Triple-negative Breast Cancer Status Research Articles

Receipt of guideline-concordant care and survival among young adult women with non-metastatic breast cancer.

Adolescent and young adults (AYA) with breast cancer, compared to older adults, are diagnosed with more aggressive cancers, at more advanced stages and may undergo more aggressive treatment but have worse survival. Despite this, no research has studied the effects of the receipt of National Comprehensive Cancer Network (NCCN) defined guideline-concordant care (GCC) for breast cancer on AYA survival. We examined the association of GCC with survival among young adult (20-39 years old) breast cancer survivors. We used the Patterns of Care Study; a stratified random sample of 952 young adult women diagnosed with Stage I-III breast cancer in 2013. NCCN guidelines were used to categorize treatment as GCC or non-GCC. We used Kaplan-Meier curves, log-rank tests, and Cox-proportional hazards models to evaluate the effect of GCC on breast cancer-specific survival, stratifying by triple-negative breast cancer (TNBC) and non-TNBC, and adjusting for sociodemographic and clinical factors. All univariate analyses showed that non-GCC was associated with worse survival than GCC. The association was statistically significant for non-TNBC (Hazard ratio: 3.45, CI 1.64-7.29) and TNBC (Hazard ratio: 3.70, CI 1.02-13.43) in multivariable Cox models adjusted for sociodemographic variables and for non-TNBC (Hazard ratio: 3.13, CI 1.13-8.72) when the model was adjusted for sociodemographic and clinical variables. Among young adult women with non-metastatic breast cancer, while receipt of NCCN GCC is univariately associated with better survival for both TNBC and non-TNBC, the effect of sociodemographic and clinical factors on the association differs by TNBC status. Further investigation with larger TNBC samples is needed.

Baseline sLAG-3 levels in Caucasian and African-American breast cancer patients.

Worse survival persists for African-Americans (AA) with breast cancer compared to other race/ethnic groups despite recent improvements for all. Unstudied in outcomes disparities to date is soluble LAG-3 (sLAG-3), cleaved from the LAG-3 immune checkpoint receptor which is a proposed target for deactivation in emerging immunotherapies due to its prominent immunosuppressive function in the tumoral microenvironment. A prior study has found that lower sLAG-3 baseline level was associated with poor outcomes. In a cross-sectional study of 95 patients with primary breast cancer (n = 58 Caucasian, n = 37 AA), we measured sLAG-3 (ELISA pg/ml) in pre-treatment blood samples using the non-parametric Mann-Whitney u-Test for independent samples, and, calculated Pearson r correlation coefficients of sLAG-3 with circulating cytokines by race. Mean sLAG-3 level was lower in AA compared to Caucasian patients (1377.6 vs 3690.3, P = .002), and in patients with triple-negative breast cancer (TNBC) compared to those with non-TNBC malignancies (P = .02). When patients with TNBC tumors were excluded from analyses, the difference in sLAG-3 level between AA (n = 21) and Caucasian patients (n = 40) substantially remained (1937.4 vs 4182.4, P = .06). Among Caucasian patients, sLAG-3 was correlated with IL-6, IL-8 and IL-10 (r = .69, P < .001; r = .70, P < .001; and, r = .46, P = .01; respectively). For AA patients, sLAG-3 was correlated only with IL-6 (r = .37, P = .03). We present the first report that African-American breast cancer patients might have comparatively low pre-treatment sLAG-3 levels, independent of TNBC status, along with reduced co-expression with circulating cytokines. The mechanistic and prognostic role of cleaved LAG-3, particularly in disparate outcomes, remains to be elucidated.

Systematic characterization of the expression, prognosis and immune characteristics of PLOD family genes in breast cancer.

The expression patterns and prognostic value of Procollagen-lysine, 2-oxoglutarate 5-dioxygenase (PLOD) family genes in breast cancer remain to be elucidated. The expression levels, prognostic value, and biological function of PLODs were determined using Oncomine, cBioPortal, GEPIA, Timer, UALCAN, PrognoScan, GeneMANIA, Metascape, and breast cancer tissue microarrays. The expressions of PLOD1 and PLOD3 were upregulated in breast cancer tissues, indicating worse clinical stages. High expression levels of PLOD family genes were associated with worse disease-free survival and distant metastasis-free survival, while high expression levels of PLOD1 and PLOD3 were related to worse overall survival in all breast cancer patients. The levels of PLOD family genes were all significantly higher in the age ≤51 y group, HR-negative patients, and triple negative breast cancer (TNBC) patients. They are associated with tumor-infiltrating immune cells (TIICs), including CD4+ T cells, CD8+ T cells, B cells, macrophages, neutrophils, and dendritic cells. According to co-expression gene analysis and functional enrichment, they are associated with protein hydroxylation, collagen biosynthesis and modifying enzymes, collagen metabolism, RNA splicing, extracellular matrix organization, VEGFA-VEGFR2 signaling pathway, and skeletal system development. Immunohistochemistry showed that the expressions of all PLOD family genes were significantly elevated in breast cancer tissues. PLOD1 expression was positively correlated with ER, TNBC status, and tumor grade. PLOD2 expression was positively connected with Ki-67 status. PLOD3 expression was positively related with age and tumor grade. PLOD family genes are novel potential prognostic biomarkers for breast cancer, and targeting PLOD inhibitors might be an effective strategy for breast cancer therapy.

Abstract PO1-15-04: Young Black Women With Triple-Negative Breast Cancer Molecular Subtypes: Population-Specific Patterns and Batch Effect Considerations

Abstract Introduction: While triple-negative breast cancer (TNBC) molecular subtypes have been associated with biological differences and clinical outcomes, studies have overwhelmingly been conducted in populations of European or Asian ancestry. Data collected across diverse populations is required to better leverage clinical directions from translational studies in TNBC. Through females recruited through the Black Women: Etiology and Survival of Triple-Negative Breast Cancers (BEST) study, we sought to characterize subtypes and explore associations. Methods: Our study included Black women diagnosed with early-stage (I-III) invasive TNBC at &amp;lt; = age 50 from 2005 to 2016, with recruitment from Florida and Tennessee state cancer registries. Germline DNA, tumor RNA, clinical outcomes, and risk factor data were collected. TNBC status in the BEST study was based on immunohistochemistry from pathology reports, cancer registry data, and self-reported data. Analyses on banked tumor samples, extracted at multiple time points, were conducted through PAM50 (Nanostring) and whole-transcriptome RNA-seq. TNBC subtype was assessed using TNBCtype-4 (Lehmann et al. 2016). Sources of batch effect were evaluated using PCA and corrected via limma. Genetic ancestry based on OncoArray or MEGA genotyping data was inferred using 1000Genomes continental reference samples. Immune cell characterization was performed via CIBERSORT and ESTIMATE. Statistical significance in comparing differences across subtypes was assessed via Chi-square test. Kaplan-Meier estimates and log-rank test were used for survival analysis. Results: RNA-seq from 114 self-reported Black females with TNBC was analyzed. TNBC subtype distribution in the BEST study cohort included 31.6% basal-like 1 (BL1), 28.9% basal-like 2 (BL2), 16.7% luminal androgen receptor (LAR), and 21.9% mesenchymal (M). While results were largely consistent with prior studies in patients of European or East Asian ancestry, a higher percentage of BL2 subtype was observed (28.9% vs. 21.0%, p=0.053). Based on PAM50 subtyping, 100% of tumors with BL1 and M were basal, whereas 68.8% of LAR and 86.7% of BL2 subtypes were basal. Tumors without subtype decreased from 9.8% to 1.1% after batch correcting for location at which RNA was extracted (Moffitt vs. Vanderbilt) and time between RNA banking and sequencing. All TNBC subtypes in this population demonstrated monocyte/activated dendritic cell predominance, with no significant subtype-specific associations with immune cell patterns. A lower relative proportion of M subtype was found in tumors from BRCA1/2 carriers, but this was not statistically significant due to the small number of carriers (8% vs. 18%; p=0.22). With a median follow-up time of 9 years, there was no significant difference in 10-year overall survival by TNBC subtype (p=0.18). However, patients with M subtype appeared to have worse survival relative to other subtypes combined (p=0.036). Conclusion: Our study is among the largest to date that interrogates TNBC subtypes and associated molecular/clinical data in self-reported Black females with invasive breast cancer. Our findings suggest that established TNBC subtyping can be applied in patients of African ancestry. Furthermore, data to correct for lab-based confounders remains critical; in this study, it enabled us to use many samples that could not be subtyped initially. Several population-specific patterns were observed, including no difference in 10-year overall survival across TNBC subtypes (consistent with prior data), but worse initial outcomes in M subtype; a preliminary association between BRCA1/2 carriers and non-M subtypes (in contrast to prior data); and no significant difference in immune cell distributions (in contrast to prior data mainly from patients of European ancestry). Studies of associations between TNBC subtypes, additional clinical data, and treatment data (type and response rates) are ongoing. Citation Format: Padma Rajagopal, Sonya Reid, Run Fan, Lindsay Venton, Anne Weidner, Mya Roberson, Susan Vadaparampil, Xuefeng Wang, Sean Yoder, Marilin Rosa, Jibril Hirbo, Jennifer Whisenant, Jennifer Pietenpol, Fei Ye, Tuya Pal, Brian Lehmann. Young Black Women With Triple-Negative Breast Cancer Molecular Subtypes: Population-Specific Patterns and Batch Effect Considerations [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 PO1-15-04.

Abstract PO5-09-06: Outcomes of breast cancer in a minority-enriched population treated at a large safety-net system: Is site of care a predictor of poor outcomes?

Abstract Background: Breast cancer outcomes in the United States continue to improve, but sociodemographic disparities remain prevalent and impact care delivery and outcomes in vulnerable populations. Safety-net health systems are intended to address inequities by providing access to care for the uninsured/underinsured. Care delivery in a safety-net setting is often inherently complicated by limited resources at these institutions. Here, we present the overall survival (OS) data of breast cancer patients treated at a large safety-net system, compared to the national average. Methods: Parkland Health (PH) is the safety-net system for Dallas County and is affiliated with the University of Texas Southwestern Harold C. Simmons Comprehensive Cancer Center (NCI-CCC). Electronic medical records of patients diagnosed with invasive breast cancer between 2018 to 2020 at PH were reviewed and data on demographics, treatment, and outcomes were collected. Categorical data was summarized with counts and percentages and continuous data was summarized with median and interquartile range. Statistical comparisons used Chi-square test for categorical data and rank sum test for continuous data with a significance level of 0.05. Survival data was analyzed using Cox proportional hazard model. Data from the National Cancer Database (NCDB) for the same period was used for comparison. Results: Of the 657 patients included in the study, the majority (82.5%) belonged to racial and ethnic minorities (Hispanic 50.2%; Black 32.3%). Compared to the NCDB, PH patients were more often uninsured (67% vs 1.6%) or enrolled on Medicaid (14.5% vs 6.9%), and less often on Medicare (8.5% v. 43%) (p&amp;lt; 0.01). Triple negative breast cancer (TNBC) phenotype and late-stage diagnosis (stage III/IV) rates were higher at PH compared to NCDB (TNBC 22.8% vs 10.7%, p&amp;lt; 0.01; late stage 30.6% vs 13.2%, p&amp;lt; 0.01). At a median follow up of 33 (range 24-43) months in PH and 28 (range 21-36) months in NCDB, the unadjusted OS for the PH population was 86.3% vs 92.6% for the NCDB population (p &amp;lt; 0.01). Within the PH population, Black race, higher stage, and TNBC were associated with decreased OS. There was a significant interaction between Black race and TNBC. When combining all patients (PH+NCDB), site of care (PH) was associated with a higher risk of death from breast cancer (HR 1.467, 95% CI 1.192-1.805, p&amp;lt; 0.0005), however, multivariate analysis did not show a significant difference based on site of care. Factors associated with a significantly increased risk of death in this multivariate analysis (corrected for interaction between Black race and TNBC phenotype) included age, late-stage diagnosis, TNBC, Black race, and uninsured status (Table 1). Conclusions: Breast cancer patients treated at PH were more likely to have adverse disease characteristics compared to the NCDB population. When accounting for these risk factors in multivariate analysis, patients treated at PH had similar outcomes compared to the national average. Our findings reinforce the critical role of safety-net systems in promoting health equity and eliminating disparities. Table 1 Citation Format: Sarah Kashanian, Shifa Kanjwal, L. Steven Brown, Andrea Semlow, Mary Hodges, Robyn Cobb, Brad Walsh, Umber Dickerson, Navid Sadeghi. Outcomes of breast cancer in a minority-enriched population treated at a large safety-net system: Is site of care a predictor of poor outcomes? [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 PO5-09-06.

Novel Insights Into the Epidemiological and Clinico-Pathological Profile of Triple-Negative Breast Cancer: Dissection of an Aggressive Variant.

Objective Triple-negative breast cancer (TNBC) represents an aggressive and prognostically poor variant of breast cancer. Over the years, detailed research has been conducted and published in Western literature. However, there lacks a detailed account of TNBC cases from the perspective of a low-volume institution. This study aims to assess the clinicalfeatures of TNBC, as well as their prognostic implications in a tertiary care centre. Methods and materials This prospective observational studytook place at a tertiary health centre for two years, spanning from 2021 to 2023. The study aimed to investigate various clinicopathological and epidemiological parameters, thereby highlighting the shortcomings in the existing knowledge about the subject in the context of a low-volume centre, as well as additional contributing factors in developing countries like India.A group of 150 participants diagnosed with TNBC through biopsy and immunohistochemistry and >40 years of age were included in the study. Patients who tested positive for hormonal receptors and who refused to give consent for participation were excluded from the study. The study subjectswere categorized according to their clinical TNM (cTNM) stage and eventually segregated into two primary heads, namely pre-surgery chemotherapy with breast-conserving surgery (BCS) after a good response, or modified radical mastectomy (MRM) upfront. Important demographic details, including age, socioeconomic status, and education, were also recorded. A comprehensive follow-up assessment post-treatment was performed to detect early recurrence. After data collection, the recurrence rates were correlated with the TNBC status to establish the aggressiveness of the cancer. Statistical analysis of the data was done using the Statistical Package for Social Sciences (SPSS) -16version software. Results The average age of the 150 participants in the study was 52.21 years (SD±4.89 years). The highest recorded age was 64 years, while the lowest recorded age was 45 years. In the study, it was observed that 41% of the participants diagnosed with TNBChad stage III disease, whereas 33.5% had stage I disease, 22% had stage IV disease and 3.6% had stage II disease. A total of 27.5% of individuals with TNBCexhibited metastases in various anatomical sites, whereas the other 72.5% did not show any signs of metastasis. Conclusion Triple-negative breast cancer has earned its position as a unique subtype of breast cancer due to its unusual molecular characteristics, aggressive behavior, limited treatment options, and poor prognosis. The lower per-capita income and limited knowledge pertaining to this variant, along with the absence of more specific treatment options, contribute to the already high levels of morbidity and mortality associated with this illness. To effectively address this unique and very virulent ailment and customize our strategies, it is imperative to do further comprehensive investigations, thereby enabling us to deliver the highest quality of medical attention to individuals afflicted by this pathology.

Do Habitat MRI and Fractal Analysis Help Distinguish Triple-Negative Breast Cancer From Non-Triple-Negative Breast Carcinoma.

Purpose: To determine whether multiparametric MRI-based spatial habitats and fractal analysis can help distinguish triple-negative breast cancer (TNBC) from non-TNBC. Method: Multiparametric DWI and DCE-MRI at 3T were obtained from 142 biopsy- and surgery-proven breast cancer with 148 breast lesions (TNBC = 26 and non-TNBC = 122). The contrast-enhancing lesions were divided into 3 spatial habitats based on perfusion and diffusion patterns using K-means clustering. The fractal dimension (FD) of the tumour subregions was calculated. The accuracy of the habitat segmentation was measured using the Dice index. Inter- and intra-reader reliability were evaluated with the intraclass correlation coefficient (ICC). The ability to predict TNBC status was assessed using the receiver operating characteristic curve. Results: The Dice index for the whole tumour was 0.81 for inter-reader and 0.88 for intra-reader reliability. The inter- and intra-reader reliability were excellent for all 3 tumour habitats and fractal features (ICC > 0.9). TNBC had a lower hypervascular cellular habitat and higher FD 1 compared to non-TNBC (all P < .001). Multivariate analysis confirmed that hypervascular cellular habitat (OR = 0.88) and FD 1 (OR = 1.35) were independently associated with TNBC (all P < .001) after adjusting for rim enhancement, axillary lymph nodes status, and histological grade. The diagnostic model combining hypervascular cellular habitat and FD 1 showed excellent discriminatory ability for TNBC, with an AUC of 0.951 and an accuracy of 91.9%. Conclusions: The fraction of hypervascular cellular habitat and its FD may serve as useful imaging biomarkers for predicting TNBC status.

Neighborhood and racial influences on triple negative breast cancer: evidence from Northeast Ohio.

Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer (BC) with higher recurrence rates and poorer prognoses and most prevalent among non-Hispanic Black women. Studies of multiple health conditions and care processes suggest that neighborhood socioeconomic position is a key driver of health disparities. We examined roles of patients' neighborhood-level characteristics and race on prevalence, stage at diagnosis, and mortality among patients diagnosed with BC at a large safety-net healthcare system in Northeast Ohio. We used tumor registry to identify BC cases from 2007 to 2020 and electronic health records and American Community Survey for individual- and area-level factors. We performed multivariable regression analyses to estimate associations between neighborhood-level characteristics, measured by the Area Deprivation Index (ADI), race and comparative TNBC prevalence, stage at diagnosis, and total mortality. TNBC was more common among non-Hispanic Black (53.7%) vs. non-Hispanic white patients (46.4%). Race and ADI were individually significant predictors of TNBC prevalence, stage at diagnosis, and total mortality. Race remained significantly associated with TNBC subtype, adjusting for covariates. Accounting for TNBC status, a more disadvantaged neighborhood was significantly associated with a worse stage at diagnosis and higher death rates. Our findings suggest that both neighborhood socioeconomic position and race are strongly associated with TNBC vs. other BC subtypes. The burden of TNBC appears to be highest among Black women in the most socioeconomically disadvantaged neighborhoods. Our study suggests a complex interplay of social conditions and biological disease characteristics contributing to racial disparities in BC outcomes.

Enhancing the BOADICEA cancer risk prediction model to incorporate new data on RAD51C, RAD51D, BARD1 updates to tumour pathology and cancer incidence

BackgroundBOADICEA (Breast and Ovarian Analysis of Disease Incidence and Carrier Estimation Algorithm) for breast cancer and the epithelial tubo-ovarian cancer (EOC) models included in the CanRisk tool (www.canrisk.org) provide future...

Differences in somatic TP53 mutation type in breast tumors by race and receptor status.

Somatic driver mutations in TP53 are associated with triple-negative breast cancer (TNBC) and poorer outcomes. Breast cancers in women of African ancestry (AA) are more likely to be TNBC and have somatic TP53 mutations than cancers in non-Hispanic White (NHW) women. Missense driver mutations in TP53 have varied functional impact including loss-of-function (LOF) or gain-of-function (GOF) activity, and dominant negative (DNE) effects. We aimed to determine if there were differences in somatic TP53 mutation types by patient ancestry or TNBC status. We identified breast cancer datasets with somatic TP53 mutation data, ancestry, age, and hormone receptor status. Mutations were classified for functional impact using published data and type of mutation. We assessed differences using Fisher's exact test. From 96 breast cancer studies, we identified 2964 women with somatic TP53 mutations: 715 (24.1%) Asian, 258 (8.7%) AA, 1931 (65.2%) NHW, and 60 (2%) Latina. The distribution of TP53 mutation type was similar by ancestry. However, 35.8% of tumors from NHW individuals had GOF mutations compared to 29% from AA individuals (p = 0.04). Mutations with DNE activity were positively associated with TNBC (OR 1.37, p = 0.03) and estrogen receptor (ER) negative status (OR 1.38; p = 0.005). Somatic TP53 mutation types did not differ by ancestry overall, but GOF mutations were more common in NHW women than AA women. ER-negative and TNBC tumors are less likely to have DNE+ TP53 mutations which could reflect biological processes. Larger cohorts and functional studies are needed to further elucidate these findings.

Upregulated WTAP expression appears to both promote breast cancer growth and inhibit lymph node metastasis

It is unclear as to whether Wilms’ tumor 1-associated protein (WTAP) promotes or suppresses breast cancer. This immunohistochemistry analysis explored levels of WTAP expression in 347 cases of breast cancer and analyzed the relationship between WTAP expression and the clinicopathological characteristics and prognosis of breast cancer patients. The rate of high WTAP expression was significantly higher in breast cancer tissue than in adjacent normal breast tissue (37.5% vs 0.0%; P < 0.001). WTAP expression was positively associated with tumor size and grade, and negatively associated with axillary lymph node metastasis, estrogen and progesterone receptor status. Rates of high WTAP expression were 66.1% in triple-negative breast cancer (TNBC) tissue and 31.3% in non-TNBC tissue. In multiple logistic regression analysis, independent predictors of WTAP expression in breast cancer included larger tumor size (odds ratio = 1.907; 95% confidence interval: 1.185–3.067; P = 0.008), lymph node metastasis (0.597; 0.373–0.956; P = 0.032) and TNBC status (3.735; 2.056–6.784; P < 0.001). No clear relationship was observed between patient prognosis and WTAP expression. We suggest that WTAP expression is upregulated in breast cancer and appears to both promote tumor growth and inhibit lymph node metastasis.

Vitamin D receptor (VDR) mRNA overexpression is associated with poor prognosis in breast carcinoma.

PurposeThe prognostic value of vitamin D receptor gene (VDR) expression in breast cancer development is unclear. Here, we aimed to investigate whether VDR expression can be used as a prognostic indicator of breast cancer.MethodsWe used various public bioinformatics platforms: Oncomine, GEPIA, UALCAN, Kaplan-Meier plotter, UCSC XENA, bc-GenExMiner, WebGestalt, and STRING database.ResultsWe found that VDR was upregulated in breast cancer in comparison to normal tissues. Overexpression of VDR was significantly associated with worse overall survival in breast cancer. The expression of VDR was related to age, TNM stages, estrogen receptor status, progesterone receptor status, human epidermal growth factor receptor 2 status, basal-like (PAM 50) status, triple-negative breast cancer (TNBC) status, and basal-like (PAM 50) & TNBC status (P < 0.05). Increased VDR expression in breast cancer was significantly associated with older age. The 5 hub genes for VDR were NCOA1, EP300, CREBBP, and RXRA.ConclusionOur investigation offers hints about the prognostic role of VDR in breast cancer. The findings suggest that VDR expression might be used as a marker to determine a breast cancer patient’s prognosis. Nevertheless, further validation is warranted.

Epigenetic Dysregulation of KCNK9 Imprinting and Triple-Negative Breast Cancer.

Simple SummaryGenomic imprinting is an inherited form of parent-of-origin specific epigenetic gene regulation that is dysregulated by poor prenatal nutrition and environmental toxins. Here, we showed that KCNK9 is imprinted in breast tissue and identified the differentially methylated region (DMR) controlling its imprint status. Hypomethylation at the DMR, coupled with biallelic expression of KCNK9, occurred in 63% of triple-negative breast cancers (TNBC). The association between hypomethylation and TNBC status was highly significant in African-Americans (p = 0.006), but not in Caucasians (p = 0.70). The high frequency of KCNK9 DMR hypomethylation in TNBC and non-cancerous breast tissue from high-risk women provides evidence that hypomethylation of the KNCK9 DMR/TASK3 overexpression may provide a new target for prevention of TNBC.Genomic imprinting is an inherited form of parent-of-origin specific epigenetic gene regulation that is dysregulated by poor prenatal nutrition and environmental toxins. KCNK9 encodes for TASK3, a pH-regulated potassium channel membrane protein that is overexpressed in 40% of breast cancer. However, KCNK9 gene amplification accounts for increased expression in <10% of these breast cancers. Here, we showed that KCNK9 is imprinted in breast tissue and identified a differentially methylated region (DMR) controlling its imprint status. Hypomethylation at the DMR, coupled with biallelic expression of KCNK9, occurred in 63% of triple-negative breast cancers (TNBC). The association between hypomethylation and TNBC status was highly significant in African-Americans (p = 0.006), but not in Caucasians (p = 0.70). KCNK9 hypomethylation was also found in non-cancerous tissue from 77% of women at high-risk of developing breast cancer. Functional studies demonstrated that the KCNK9 gene product, TASK3, regulates mitochondrial membrane potential and apoptosis-sensitivity. In TNBC cells and non-cancerous mammary epithelial cells from high-risk women, hypomethylation of the KCNK9 DMR predicts for increased TASK3 expression and mitochondrial membrane potential (p < 0.001). This is the first identification of the KCNK9 DMR in mammary epithelial cells and demonstration that its hypomethylation in breast cancer is associated with increases in both mitochondrial membrane potential and apoptosis resistance. The high frequency of hypomethylation of the KCNK9 DMR in TNBC and non-cancerous breast tissue from high-risk women provides evidence that hypomethylation of the KNCK9 DMR/TASK3 overexpression may serve as a marker of risk and a target for prevention of TNBC, particularly in African American women.

Abstract 788: Differences in somatic TP53 mutation type in breast tumors by race and receptor status

Abstract Somatic driver mutations in TP53 are associated with triple negative breast cancer (TNBC) and poorer outcomes. African American women with breast cancer are more likely to have TNBC and TP53 driver mutations. Missense driver mutations in TP53 can be loss-of function (LOF) or gain-of-function (GOF). Additionally, some variants show dominant negative (DNE) activity. There are a number of hot-spot TP53 mutations, many occurring at CpG sites. Given the differences in TNBC status and TP53 driver mutation frequency by race, we wanted to determine if there are differences in TP53 mutation type by race. From multiple databases and a literature review, we identified 98 studies with patient race and TP53 mutation details. We compiled TP53 mutations from 2964 breast tumors from these 98 studies. The cohort included 715 (24.1%) Asian, 258 (8.7%) African (AA), and 1931 (65.1%) European (EA) ancestry individuals. Additionally, 60 patients (2%) had Hispanic or Latina ethnicity. Of the somatic TP53 mutations, 939 (31.7%) were GOF, 1739 (58.7%) were LOF, while the remaining 286 (9.6%) were not able to be classified by existing literature. With respect to DNE activity, 1246 (42%) showed DNE, 1190 (40.1%) showed no DNE, and 528 (17.8%) were unknown DNE status. CpG hotspots accounted for 613 (20.6%) mutations. Using Fisher's Exact test, we did not identify significant associations between any of the functional categories and overall race. However, 35.8% of tumors from EA individuals had GOF mutations compared to 29% in AA individuals (p=0.04). Mutations with DNE activity were positively associated with TNBC (OR=1.37, p=0.03) and negative estrogen receptor (ER) status (OR=1.38; p=0.005). Patients with hotspot mutations were slightly younger (mean age 53.61) compared to those with non-hotspot mutations (mean age 55.04), and this association neared significance (p=0.065). Larger cohorts are needed, in particular the AA cohort, to further elucidate some of these borderline findings. In summary, ER negative and TNBC breast tumors are more likely to have DNE TP53 mutations which could reflect biological processes. Citation Format: Nijole Pollock, Johnny Ramroop, Joseph McElroy, Amanda E. Toland. Differences in somatic TP53 mutation type in breast tumors by race and receptor status [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 788.

Abstract 2725: Immune-cell profiling in breast cancer patients by race and triple negative breast cancer status

Abstract Breast cancer is the most frequently diagnosed cancer and the second leading cause of cancer-related death among American women. Underserved minorities suffer from higher mortality rates and a higher prevalence of triple-negative breast cancer (TNBC). Previous studies showed that African Americans (AA) have a higher risk for TNBC, advanced tumor, pro-inflammatory tumor microenvironment, and worse survival. Immune responses and inflammatory processes play critical roles in cancer initiation, progression, and suppression. Therefore, this study was designed to investigate the complex interplay among the tumor, microenvironment, and immune response by race and TNBC status. Tumor biopsy samples from 96 breast cancer cases were used for the NanoString PanCancer IO 360 Gene Expression Panel analysis. The panel is a 770 gene Code Set that is designed for profiling tumor biopsies and characterizing gene expression patterns associated with the tumor, the immune response, and the microenvironment that shape tumor-immune interactions. After quality control for 20 reference genes, gene expression data from 93 samples were used for subsequent data analysis with nSolver 4.0 to calculate expression changes, pathway mapping based on annotations, sample clustering (heat maps), and 14 immune cell type abundance signatures. Then we conducted a Student's t-test and ANOVA for comparison of immune cell types by patient demographics and clinical characteristics using SAS University Edition 2019. In 10 TNBC and 83 non-TNBC, the expression of 12 genes was significantly different, 4 were higher and 8 were lower in TNBC (False Discovery Rate adjusted p&amp;lt;0.05). AA patients had significantly higher B cells (p=0.02), T cells (p=0.03), macrophages (p=0.02), CD45 (p=0.02), and Treg cells (p=0.04) than white patients. Patients diagnosed at a young age had significantly lower neutrophil counts (p=0.02). Nonsmokers had significantly higher mast cell scores than smokers (p=0.03). Mast cells were significantly lower in ER-negative (p&amp;lt;0.01), PR-negative (p&amp;lt;0.01), and TNBC (p&amp;lt;0.01) patients than ER-positive, PR-positive, and non-TNBC patients. Our study results suggest that gene expression and immune cell types are different by race and TNBC status. Future data analysis will focus on 18-gene tumor inflammation signature, 15+ proprietary gene expression signatures, and 14 immune cell type abundance signatures with a single sample and dataset analysis report, and automatic predictive algorithm training. With the recent FDA accelerated approval of PD-L1 inhibitor atezolizumab as a treatment for PD-L1-positive metastatic TNBC, the NanoString PanCancer IO 360 panel may provide the capability to both discover new biomarkers and create potentially predictive signatures for immunotherapies, which in turn may help improve minority health and reduce survival disparities. Citation Format: Xiaozhuang Hu, Fernando Collado-Mesa, George R. Yang, Isildinha M. Reis, Jennifer J. Hu. Immune-cell profiling in breast cancer patients by race and triple negative breast cancer status [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2725.

Abstract 897: Association between transforming growth factor beta 1 and progression-free survival in breast cancer

Abstract Transforming growth factor-beta 1 (TGF-β1) has a dual role in cancer progression, with inhibitory functions in early tumor stages and tumor promotion in advanced stages. However, most prior studies have studied survival in either all tumor stages together or a subset of stages. Therefore, this study examined the effect of TGF-β1 before and after radiotherapy (RT) on progression-free survival (PFS) in a racially/ethnically diverse breast cancer patient population and stratified by cancer stage (n=488). Plasma samples were collected on the first (pre-RT) and last day of RT (post-RT), and patients were followed for up to 10 years through electronic medical records. TGF-β1 was assayed with the ELISA kit (R&amp;D Systems, Inc, Minneapolis, MN). PFS was calculated as the time between the date of diagnosis to the first event (death, recurrence, or metastasis) or last follow-up if no event occurred. TGF-β1 was treated as a continuous variable (per 1,000 pg/mL increase). Univariable and multivariable Cox proportional hazards regression models were used to evaluate the association between TGF-β1 and PFS, adjusted for age at diagnosis, clinical tumor stage, and triple-negative breast cancer status in total, stage 0-II, or stage III-IV patients. The study population was comprised of 306 Hispanic white (63%), 102 Black/African American (21%), 64 non-Hispanic white (13%), and 16 other patients (3%). In univariable analyses, stage 0-II patients had 12% and 11% higher risk for event for every 1,000 pg/mL increase in pre-RT (HR=1.12, 95%CI=1.05-1.19, p&amp;lt;0.001) and post-RT (HR=1.11, 95%CI=1.01-1.23, p=0.034) TGF-β1, respectively. In multivariable models, increases in pre-RT (HR=1.11, 95%CI=1.04-1.19, p&amp;lt;0.001) and post-RT TGF-β1 (HR=1.12, 95%CI=1.01-1.24, p=0.031) remained significantly associated with worse PFS in stage 0-II patients, and post-RT TGF-β1 became significantly associated with worse PFS in all patients (HR=1.10, 95%CI=1.00-1.20, p=0.048). In a subset of 107 patients, there was a significant drop of TGF-β1 levels from baseline before any treatment to pre-RT (mean±SD: 6269±3296 to 3787±2810, p&amp;lt;0.001). The implication is that other treatment(s) before RT reduced the tumor burden that may contribute to decreasing circulating TGF-β1 levels. However, TGF-β1 levels were not different between pre- and post-RT. In summary, our current data suggest that higher pre- and post-RT TGF-β1 levels were associated with worse PFS in early-stage patients. Although plasma TGF-β1 levels may not represent the amount of TGF-β1 in tumor, it may still serve as a surrogate prognostic marker and a potential target in cancer therapy. Future larger studies are warranted to validate our findings that circulating TGF-β1 may predict progression-free survival of breast cancer, especially in patients with early stage tumors. Citation Format: George Ruochen Yang, Cristiane Takita, Jean L. Wright, Isildinha M. Reis, Eunkyung Lee, Jennifer J. Hu. Association between transforming growth factor beta 1 and progression-free survival in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 897.

A network approach reveals driver genes associated with survival of patients with triple-negative breast cancer

SummaryWe aimed to identify triple-negative breast cancer (TNBC) drivers that regulate survival time as predictive signatures that improve TNBC prognostication. Breast cancer (BrCa) transcriptomic tumor biopsies were analyzed, identifying network communities enriched with TNBC-specific differentially expressed genes (DEGs) and correlated strongly to TNBC status. Two anticorrelated modules correlated strongly to TNBC subtype and survival. Querying module-specific hubs and DEGs revealed transcriptional changes associated with high survival. Transcripts were nominated as biomarkers and tested as combinatoric ratios using receiver operator characteristic (ROC) analysis to assess survival prediction. ROC test rounds integrated genes with established interactions to hubs and DEGs of key modules, improving prediction. Finally, we tested whether integration of literature-derived genes for implicated hallmark cancer processes could improve prediction of survival. Complementary coexpression, differential expression, genetic interaction, and survival stratification integrated by ROC optimization uncovered a panel of “linchpin survival genes” predictive of patient survival, representing gene interactions in hallmark cancer processes.

CLIP4 Shows Putative Tumor Suppressor Characteristics in Breast Cancer: An Integrated Analysis.

Background: CAP-Gly domain containing linker protein family member 4 (CLIP4) plays an important role in cancers. However, its expression, prognostic value, and biological effect in breast cancer remain unclear. Methods: Data on patients diagnosed with breast cancer were retrieved from the TCGA-BRCA and other public omics databases. The expression profile of CLIP4 was analyzed using Oncomine, bc-GenExMiner, and TCGA. The prognostic value of CLIP4 was determined by Kaplan-Meier Plotter and Human Protein Atlas. Identification of genes co-expressed with CLIP4 and potential mechanism analyses were performed using UALCAN, STRING, Metascape, and GSEA. The epigenetic characteristics of CLIP4 were determined by DiseaseMeth and MEXPRESS. Results: CLIP4 was downregulated and its expression was negatively correlated with estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor type 2 (HER2) status, Nottingham prognostic index (NPI), and Scarff-Bloom-Richardson (SBR) grade in breast cancer, whereas it was positively linked to basal-like and triple negative breast cancer status. Ectopic expression of CLIP4 was related with poor prognosis. In the analysis of genes co-expressed with CLIP4, GSEA showed that the Hedgehog (Hh), JAK-STAT, ERBB, Wnt signaling pathway, cell adhesion molecules, and pathways in cancer were dissimilarly enriched in the CLIP4 expression high phenotype. Analysis of the genetics and epigenetics of CLIP4 indicated that its expression was negatively correlated with DNA methylation. Conclusion: Methylated CLIP4 may be a novel prognostic and therapeutic biomarker for breast cancer.

Pembrolizumab plus chemotherapy versus placebo plus chemotherapy for previously untreated locally recurrent inoperable or metastatic triple-negative breast cancer (KEYNOTE-355): a randomised, placebo-controlled, double-blind, phase 3 clinical trial

Pembrolizumab monotherapy showed durable antitumour activity and manageable safety in patients with metastatic triple-negative breast cancer. We aimed to examine whether the addition of pembrolizumab would enhance the antitumour activity of chemotherapy in patients with metastatic triple-negative breast cancer. In this randomised, placebo-controlled, double-blind, phase 3 trial, done in 209 sites in 29 countries, we randomly assigned patients 2:1 with untreated locally recurrent inoperable or metastatic triple-negative breast cancer using a block method (block size of six) and an interactive voice-response system with integrated web-response to pembrolizumab (200 mg) every 3 weeks plus chemotherapy (nab-paclitaxel; paclitaxel; or gemcitabine plus carboplatin) or placebo plus chemotherapy. Randomisation was stratified by type of on-study chemotherapy (taxane or gemcitabine-carboplatin), PD-L1 expression at baseline (combined positive score [CPS] ≥1 or <1), and previous treatment with the same class of chemotherapy in the neoadjuvant or adjuvant setting (yes or no). Eligibility criteria included age at least 18 years, centrally confirmed triple-negative breast cancer; at least one measurable lesion; provision of a newly obtained tumour sample for determination of triple-negative breast cancer status and PD-L1 status by immunohistochemistry at a central laboratory; an Eastern Cooperative Oncology Group performance status score 0 or 1; and adequate organ function. The sponsor, investigators, other study site staff (except for the unmasked pharmacist), and patients were masked to pembrolizumab versus saline placebo administration. In addition, the sponsor, the investigators, other study site staff, and patients were masked to patient-level tumour PD-L1 biomarker results. Dual primary efficacy endpoints were progression-free survival and overall survival assessed in the PD-L1 CPS of 10 or more, CPS of 1 or more, and intention-to-treat populations. The definitive assessment of progression-free survival was done at this interim analysis; follow-up to assess overall survival is continuing. For progression-free survival, a hierarchical testing strategy was used, such that testing was done first in patients with CPS of 10 or more (prespecified statistical criterion was α=0·00411 at this interim analysis), then in patients with CPS of 1 or more (α=0·00111 at this interim analysis, with partial alpha from progression-free survival in patients with CPS of 10 or more passed over), and finally in the intention-to-treat population (α=0·00111 at this interim analysis). This study is registered with ClinicalTrials.gov, NCT02819518, and is ongoing. Between Jan 9, 2017, and June 12, 2018, of 1372 patients screened, 847 were randomly assigned to treatment, with 566 patients in the pembrolizumab-chemotherapy group and 281 patients in the placebo-chemotherapy group. At the second interim analysis (data cutoff, Dec 11, 2019), median follow-up was 25·9 months (IQR 22·8-29·9) in the pembrolizumab-chemotherapy group and 26·3 months (22·7-29·7) in the placebo-chemotherapy group. Among patients with CPS of 10 or more, median progression-free survival was 9·7 months with pembrolizumab-chemotherapy and 5·6 months with placebo-chemotherapy (hazard ratio [HR] for progression or death, 0·65, 95% CI 0·49-0·86; one-sided p=0·0012 [primary objective met]). Median progression-free survival was 7·6 and 5·6 months (HR, 0·74, 0·61-0·90; one-sided p=0·0014 [not significant]) among patients with CPS of 1 or more and 7·5 and 5·6 months (HR, 0·82, 0·69-0·97 [not tested]) among the intention-to-treat population. The pembrolizumab treatment effect increased with PD-L1 enrichment. Grade 3-5 treatment-related adverse event rates were 68% in the pembrolizumab-chemotherapy group and 67% in the placebo-chemotherapy group, including death in <1% in the pembrolizumab-chemotherapy group and 0% in the placebo-chemotherapy group. Pembrolizumab-chemotherapy showed a significant and clinically meaningful improvement in progression-free survival versus placebo-chemotherapy among patients with metastatic triple-negative breast cancer with CPS of 10 or more. These findings suggest a role for the addition of pembrolizumab to standard chemotherapy for the first-line treatment of metastatic triple-negative breast cancer. Merck Sharp & Dohme Corp, a subsidiary of Merck & Co, Inc.

Simultaneous Targeting of RSK and AKT Efficiently Inhibits YB-1-Mediated Repair of Ionizing Radiation-Induced DNA Double-Strand Breaks in Breast Cancer Cells

Y-box binding protein 1 (YB-1) overexpression is associated with chemotherapy- and radiation therapy resistance. Ionizing radiation (IR), receptor tyrosine kinase ligands, and mutation in KRAS gene stimulate activation of YB-1. YB-1 accelerates the repair of IR-induced DNA double-strand breaks (DSBs). Ribosomal S6 kinase (RSK) is the main kinase inducing YB-1 phosphorylation. We investigated the impact of RSK targeting on DSB repair and radiosensitivity. The triple negative breast cancer (TNBC) cell lines MDA-MB-231, MDA-MB-468, and Hs 578T, in addition to non-TNBC cell lines MCF7, HBL-100, and SKBR3, were used. MCF-10A cells were included as normal breast epithelial cells. The RSK inhibitor LJI308 was used to investigate the role of RSK activity in S102 phosphorylation of YB-1 and YB-1-associated signaling pathways. The activation status of the underlying pathways was investigated by Western blotting after treatment with pharmacologic inhibitors or transfection with siRNA. The impact of LJI308 on DSB repair and postirradiation cell survival was tested by the γH2AX foci and the standard clonogenic assays, respectively. LJI308 inhibited the phosphorylation of RSK (T359/S363) and YB-1 (S102) after irradiation, treatment with EGF, and in cells expressing a KRAS mutation. LJI308 treatment slightly inhibited DSB repair only in some of the cell lines tested. This was shown to be due to PI3K-dependent stimulation of AKT or constitutive AKT activity mainly in cancer cells but not in normal breast epithelial MCF-10A cells. Simultaneous targeting of AKT and RSK strongly blocked DSB repair in all cancer cell lines, independent of TNBC status or KRAS mutation, with a minor effect in MCF-10A cells. Cotargeting of RSK- and AKT-induced radiation sensitivity in TNBC MDA-MB-231 and non-TNBC MCF7 cells but not in MCF-10A cells. Simultaneous targeting of RSK and AKT might be an efficient approach to block the repair of DSBs after irradiation and to induce radiosensitization of breast cancer cells.