartha Yadav, Nicholas J. Boddicker, Jie Na, Eric C. Polley, Chunling Hu, Steven N. Hart, Rohan D. Gnanaolivu, Nicole Larson, Carolyn Dunn, Susan Holtegaard, Huaizhi Huang, Lauren R. Teras, Alpa V. Patel, James V. Lacey Jr., Susan Neuhausen, Leslie Bernstein, Elena Martinez, Christopher Haiman, Fei Chen, Kathryn Ruddy, Janet Olson, Esther John, Allison W. Kurian, Dale P. Sandler, Katie M. O’Brien, Jack A. Taylor, Clarice R. Weinberg, Hoda Anton-Culver, Argyrios Ziogas, Gary R. Zirpoli, David E. Goldgar, Katherine L. Nathanson, Susan Domchek, Julie R. Palmer, Jeffrey Weitzel, Peter Kraft, Fergus J. Couch. Population-based Estimates of contralateral Breast Cancer Risk among Carriers of Germline Pathogenic Variants in ATM, BRCA1, BRCA2, CHEK2, and PALB2 [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 GS4-04.

Abstract P2-07-01: Hydroxytyrosol, a Component of Olive Oil for Breast Cancer Prevention in Women at High Risk

Abstract Background Breast cancer is the most frequently diagnosed cancer in women in developed countries with increased incidence in women at high risk such as those with strong family history, BRCA mutations, atypical hyperplasia etc. Chemoprevention with drugs like tamoxifen and aromatase inhibitors come with challenges of intolerance and limited efficacy in estrogen receptor negative breast cancers. The purpose of our study was to evaluate the effects of hydroxytyrosol (HT), a component of olive oil on mammographic breast density reduction, which is a validated surrogate biomarker for breast cancer prevention as well as to explore on-target effects on Wingless-related integration site (Wnt) pathway, and alterations in key pathways of proliferation, DNA damage repair and stem cell function. Methods This study was conducted using 25mg/day oral dose of HT for 12 months in both pre- and post-menopausal women who are at increased risk of breast cancer with Gail 5-year risk score ≥ 1.67 and/or &amp;gt; 10% probability of BRCA mutation and had declined standard chemoprevention. These women underwent annual mammograms as well as had the option to have a biopsy of normal breast tissue before and after HT. Maximum volumetric breast density (Max VBD%) was assessed quantitatively using Volpara software (VIS 3.2) and the annualized percent decrease in max VBD% between baseline (BL) and end of treatment (EOT) with HT was analyzed in the study. RNA was extracted from the breast biopsies and RNA sequencing (RNA-Seq) and multiplex analysis of 28 proteins using NanoString nCounter analysis was performed to compare the BL with EOT samples. Results A total of 61 women were screened for the trial, 51 were enrolled and 41 patients completed the study. Median age for the study population was 54 years (range 35-76 years). There were 26 patients with paired BL and EOT mammograms. There were 14 patients with paired BL and EOT breast biopsies and seven patients with only BL and seven with only EOT biopsies. Mammographic density as measured by max VBD% showed a non-significant change of -0.038%, p = 0.49. However, on subgroup analysis in women 60-years or older, the mean decrease in max VBD% was - 3.7% (p = 0.0391), especially in those with high baseline mammographic density (&amp;gt; 10% baseline max VBD%). RNAseq data was generated from 15 BL and 17 EOT samples and pathways were confirmed by NanoString. Our bioinformatics pipeline identified 190 transcripts that were upregulated and 90 that were downregulated in EOT vs. BL by at least 1.5 fold. Reactome pathway R-HSA-195721 (Signaling by WNT) is identified as the top pathway enriched with EOT downregulated genes. On further review of the expression profiles, 82 of 261 member genes had p-value &amp;lt; 0.05 for EOT vs. BL, among which only 4 were upregulated and 78 were downregulated in EOT samples. These 82 genes were highly interactive with each other, with 379 high confidence protein-protein interactions (PPIs) (confidence score &amp;gt; 0.7 from STRING database) involving 81 out of 82 genes. In addition, mitotic telophase/cytokinesis (p = 0.004), ATM signaling (p = 0.007) were some pathways that were upregulated whereas, NOTCH signaling (p = 0.007), oxidative stress induced senescence (p = 0.0001) pathways were downregulated in EOT vs. BL. Conclusions Hydroxytyrosol, a component of olive oil did not show a decrease in breast density in the intention to treat population but showed some reduction in breast density in women 60-years of age and above, especially in those with high baseline breast density. HT showed on target effects by affecting Wnt signaling pathway, as well as decrease in proliferation. This study lays the foundation for future larger studies in exploring a natural compound such as HT for chemoprevention of breast cancer. Citation Format: Akshjot Puri, Zheng Yin, Sergio Granados-Principal, Joe Ensor, Liliana Guzman, Roberto Rosato, Hong Zhao, Stephen Wong, Tejal Patel, Jenny Chang. Hydroxytyrosol, a Component of Olive Oil for Breast Cancer Prevention in Women at High Risk [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 P2-07-01.

Abstract P6-11-03: Lipid-rich environment induces epigenetic reprogramming in non-transformed breast epithelial cells enhancing a mammary cell plasticity

Abstract Introduction. The identification of women specifically at risk for estrogen receptor negative breast cancer (ER- BC) and the targeted treatment of this disease are significantly unmet clinical needs. We analyzed the gene expression profiles of epithelial cells from the contralateral unaffected breasts (CUBs) of BC patients and identified a lipid metabolism gene signature enriched in the CUBs of women with ER- BC (PMID: 28263391). Subsequently, we observed that exposure of non-transformed breast epithelial cells to lipids results in significant changes in gene expression and histone posttranslational modifications; and increased flux through multiple metabolic reactions, including those of serine and methionine (PMID: 35508495). Interestingly, the upregulated genes are involved in neural development, axon guidance, neural crest pathways and stemness. Neural genes are highly expressed in Triple Negative Breast Cancers (TNBCs) especially in the C2 cluster. Given that mammary stem/progenitor cells have distinct metabolic properties compared to other mammary cell subsets, we hypothesized that upon lipid exposure, stem-like cells have a survival advantage, and that lipid induces epigenetic reprogramming into neural-like cells which may foster a malignant transformation. Methods. To interrogate potential mechanisms linking lipids and epigenetic reprogramming, we performed CUT&amp;RUN for H3K4me3 and H3K27me3 in non-transformed, estrogen and progesterone receptor negative MCF-10A cells cells exposed to vehicle or octanoic acid (OA) for 24 hrs. Peaks were called using MACS2 and differential peaks identified with DiffBind. Differentially expressed genes affected by OA (PMID: 28263391) were compared with target genes from the CUT&amp;RUN. To determine the contribution in OA exposed cells of serine and methionine metabolism to S-adenosyl methionine (SAM), 13C-glucose tracing was performed. The Aldefluour assay was used to identify stem-like (ALDH+) cells in lipid-exposed MCF-10A cells. To determine if lipid-exposed cells adopt a neural-like phenotype, MCF-10A cells were grown on Poly-D-Lysine/Laminin (PDL/LM) coated plates. Results. A total of 661 differential peaks were identified for H3K4me3 (FDR &amp;lt; 0.05) encompassing TNBC C2 markers (NRTN, CHRM3) and genes involved in neuron differentiation, axonogenesis (NGFR, NGF, FOXA2), neural crest migration (NTRK2, MMP2) and EMT (DLL4, MMP15). Approximately 73% of H3K4me3 OA-associated peaks encompass regulatory regions of genes that experienced a significant increase in gene expression with OA exposure (FDR &amp;lt; 0.01), including NGFR (log2 FC = 11.7), FOXA2 (log2 FC = 11.6), NGF (log2 FC = 8). Twelve H3K27me3 peaks were significantly enriched in vehicle (FDR &amp;lt; 0.05) and associated with increased gene expression in OA, among them were the stem cell markers LGR6 (log2 FC = 1.9) and PLAG1 (log2 FC = 2.8). 13C stable isotope tracing revealed that in presence of OA, glucose contributes to increased fractional abundance of the SAM M+1 isotopologue (adj p = 0.003) indicating that carbons derived from the serine synthesis pathway are used for re-methylation of homocysteine to methionine. Vehicle treated cells growing on PDL/LM plates assumed an epithelial phenotype while OA-treated cells adopted a neurite-like phenotype. Upon OA treatment the percentage of ALDH+ cells increased by a minimum of 10%. Conclusions. The increase of fractional abundance of SAM and the upregulation of neural genes regulated by H3K4me3 as well as the enrichment of ALDH+ cells and the development of a neural-like phenotype in a rich lipid environment, suggests that lipid exposure affects the production of SAM, which results in epigenetic fostered plasticity, leading to reprogramming/selecting cells with a multi-potential embryonic or stem-like state and/or differentiation to a neural/neural crest-like state. Citation Format: Mariana Bustamante Eduardo, Gannon Cottone, Seema Khan, Susan Clare. Lipid-rich environment induces epigenetic reprogramming in non-transformed breast epithelial cells enhancing a mammary cell plasticity [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 P6-11-03.

Abstract P6-11-08: Non-transformed breast epithelial cells show neural-like gene signature after lipid exposure

Abstract Introduction: The identification of women specifically at risk for estrogen receptor negative breast cancer (ER-BC) and the targeted treatment of this disease are significantly unmet clinical needs. To that end, we analyzed the gene expression profiles of epithelial cells from the contralateral unaffected breasts (CUBs) of BC patients and identified a lipid metabolism gene signature, which was enriched in the CUBs of women with ER-negative BC (PMID: 28263391). Subsequent experiment revealed that exposure of non-transformed breast epithelial cells to lipid results in significant changes gene expression, chromatin accessibility and histone posttranslational modifications (PMID: 35508495). Several of the upregulated genes are hallmarks of the various fates of vagal neural crest: Neural, neurogenic and mesenchymal lineages. We hypothesize that lipid exposure imparts a survival advantage of stem-like cells, that lipid-induced epigenetic changes lead to a neural crest-like transcription signature and that these genes are not expressed normally in the breast. Methods: MCF10A cells were exposed to vehicle or octanoic acid (OA) for 24 hours. Gene expression was assayed by RNA-seq and OA responsive genes were identified (PMID: 35508495). Single-cell RNA sequencing (scRNA-seq) data from 14 human reduction mammoplasties (RM) was obtained from a publicly accessible data set (PMID: 34031589). The scRNA data was clustered and identified by unsupervised clustering (Seurat, v3.4.1) using cell-type markers curated using Supplementary table 2 from (PMID: 34031589). The bulk RNA-seq data from the OA treated cells was deconvoluted to cell-lineages using Bisque. The most significant upregulated VNC neural/neuronal/mesenchymal genes from the gene expression analysis were then plotted on the lineage clusters using FeaturePlot to determine if these markers are found in the normal breast epithelium, or other cell lineages. The plots were then filtered and re-clustered to look at basal-luminal cell types only. We utilized a second resource, a web-application for snATAC-seq data from various stages of mouse mammary development developed by the Wahl lab (https://wahl-lab-salk.shinyapps.io/Mammary_snATAC/), to query these same genes. Results: Deconvolution of the bulk RNA-sequencing data revealed a transition to a pericyte transcription program following exposure to OA. Nerve growth factor (NGF) was found to be expressed in pericytes while nerve growth factor receptor (NGFR) was found within the basal epithelial cell lineage. Genes overexpressed in the VNC neural cluster and overexpressed in the OA-exposed MCF10 cells, PPP1R1C (2.39x, adj p=1.6E-5), FOXD3 (6.7x, adj p=6.7E-10), DIO3 (5.9x, adj p=3.9E-6) and MOXD3 (4.2x, adj p=1.5E-23) all evidence little to no expression in the normal breast but are observed in murine fetal mammary stem cells. Schwann cell precursor (SCP) markers, CDH19 and ROPN1, significantly upregulated in OA treated cells: 5.4x and 1.6x, respectively, exhibited low expression in luminal progenitors but in the mouse were observed in the mammary stroma. CDH19, a gene exclusive to SCPs, is expressed in stroma following murine birth. PRRX1, a key regulator of the VNC mesenchymal cell fate cluster, is 9.3-fold (p adj=4.9E-49) overexpressed in the OA treated cells, expressed strongly in pericytes and stroma and to a lesser extent in basal epithelial cells of the normal human breast and stroma of the murine mammary gland. Conclusions: Treatment of non-transformed mammary epithelial cells with lipid, specifically OA, shows significant upregulation of multiple VNC genes associated with both neural and mesenchymal fate. scRNA-seq from RM patients reveals that many of these same markers are either found in non-epithelial cell clusters or are found with low expression in luminal mammary lineages (both progenitors and mature). Citation Format: Gannon Cottone, Mariana Bustamante Eduardo, Shivangi Yadav, Seema Khan, Susan Clare. Non-transformed breast epithelial cells show neural-like gene signature after lipid exposure [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 P6-11-08.

Abstract P2-23-06: Estrogen receptor-negative progesterone receptor-positive breast cancer is a molecularly distinct group characterized by the down-regulation of genes controlled by ESR1 and SUZ12

Abstract Background: Single hormone receptor-positive breast cancers (BCs) display two distinct phenotypes: ER+/PgR– and ER–/PgR+ further stratified by their HER2 status. Their molecular features are not well defined. Our study aimed to identify differentially expressed genes in ER–/PgR+ BCs compared to other phenotypes. Methods: Our cohort comprised 15 ER+/PgR–/HER2–, 11 ER+/PgR–/HER2+, 17 ER–/PgR+/HER2–, 9 ER–/PgR+/HER2+, 5 ER+/PgR+/HER2–, and 5 ER–/PgR–/HER2– invasive BCs collected from 9 Polish and 2 Hungarian centers. The cases were selected from a larger cohort after being matched according to grade, HER2 status, lymph nodes, and distant metastasis status. ER–/PgR+ group was thoroughly validated via immunohistochemistry [Kunc et al. 2022]. The expression of 776 genes was profiled with nCounter® Breast Cancer 360™ Panel in archival formalin-fixed paraffin-embedded tissue samples. A gene was defined as differentially expressed between groups if it met the following criteria: the log2 fold-change in the expression of &amp;gt;1 or ←1 and the p-value &amp;lt; 0.05 (Mann-Whitney U test). Additionally, weighted correlation network analysis (WGCNA) was performed to identify modules of at least 15 highly correlated genes. Subsequently, the association between gene modules and PgR status in ER– subgroup was performed. Identified mRNAs were subjected to functional annotation analysis to determine the top enriched pathways. Results: ER–/PgR+ BCs were characterized by significantly lower expression of ESR1 compared to double-positive (p&amp;lt; 0.001) and ER+/PgR– tumors (p&amp;lt; 0.001), whereas PGR expression was higher compared to ER+/PgR– (p&amp;lt; 0.001), and no significantly different from ER+/PgR+ BCs (p=0.14). Triple-negative BCs had no detectable PGR mRNA. Four genes (MIA, ID4, FOXC1, CDC20) were consistently up-regulated and six genes (FAM214A, MLPH, NFKBIZ, FOS, SLC44A4, SPDEF) were down-regulated in ER–/PgR+/HER2– tumors compared to other HER2– subgroups. Compared to ER+/HER2– BCs, ER–/PgR+/HER2– cases showed up-regulation of 15 genes associated with response to vitamin D, response to ketone, and regulation of transcription, and downregulation of 33 genes involved in response to estrogen, negative regulation of cell population proliferation, regulation of epithelial-mesenchymal transition, and controlled by ESR1 and SUZ12. In WGCNA analysis of the ER– subgroup, PgR status was negatively correlated with 4 gene modules and positively correlated with 1 gene module. In line with differential gene expression analysis, genes negatively correlated with ER–/PgR+ status are regulated by ESR1 and SUZ12 and are involved in the regulation of cell proliferation, extracellular matrix organization, and NOTCH1 signaling. Genes positively correlated with ER–/PgR+ status are regulated by E2F4, FOXM1, SIN3A, NFYB, E2F1, FOS, IRF1, ZMIZ1, and UBTF and participate in cell cycle, regulation of mitosis, and microtubule cytoskeleton regulation. Conclusions: ER–/PgR+ BCs display a distinct mRNA expression profile characterized by the down-regulation of genes controlled by ESR1 and SUZ12. The latter as a part of Polycomb Repressive Complex 2 contributes to chromatin silencing, and some previous studies suggested its role in the regulation of steroid hormone receptors expression. Additionally, ER–/PgR+ BCs overexpress FOXC1 which is linked to more aggressive, high-grade, and treatment-resistant breast cancers. Our data indicate the need to unravel the mechanism of epigenetic regulation of PGR expression, especially its methylation status, in ER–/PgR+ breast cancer. Citation Format: Michał Kunc, Marta Popęda, Michał Bieńkowski, Marcin Braun, Aleksandra Łacko, Barbara Radecka, Joanna Pikiel, Maria Litwiniuk, Katarzyna Pogoda, Magdalena Niemira, Anna Szałkowska, Ewa Iżycka-Świeszewska, Gabor Cserni, Wojciech Biernat, Elżbieta Senkus. Estrogen receptor-negative progesterone receptor-positive breast cancer is a molecularly distinct group characterized by the down-regulation of genes controlled by ESR1 and SUZ12 [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 P2-23-06.

Abstract OT2-18-02: OlympiaN: a phase 2, multicenter, open-label study to assess the efficacy and safety of neoadjuvant olaparib monotherapy and olaparib plus durvalumab in patients with BRCA mutations and early-stage HER2-negative breast cancer

Abstract Background: In early breast cancer (BC), neoadjuvant therapy can promote de-escalation of surgery or treat micrometastases. Pathological complete response (pCR) is a standard primary endpoint in neoadjuvant BC studies, and correlates with long-term outcomes such as event-free survival (EFS). The poly(ADP-ribose) polymerase (PARP) inhibitors olaparib and talazoparib are approved in the metastatic setting for patients with BC and a germline BRCA1 and/or BRCA2 pathogenic/likely pathogenic mutation (gBRCAm). Following the phase 3 OlympiA trial primary analysis (data cut-off [DCO] Mar 2020), the PARP inhibitor olaparib was FDA approved for the adjuvant treatment of patients with gBRCAm and HER2-negative, high-risk early BC who have been treated with neoadjuvant/adjuvant chemotherapy. At the second pre-specified OlympiA analysis (DCO Jul 2021), olaparib treatment sustained improvements in invasive disease-free survival (IDFS HR 0.63, 95% CI 0.50–0.78) and distant disease-free survival (DDFS HR 0.61, 95% CI 0.48–0.77), and also significantly reduced the risk of death (overall survival [OS] HR 0.68, 98.5% CI 0.47–0.97) vs placebo. PARP inhibitors have also shown efficacy in the neoadjuvant setting in smaller studies. Durvalumab, a programmed death-ligand 1 inhibitor, has been studied as neoadjuvant therapy in triple negative BC and showed improved IDFS, DDFS and OS vs placebo (GeparNuevo). For BRCAm carriers at low risk of recurrence, olaparib monotherapy may provide adequate neoadjuvant treatment, allowing de-escalation or omission of chemotherapy. For those at high risk of recurrence, it is hypothesized that addition of the immune checkpoint inhibitor durvalumab will enhance immunogenicity provided through cell death following PARP inhibition. Feasibility for a larger study will be assessed. Methods: OlympiaN is a phase 2, international, multicenter, open-label trial examining the efficacy and safety of neoadjuvant olaparib monotherapy and olaparib plus durvalumab in adults with deleterious/suspected deleterious BRCAm and operable, early-stage, HER2-negative, ER-negative or ER-low BC (immunohistochemistry nuclear staining &amp;lt; 10%). Patients will be enrolled in two cohorts: (A) patients at lower risk of recurrence: tumor size &amp;gt;5 to &amp;lt; 20 mm and nodal status N0 will receive olaparib 300 mg orally twice daily continuously in 28-day cycles; (B) patients at higher risk of recurrence: tumor size &amp;gt;20 to ≤50 mm and N0, or T1 and N1 will receive olaparib 300 mg orally twice daily continuously in 28-day cycles plus durvalumab 1500 mg IV infusion every four weeks. After four to six cycles, patients will undergo definitive surgery, then systemic and radiation therapy in accordance with local standard of care. Patients who achieve pCR at surgery will be able to receive adjuvant olaparib monotherapy in lieu of standard adjuvant systemic therapy for a total of twelve 28-day cycles of neoadjuvant and adjuvant olaparib therapy. The primary endpoint is the pCR rate after completion of neoadjuvant systemic therapy, assessed by central pathology review. Secondary endpoints include the pCR rate assessed by local pathology review, residual cancer burden, tumor volumetric analysis, EFS, safety and tolerability. Tumor tissue will be collected pre-treatment and at surgery to evaluate the mechanism of action of therapy. Baseline and longitudinal assessment of ctDNA and its association with clinical outcomes will also be performed. The primary pCR analysis will occur ~6 months after last participant enrollment; the final DCO will be 3 years after last participant dosed or once all participants have had an EFS event. No formal statistical analyses are planned, data will be summarized descriptively. Enrollment is planned to commence by late 2022, across ~65 sites in 10 countries; the target accrual is ~25 patients per cohort to ensure adequate precision in the estimated pCR rate. Funding: This study is funded by AstraZeneca UK Plc. Citation Format: Judith Balmaña, Mike Dymond, Elizabeth S. Lowe, Natalia Lukashchuk, Maria Winter, Nadine Tung. OlympiaN: a phase 2, multicenter, open-label study to assess the efficacy and safety of neoadjuvant olaparib monotherapy and olaparib plus durvalumab in patients with BRCA mutations and early-stage HER2-negative breast cancer [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 OT2-18-02.

Abstract PD10-08: PD10-08 Immune cell infiltration associated with poor anti-proliferative response to aromatase inhibitors in postmenopausal women with primary ER-positive HER2-negative breast cancer

Abstract Background: Aromatase inhibitors (AIs) are one of the main treatment strategies for the clinical management of estrogen receptor-positive (ER+) breast cancer (BC). Despite prolonged time to recurrence and initial clinical responses, &amp;gt;20% of patients eventually relapse, and previous studies have shown an association of poor anti-proliferative response to AIs and worse outcome. High immune activity in ER+ tumors may be associated with worse outcome, in contrast to ER-negative BC where immune infiltration is a feature associated with better outcome. Our work focused on understanding the correlations between immune cell infiltration and response to AI. Methods: All patients with ER+ HER2- tumors within the bottom 15% of Ki67 anti-proliferative responders to AIs (poor responders [PRs]; n=177) were selected from the PeriOperative Endocrine Therapy for Individualizing Care (POETIC) trial and matched on baseline Ki67 levels to good responders (GRs) within the 50% showing the best response (n=190). Response to AI was measured by the Ki67 percentage change after 2 weeks of treatment. PRs were further divided into groups expressing high ESR1 (PRs ESR1HIGH; n=119) and low ESR1 (PRs ESR1LOW; n=58) levels to represent PR subgroups that showed partial or no response to AIs. The percentage of stromal tumor-infiltrating lymphocytes (TILs) was assessed. Multiple immunofluorescence was performed for ER, CD3, CD20, CD68, FOXP3, and CD3/FOXP3 in 15 baseline samples from each of the GR, PR ESR1HIGH, and PR ESR1LOW populations and immune cell density in stromal or tumor compartments was estimated. Spearman correlations of TILs with Consensus tumor microenvironment (TME) deconvolution and Molecular Signatures Database hallmark gene sets were conducted. The relationship between the immune markers’ density and genes, hallmark gene sets and Consensus TME was assessed. Results: The percentage of TILs was significantly higher in the PR ESR1HIGH and PR ESR1LOW compared to the GRs (adjusted p&amp;lt; 0.05). As expected, TILs were highly correlated with T cells (particularly T-regulatory cells) and immune hallmark gene sets. There was a tendency for higher density of each of the immune markers in PRs compared to GRs, with significant differences being observed in stromal B-cell marker CD20 density (p&amp;lt; 0.05). Analysis showed a significant correlation between TILs and stromal FOXP3 marker density (FDR&amp;lt; 0.05), and stromal biomarker density was highly correlated to the gene expression of the encoding genes of the same tumors (CD3/CD3D, FOXP3/FOXP3, and CD20/MS4A1) (FDRs&amp;lt; 0.05). There was also a strong and significant correlation between the stromal expression of CD20, CD3, FOXP3, and CD3/FOXP3 with the immune hallmark gene sets (FDRs&amp;lt; 0.05). Finally, the immune phenotyping showed the expected correlations with TME deconvolution, with particularly strong correlations of CD20 and CD3 with B- and T-cell gene signatures, respectively (FDRs&amp;lt; 0.05). Conclusions: Different immune features indicated a broad involvement of several immune cell types in PRs to AIs, suggesting that the immune system might be associated with resistance of ER+ breast tumors to AI treatment. Spatial gene expression profiling is ongoing to characterize these tumors further and investigate potential mechanisms of AI resistance. Citation Format: Anastasia Alataki, Gene Schuster, Lila Zabaglo, Perry Maxwell, Elena López-Knowles, Elizabeth Folkerd, David Evans, Kally Sidhu, Holly Tovey, Nicholas Turner, Stephen Johnston, Maggie Chon U Cheang, John Robertson, Manuel Salto-Tellez, Ian Smith, Judith Bliss, Mitch Dowsett. PD10-08 Immune cell infiltration associated with poor anti-proliferative response to aromatase inhibitors in postmenopausal women with primary ER-positive HER2-negative breast cancer [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 PD10-08.

Plexin-B3 expression stimulates MET signaling, breast cancer stem cell specification, and lung metastasis

Intratumoral hypoxia is a microenvironmental feature that promotes breast cancer progression and is associated with cancer mortality. Plexin B3 (PLXNB3) is highly expressed in estrogen receptor-negative breast cancer, but the underlying mechanisms and consequences have not been thoroughly investigated. Here, we report that PLXNB3 expression is increased in response to hypoxia and that PLXNB3 is a direct target gene of hypoxia-inducible factor 1 (HIF-1) in human breast cancer cells. PLXNB3 expression is correlated with HIF-1α immunohistochemistry, breast cancer grade and stage, and patient mortality. Mechanistically, PLXNB3 is required for hypoxia-induced MET/SRC/focal adhesion kinase (FAK) and MET/SRC/STAT3/NANOG signaling as well as hypoxia-induced breast cancer cell migration, invasion, and cancer stem cell specification. PLXNB3 knockdown impairs tumor formation and lung metastasis in orthotopic breast cancer mouse models.

Variation in second breast cancer risk after primary invasive cancer by time since primary cancer diagnosis and estrogen receptor status.

In women with previously treated breast cancer, occurrence and timing of second breast cancers have implications for surveillance. The authors examined the timing of second breast cancers by primary cancer estrogen receptor (ER) status in the Breast Cancer Surveillance Consortium. Women who were diagnosed with American Joint Commission on Cancer stage I-III breast cancer were identified within six Breast Cancer Surveillance Consortium registries from 2000 to 2017. Characteristics collected at primary breast cancer diagnosis included demographics, ER status, and treatment. Second breast cancer events included subsequent ipsilateral or contralateral breast cancers diagnosed >6months after primary diagnosis. The authors examined cumulative incidence and second breast cancer rates by primary cancer ER status during 1-5 versus 6-10years after diagnosis. At 10years, the cumulative second breast cancer incidence was 11.8% (95% confidence interval [CI], 10.7%-13.1%) for women with ER-negative disease and 7.5% (95% CI, 7.0%-8.0%) for those with ER-positive disease. Women with ER-negative cancer had higher second breast cancer rates than those with ER-positive cancer during the first 5 years of follow-up (16.0 per 1000 person-years [PY]; 95% CI, 14.2-17.9 per 1000 PY; vs. 7.8 per 1000 PY; 95% CI, 7.3-8.4 per 1000 PY, respectively). After 5 years, second breast cancer rates were similar for women with ER-negative versus ER-positive breast cancer (12.1 per 1000 PY; 95% CI, 9.9-14.7; vs. 9.3 per 1000 PY; 95% CI, 8.4-10.3 per 1000 PY, respectively). ER-negative primary breast cancers are associated with a higher risk of second breast cancers than ER-positive cancers during the first 5 years after diagnosis. Further study is needed to examine the potential benefit of more intensive surveillance targeting these women in the early postdiagnosis period.

Estimation of ALU Repetitive Elements in Plasma as a Cost-Effective Liquid Biopsy Tool for Disease Prognosis in Breast Cancer

Liquid biopsy is widely recognized as an efficient diagnostic method in oncology for disease detection and monitoring. Though the examination of circulating tumor cells (CTC) is mostly implemented for the assessment of genomic aberrations, the need of complex methodologies for their detection has impeded its acceptance in low-resource settings. We evaluated cell-free DNA (cfDNA) as a liquid biopsy tool and investigated its utility in breast cancer patients. Total cell-free DNA was extracted from the plasma of breast cancer patients (n = 167) with a median follow-up of more than 5 years, at various stages of the disease. Quantitative PCR was performed to estimate the copy numbers of two fractions of ALU repetitive elements (ALU 115 and ALU 247), and DNA integrity (DI) was calculated as the ratio of ALU 247/115. Mutations in TP53 and PIK3CA in the cfDNA were estimated by next-gen sequencing (NGS) in a subset of samples. Associations of the levels of both the ALU fragments with various clinico-pathological factors and disease-free survival at various stages were examined. Nomogram models were constructed with clinical variables and ALU 247 levels to predict disease-free survival and the best performing model was evaluated by decision curve analysis. DI and ALU 247 levels were significantly lower (p < 0.0001) in the post-operative plasma when compared to their pre-surgery levels. DI and ALU 247 were found to be significantly higher in patients with metastasis (p < 0.05). Patients with higher levels of ALU 247 in their post-operative plasma had significant poor disease-free survival (p = 0.005). Higher levels of ALU 247 in the circulation also correlated with low tumor-infiltrating lymphocytes (TIL) within their primary tumors in the ER-negative breast cancer subtype (p = 0.01). Cox proportional hazard analysis confirmed ALU 247 as an independent variable of disease-free survival both in univariate and multivariate analysis [HR 1.3 (95% CI 1.047 to 1.613, p = 0.017)]. The nomogram model showed that the addition of ALU 247 with other variables significantly improved (C-index 0.823) the predictive ability of the model. Our results confirm the utility of cfDNA as an evolving liquid biopsy tool for molecular analysis. Evaluation of larger fragments of cfDNA estimated through ALU 247 can provide vital information concurrent with the pathological process of disease evolution in breast cancer and warrants expansion to other cancer types.

Cationic lipid-conjugated bis-arylidene oxindole derivatives as broad-spectrum breast cancer-selective therapeutics

Breast cancer is a heterogeneous malignancy with wide-ranging variations in therapeutic responses, overall survival etc. Major challenges for available chemotherapeutic agents in achieving clinical success are in maintaining systemic bio-distribution and avoiding non-specific adverse effects. Bis-arylidene oxindoles are estrogen receptor (ER)-selective bioactive molecules with moderate potency. In here, we have designed, synthesized and evaluated a series of twin aliphatic chain cationic lipid-conjugated bis-arylidene oxindole molecules with variations in nature of linker, lengths of carbon spacer and hydrophobic twin chains. We observed that among the various structural analogues, C8 twin-chain containing molecules, PGC8, S2C8 and S3C8 showed effective cancer cell-selective cytotoxicity in different cancer cell lines with an IC50 ranging from 4 to 7 µM. These molecules selectively induced apoptosis, ROS production and cell cycle inhibition at G1/S phase in ER + breast cancer cells but not in non-cancer cells. Additionally, these molecules formed homogenous self-assemblies exhibiting effective hydrodynamic diameter with positive surface charge. The self-assemblies also showed prominent cancer cell-selective uptake and DNA-binding abilities. Hence, we have shown successful incorporation of dexamethasone to the self-assemblies, and its enhanced cytotoxicity even in ER-negative breast cancer cells. All these results indicate that PGC8, S2C8 and S3C8 molecules, albeit their potent and selective ER-positive anti-breast cancer activity, can be repurposed as targeted delivery systems and hold promise as unique, broader spectrum breast cancer cell-selective therapeutic payloads.

FANCM missense variants and breast cancer risk: a case-control association study of 75,156 European women

Evidence from literature, including the BRIDGES study, indicates that germline protein truncating variants (PTVs) in FANCM confer moderately increased risk of ER-negative and triple-negative breast cancer (TNBC), especially for women with a family history of the disease. Association between FANCM missense variants (MVs) and breast cancer risk has been postulated. In this study, we further used the BRIDGES study to test 689 FANCM MVs for association with breast cancer risk, overall and in ER-negative and TNBC subtypes, in 39,885 cases (7566 selected for family history) and 35,271 controls of European ancestry. Sixteen common MVs were tested individually; the remaining rare 673 MVs were tested by burden analyses considering their position and pathogenicity score. We also conducted a meta-analysis of our results and those from published studies. We did not find evidence for association for any of the 16 variants individually tested. The rare MVs were significantly associated with increased risk of ER-negative breast cancer by burden analysis comparing familial cases to controls (OR = 1.48; 95% CI 1.07–2.04; P = 0.017). Higher ORs were found for the subgroup of MVs located in functional domains or predicted to be pathogenic. The meta-analysis indicated that FANCM MVs overall are associated with breast cancer risk (OR = 1.22; 95% CI 1.08–1.38; P = 0.002). Our results support the definition from previous analyses of FANCM as a moderate-risk breast cancer gene and provide evidence that FANCM MVs could be low/moderate risk factors for ER-negative and TNBC subtypes. Further genetic and functional analyses are necessary to clarify better the increased risks due to FANCM MVs.

Contralateral Breast Cancer Risk Among Carriers of Germline Pathogenic Variants in ATM, BRCA1, BRCA2, CHEK2, and PALB2.

To estimate the risk of contralateral breast cancer (CBC) among women with germline pathogenic variants (PVs) in ATM, BRCA1, BRCA2, CHEK2, and PALB2. The study population included 15,104 prospectively followed women within the CARRIERS study treated with ipsilateral surgery for invasive breast cancer. The risk of CBC was estimated for PV carriers in each gene compared with women without PVs in a multivariate proportional hazard regression analysis accounting for the competing risk of death and adjusting for patient and tumor characteristics. The primary analyses focused on the overall cohort and on women from the general population. Secondary analyses examined associations by race/ethnicity, age at primary breast cancer diagnosis, menopausal status, and tumor estrogen receptor (ER) status. Germline BRCA1, BRCA2, and CHEK2 PV carriers with breast cancer were at significantly elevated risk (hazard ratio > 1.9) of CBC, whereas only the PALB2 PV carriers with ER-negative breast cancer had elevated risks (hazard ratio, 2.9). By contrast, ATM PV carriers did not have significantly increased CBC risks. African American PV carriers had similarly elevated risks of CBC as non-Hispanic White PV carriers. Among premenopausal women, the 10-year cumulative incidence of CBC was estimated to be 33% for BRCA1, 27% for BRCA2, and 13% for CHEK2 PV carriers with breast cancer and 35% for PALB2 PV carriers with ER-negative breast cancer. The 10-year cumulative incidence of CBC among postmenopausal PV carriers was 12% for BRCA1, 9% for BRCA2, and 4% for CHEK2. Women diagnosed with breast cancer and known to carry germline PVs in BRCA1, BRCA2, CHEK2, or PALB2 are at substantially increased risk of CBC and may benefit from enhanced surveillance and risk reduction strategies.