Luminal Tumors Research Articles

Pparg signaling controls bladder cancer subtype and immune exclusion

Pparg, a nuclear receptor, is downregulated in basal subtype bladder cancers that tend to be muscle invasive and amplified in luminal subtype bladder cancers that tend to be non-muscle invasive. Bladder cancers derive from the urothelium, one of the most quiescent epithelia in the body, which is composed of basal, intermediate, and superficial cells. We find that expression of an activated form of Pparg (VP16;Pparg) in basal progenitors induces formation of superficial cells in situ, that exit the cell cycle, and do not form tumors. Expression in basal progenitors that have been activated by mild injury however, results in luminal tumor formation. We find that these tumors are immune deserted, which may be linked to down-regulation of Nf-kb, a Pparg target. Interestingly, some luminal tumors begin to shift to basal subtype tumors with time, down-regulating Pparg and other luminal markers. Our findings have important implications for treatment and diagnosis of bladder cancer.

A DOCK1 Gene-Derived Circular RNA Is Highly Expressed in Luminal Mammary Tumours and Is Involved in the Epithelial Differentiation, Growth, and Motility of Breast Cancer Cells.

Simple SummaryIn the present study, we define the expression of 15 selected circular RNAs, using a panel of 18 breast cancer cell lines recapitulating the heterogeneity of tumors and consisting of three distinct groups according to the mesenchymal/epithelial phenotype. We identify circDOCK1-1 generated from the DOCK1 gene as an important regulator of the epithelial differentiation of mammary tumour cells. In addition, our high-throughput transcriptomic studies provide information on the genes that are regulated by circDOCK1-1, casting new light on the gene networks involved in the processes underlying the development of mammary tumours. Our results further indicate that circDOCK1-1 controls cell–cell adhesion and consequently random as well as directional motility. Besides their interest from a basic point of view, the data generated may be of translational relevance, as they suggest that strategies aimed at increasing the amounts or at stimulating the activity of circDOCK1-1 may be of significance for the treatment of a subset of triple-negative breast cancer patients.Circular RNAs are regulatory molecules involved in numerous cellular processes and may be involved in tumour growth and diffusion. Here, we define the expression of 15 selected circular RNAs, which may control the process of epithelial-to-mesenchymal transition, using a panel of 18 breast cancer cell lines recapitulating the heterogeneity of these tumours and consisting of three groups according to the mesenchymal/epithelial phenotype. A circular RNA from the DOCK1 gene (hsa_circ_0020397) shows low/undetectable levels in triple-negative mesenchymal cell lines, while its content is high in epithelial cell lines, independent of estrogen receptor or HER2 positivity. RNA-sequencing experiments performed on the triple-negative/mesenchymal MDA-MB-231 and MDA-MB-157 cell lines engineered to overexpress hsa_circ_0020397 demonstrate that the circRNA influences the expression of 110 common genes. Pathway analysis of these genes indicates that overexpression of the circular RNA differentiates the two mesenchymal cell lines along the epithelial pathway and increases cell-to-cell adhesion. This is accompanied by growth inhibition and a reduction in the random/directional motility of the cell lines. The upregulated AGR2, ENPP1, and PPP1R9A genes as well as the downregulated APOE, AQP3, CD99L2, and IGFBP4 genes show an opposite regulation by hsa_circ_0020397 silencing in luminal CAMA1 cells. The results provide novel insights into the role played by specific circular RNAs in the generation/progression of breast cancer.

Halophilic Carotenoids and Breast Cancer: From Salt Marshes to Biomedicine.

Breast cancer is the leading cause of death among women worldwide. Over the years, oxidative stress has been linked to the onset and progression of cancer. In addition to the classical histological classification, breast carcinomas are classified into phenotypes according to hormone receptors (estrogen receptor—RE—/progesterone receptor—PR) and growth factor receptor (human epidermal growth factor receptor—HER2) expression. Luminal tumors (ER/PR-positive/HER2-negative) are present in older patients with a better outcome. However, patients with HER2-positive or triple-negative breast cancer (TNBC) (ER/PR/HER2-negative) subtypes still represent highly aggressive behavior, metastasis, poor prognosis, and drug resistance. Therefore, new alternative therapies have become an urgent clinical need. In recent years, anticancer agents based on natural products have been receiving huge interest. In particular, carotenoids are natural compounds present in fruits and vegetables, but algae, bacteria, and archaea also produce them. The antioxidant properties of carotenoids have been studied during the last years due to their potential in preventing and treating multiple diseases, including cancer. Although the effect of carotenoids on breast cancer during in vitro and in vivo studies is promising, clinical trials are still inconclusive. The haloarchaeal carotenoid bacterioruberin holds great promise to the future of biomedicine due to its particular structure, and antioxidant activity. However, much work remains to be performed to draw firm conclusions. This review summarizes the current knowledge on pre-clinical and clinical analysis on the use of carotenoids as chemopreventive and chemotherapeutic agents in breast cancer, highlighting the most recent results regarding the use of bacterioruberin from haloarchaea.

Patients with Muscle-Invasive Bladder Cancer with Nonluminal Subtype Derive Greatest Benefit from Platinum Based Neoadjuvant Chemotherapy.

Neoadjuvant chemotherapy (NAC) prior to radical cystectomy (RC) in patients with nonmetastatic muscle-invasive bladder cancer (MIBC) confers an absolute survival benefit of 5%-10%. There is evidence that molecular differences between tumors may impact response to therapy, highlighting a need for clinically validated biomarkers to predict response to NAC. Four bladder cancer cohorts were included. Inverse probability weighting was used to make baseline characteristics (age, sex and clinical tumor stage) between NAC-treated and untreated groups more comparable. Molecular subtypes were determined using a commercial genomic subtyping classifier. Survival rates were estimated using weighted Kaplan-Meier curves. Cox proportional hazards models were used to evaluate the primary and secondary study end points of overall survival (OS) and cancer-specific survival, respectively. A total of 601 patients with MIBC were included, of whom 247 had been treated with NAC and RC, and 354 underwent RC without NAC. With NAC, the overall net benefit to OS and cancer-specific survival at 3 years was 7% and 5%, respectively. After controlling for clinicopathological variables, nonluminal tumors had greatest benefit from NAC, with 10% greater OS at 3 years (71% vs 61%), while luminal tumors had minimal benefit (63% vs 65%) for NAC vs non-NAC. In patients with MIBC, a commercially available molecular subtyping assay revealed nonluminal tumors received the greatest benefit from NAC, while patients with luminal tumors experienced a minimal survival benefit. A genomic classifier may help identify patients with MIBC who would benefit most from NAC.

Evaluation of the Histopathological Features of Early-stage Invasive Ductal Breast Carcinoma by 18Fluoride-fluorodeoxyglucose Positron Emission Tomography/Computed Tomography

Objectives:This study investigates the relationship between 18fluoride-fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET/CT) parameters and histopathological features in patients with early-stage invasive ductal breast carcinoma (IDBC).Methods:Patients with early-stage IDBC who underwent 18F-FDG PET/CT scan for staging were included in this retrospective study. The status of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2, Ki-67 proliferative index, and grades of tumors were recorded. The conventional metabolic parameters [maximum standard uptake value (SUVmax) and average standard uptake value] and volume-based parameters [metabolic tumor volume (MTV) and total lesion glycolysis] of the primary tumor were obtained from the 18F-FDG PET/CT images. The associations and correlations between the 18F-FDG PET/CT parameters and histopathological features were assessed.Results:One hundred forty-three patients were included. 18F-FDG PET/CT parameters, other than MTV, were significantly associated with the ER and PR status and Ki-67 index, while T-staging was significantly associated with all 18F-FDG PET/CT parameters. In the axillary lymph node (ALN) involvement, no significant difference was found in the 18F-FDG PET/CT parameters. In terms of the pathological stage, a significant difference was found in all 18F-FDG PET/CT parameters. 18F-FDG PET/CT parameters, other than MTV, were significantly higher in non-luminal breast tumors than luminal tumors and in high-grade tumors than low-grade ones. Triple-negative tumors had the highest 18F-FDG PET/CT parameter, but the difference was insignificant for MTV. The SUVmax had the strongest correlation with Ki-67 proliferative index.Conclusion:Tumors with aggressive histopathological features had higher 18F-FDG PET/CT parameter values. This study suggests that 18F-FDG PET/CT may provide prognostic information in patients with early-stage IDBC.

Prognosis Associated With Luminal and Basal Subtypes of Metastatic Prostate Cancer

Luminal and basal subtypes of primary prostate cancer have been shown to be molecularly distinct and clinically important in predicting response to therapy. These subtypes have not been described in metastatic prostate cancer. To identify clinical and molecular correlates of luminal and basal subtypes in metastatic castration-resistant prostate cancer (mCRPC) and investigate differences in survival, particularly after treatment with androgen-signaling inhibitors (ASIs). In this cohort study, a retrospective analysis was conducted of 4 cohorts with mCRPC (N = 634) across multiple academic centers. Treatment was at the physicians' discretion. Details of the study cohorts have been published elsewhere between 2016 and 2019. Data were analyzed from March 2018 to February 2021. The primary clinical end point was overall survival from the date of tissue biopsy/molecular profiling. Luminal and basal subtypes were also stratified by postbiopsy ASI treatment. The primary molecular analyses included associations with small cell/neuroendocrine prostate cancer (SCNC), molecular pathways, and DNA alterations. In the 634 patients, 288 (45%) had tumors classified as luminal, and 346 (55%) had tumors classified as basal. However, 53 of 59 (90%) SCNC tumors were basal (P < .001). Similar to primary prostate cancer, luminal tumors exhibited overexpression of AR pathway genes. In basal tumors, a significantly higher rate of RB1 loss (23% basal vs 4% luminal; P < .001), FOXA1 alterations (36% basal vs 27% luminal; P = .03) and MYC alterations (73% basal vs 56% luminal; P < .001) were identified. Patients with basal tumors had worse overall survival compared with those with luminal tumors only in patients treated with an ASI postbiopsy (East Coast Dream Team: hazard ratio [HR], 0.39; 95% CI, 0.20-0.74; P = .004; West Coast Dream Team: HR, 0.57; 95% CI, 0.33-0.97; P = .04). Among patients with luminal tumors, those treated with an ASI had significantly better survival (HR, 0.27; 95% CI, 0.14-0.53; P < .001), whereas patients with basal tumors did not (HR, 0.62; 95% CI, 0.36-1.04, P = .07). The interaction term between subtype and ASI treatment was statistically significant (HR, 0.42; 95% CI, 0.20-0.89; P = .02). These findings represent the largest integrated clinical, transcriptomic, and genomic analysis of mCRPC samples to date, and suggest that mCRPC can be classified as luminal and basal tumors. Analogous to primary prostate cancer, these data suggest that the benefit of ASI treatment is more pronounced in luminal tumors and support the use of ASIs in this population. In the basal tumors, a chemotherapeutic approach could be considered in some patients given the similarity to SCNC and the diminished benefit of ASI therapy. Further validation in prospective clinical trials is warranted.

Activation of epithelial-mesenchymal transition process during breast cancer progression - the impact of molecular subtype and stromal composition.

Breast cancer (BC) is a heterogeneous disease with different molecular subtypes, which can be defined by oestrogen (ER), progesterone (PR) and human epidermal growth factor (HER2) receptors' status as luminal, HER2+ and triple negative (TNBC). Molecular subtypes also differ in their epithelial-mesenchymal phenotype, which might be related to their aggressiveness, as activation of the epithelial-mesenchymal transition (EMT) is linked with increased ability of cancer cells to survive andmetastasize. Nevertheless, the reverse process of mesenchymal-epithelial transition was shown tobe required to sustain metastatic colonization. In this study we aimed to analyse activation of the EMT process in primary tumours (PT), which have (N+) or have not (N-) colonized the lymph nodes, as well as the lymph nodes metastases (LNM) themselves in 88 BC patients. We showed that luminal N- PT have the lowest activation of the EMT process (27%), in comparison to N+ PT (48%,p=0.06). On the other hand, TNBC do not show statistically significant EMT activation at the stage before lymph colonization (N-, 83%) and after colonization of the lymph nodes (N+, 63%, p=0.58). TNBC are also the least plastic (unable to change the EMT phenotype) in terms of turning EMT on or off between matched PT and LNM (0% EMT plasticity in TNBC vs 36% plasticity in luminal tumours). Moreover, in TNBC activation of EMT was correlated with increased cell division rate of the PT- inmesenchymal TNBC PT median Ki-67 was 45% in comparison to 10% in epithelial TNBC PT (p=0.002), whereas in PT of luminal subtypes Ki-67 did not differ between epithelial andmesenchymal phenotypes. Profiling of immunotranscriptome of epithelial and mesenchymal luminal BC with Nanostring technology revealed that N- PT with epithelial phenotype were enriched ininflammatory response signatures, whereas N+ mesenchymal cancers showed elevated MHC class II antigen presentation. Overall, activation of EMT changes during cancer progression and metastatic colonization of the lymph nodes depending on the PT molecular subtype and is related to differences in stromal signatures. Activation of EMT is associated with colonizing phenotype in luminal PT andproliferative phenotype of TNBC.

Integrative Transcriptomic, Lipidomic, and Metabolomic Analysis Reveals Potential Biomarkers of Basal and Luminal Muscle Invasive Bladder Cancer Subtypes.

Muscle invasive bladder cancer (MIBC) is a heterogeneous disease with a high recurrence rate and poor clinical outcomes. Molecular subtype provides a new framework for the study of MIBC heterogeneity. Clinically, MIBC can be classified as basal and luminal subtypes; they display different clinical and pathological characteristics, but the molecular mechanism is still unclear. Lipidomic and metabolomic molecules have recently been considered to play an important role in the genesis and development of tumors, especially as potential biomarkers. Their different expression profiles in basal and luminal subtypes provide clues for the molecular mechanism of basal and luminal subtypes and the discovery of new biomarkers. Herein, we stratified MIBC patients into basal and luminal subtypes using a MIBC classifier based on transcriptome expression profiles. We qualitatively and quantitatively analyzed the lipids and metabolites of basal and luminal MIBC subtypes and identified their differential lipid and metabolite profiles. Our results suggest that free fatty acids (FFAs) and sulfatides (SLs), which are closely associated with immune and stromal cell types, can contribute to the diagnosis of basal and luminal subtypes of MIBC. Moreover, we showed that glycerophosphocholine (GCP)/imidazoles and nucleosides/imidazoles ratios can accurately distinguish the basal and luminal tumors. Overall, by integrating transcriptomic, lipidomic, and metabolomic data, our study reveals specific biomarkers to differentially diagnose basal and luminal MIBC subtypes and may provide a basis for precision therapy of MIBC.

African-American Patients Differentially Express Genes in Luminal Tumors

African-American Patients Differentially Express Genes in Luminal Tumors

Molecular subtyping and immune-gene signatures identify a subset of early bladder tumors as candidates for single-agent immune-checkpoint inhibition

PurposeClinical high-grade (HG) T1 non-muscle invasive bladder cancer (NMIBC) represents a significant risk to patients, but these patients are not typically offered neoadjuvant therapies, including immune therapy. In this study, we determine whether patients with HG clinical T1 or T2 bladder urothelial carcinoma (UC) have profiles that predict the potential effectiveness of immune-checkpoint inhibitors (ICI). Materials and MethodsData from transurethral resection of bladder tumor (TURBT) specimens from 2 studies was evaluated. The molecular upstaging (MOL) cohort included HG cT1N0M0 (n = 87) and cT2N0M0 (n = 119) bladder UC who underwent radical cystectomy (RC) without any neoadjuvant therapy. The PURE-01 cohort (n = 102) was used as ICI-treated reference. Specimen collection and sample processing were conducted using a clinical-grade whole-transcriptome assay (Decipher). Immune-signatures scores and molecular subtyping were evaluated. Kaplan-Meier curves and log-rank tests were used for exploratory analyses of recurrence-free survival (RFS) and overall survival (OS). ResultsIn both the PURE-01 and MOL cohorts, the Immune190 signature, stratified by subtype, showed the highest scores in basal-type, but also in luminal-infiltrated tumors, but the lowest scores in the luminal tumors. However, in HG cT1 tumors the Immune190 scores were the lowest for luminal papillary tumors (Consensus, TCGA) and luminal tumors (GSC), with less distinct differences between other subtypes. RFS was significantly longer for luminal vs non-luminal tumors in MOL (P = 0.04) but not in PURE-01 (P = 0.8). In the MOL cohort, OS was inferior in HG cT1 tumors for Immune190-high vs low tumors (median split, P = 0.042). ConclusionWe identified a population of cT1-T2N0M0 tumors in the MOL cohort that shared molecular features with tumors included in PURE-01. These profiles suggest that treatment with ICI could be proposed to more selected HG cT1N0M0 tumors, identified with a gene expression assay.

Detection of secondary metastatic breast cancer by measurement of plasma CA 15.3

BackgroundMost current guidelines do not recommend the serial analysis of tumour marker CA 15.3 in the follow-up of asymptomatic patients treated for early breast cancer (EBC). These guidelines are based on small-scale studies carried out in an era with more limited treatment options than today. In our large academic centre, serial measurements of CA 15.3 are used routinely in the follow-up of EBC, whereas imaging for distant metastases is only carried out on indication.Patients and methodsIn this retrospective single-centre study, patients were included if they were treated for EBC between 1 January 2000 and 1 January 2018, diagnosed with secondary metastatic disease at least 6 months after initial surgery and had CA 15.3 available at the time of diagnosis of metastases. The primary objective was to evaluate the proportion of patients in whom metastatic disease was discovered by an increasing CA 15.3. Information on the method of metastases detection, CA 15.3 evolution and survival was collected after approval of the ethics committee.ResultsAt the moment of diagnosis of metastases, 451 of 730 included patients (62%) had CA 15.3 levels above the upper limit of normal (>30 kU/l). In 269 patients (37%), an increasing CA 15.3 was the first sign that led to the diagnosis of metastases. This was most frequent in luminal A-like tumours (48%) and in liver (45%) and bone (41%) localisation of metastases. By contrast, reported symptoms triggered the diagnosis of metastatic disease in 48% of the patients. Median overall survival was significantly longer when the relapse was discovered by CA 15.3 elevation versus those discovered by another trigger (abnormal clinical examination or history, abnormal laboratory tests or an incidental finding) (35 versus 22 months; P = 0.0027).ConclusionWhen CA 15.3 is systematically used in the follow-up of EBC patients, the diagnosis of metastatic disease is made in 37% by a CA 15.3 increase.

High prevalence of APOA1/C3/A4/A5 alterations in luminal breast cancers among young women in East Asia

In East Asia, the breast cancer incidence rate among women aged <50 years has rapidly increased. Emerging tumors are distinctly characterized by a high prevalence of estrogen receptor (ER)–positive/human epidermal growth factor receptor (HER2)–negative cancer. In the present study, we identified unique genetic alterations in these emerging tumors. We analyzed gene copy number variations (CNVs) in breast tumors from 120 Taiwanese patients, and obtained public datasets of CNV and gene expression (GE). The data regarding CNV and GE were separately compared between East Asian and Western patients, and the overlapping genes identified in the comparisons were explored to identify the gene–gene interaction networks. In the age <50 years/ER + /HER2– subgroup, tumors of East Asian patients exhibited a higher frequency of copy number loss in APOA1/C3/A4/A5, a lipid-metabolizing gene cluster (33 vs. 10%, P < .001) and lower APOA1/C3/A4/A5 expressions than tumors of Western patients. These copy number loss related– and GE–related results were validated in another Taiwanese cohort and in two GE datasets, respectively. The copy number loss was significantly associated with poor survival among Western patients, but not among East Asian patients. Lower APOA1, APOC3, and APOA5 expressions were associated with higher ESTIMATE immune scores, indicating an abundance of tumor-infiltrating immune cells. In conclusion, APOA1/C3/A4/A5 copy number loss was more prevalent in luminal breast tumors among East Asian women aged <50 years, and its immunomodulatory effect on the tumor microenvironment possibly plays various roles in the tumor biology of East Asian patients.

CPT1A and fatty acid β-oxidation are essential for tumor cell growth and survival in hormone receptor-positive breast cancer.

Chromosome 11q13-14 amplification is a defining feature of high-risk hormone receptor-positive (HR+) breast cancer; however, the mechanism(s) by which this amplicon contributes to breast tumorigenesis remains unclear. In the current study, proteogenomic analyses of >3000 breast tumors from the TCGA, METABRIC and CPTAC studies demonstrated that carnitine palmitoyltransferase 1A (CPT1A), which is localized to this amplicon, is overexpressed at the mRNA and protein level in aggressive luminal tumors, strongly associated with indicators of tumor proliferation and a predictor of poor prognosis. In vitro genetic studies demonstrated that CPT1A is required for and can promote luminal breast cancer proliferation, survival, as well as colony and mammosphere formation. Since CPT1A is the rate-limiting enzyme during fatty acid oxidation (FAO), our data indicate that FAO may be essential for these tumors. Pharmacologic inhibition of FAO prevented in vitro and in vivo tumor growth and cell proliferation as well as promoted apoptosis in luminal breast cancer cells and orthotopic xenograft tumor models. Collectively, our data establish an oncogenic role for CPT1A and FAO in HR+ luminal tumors and provide preclinical evidence and rationale supporting further investigation of FAO as a potential therapeutic opportunity for the treatment of HR+ breast cancer.

Abstract 2729: Cell infiltration prediction of breast cancer tissues identifies basophils as potentially important immune cells in the tumor microenvironment

Abstract Background: Breast cancer (BC) is the most common and deadliest cancer in women worldwide. Hormone receptor (HR) positive tumors (luminal) are the most frequent while HER-2 overexpressing (HR-, HER2+) and triple negative (TNBC, HR-/HER2-) are most deadly. HR+ subtypes are more common in non-Hispanic White women while HER-2 and TNBC present either higher prevalence or risk of mortality in minority groups. Hispanic/Latinos (H/L) is the largest and fastest growing minority in the U.S.A. However, H/L women have been underrepresented in BC studies. We reported that luminal B is the most common BC subtype in a group of H/L and that ERBB2, GRB7 and MIEN1 genes have increased expression in women with higher Indigenous American ancestry. Immune infiltration of tumors has become a hallmark for the prediction of outcome. However, contrary to TNBC, luminal tumors have been considered low immunogenic. Immune infiltration of luminal B tumors in H/L has not yet been investigated. Our goal was to predict immune infiltration of luminal B tumors based on RNA-seq data and according to the levels of body mass index (BMI) and ancestral fractions (West African, European, Indigenous American). Methods: We performed RNA-seq in 70 luminal B tumors of H/L from Puerto Rico. Ancestry was estimated by genotyping on the Affymetrix U.K. Biobank array and global ancestry proportions determined with Admixture Software v1.3, using 1000 Genomes reference population for anchoring. BMI, age at diagnosis and tumor size were extracted from the Electronic Medical Records. We used Partek Flow for the RNA-seq analysis based on ancestry fraction and BMI. Prediction of cell populations was done in xCell. Correlation analysis was done in GraphPad Prism v7.05. Results: We found an inverse correlation between Indigenous American and γδ-T cell infiltration (R2= -0.3; p=0.03) while BMI was associated with increased infiltration of basophils (R2=0.3; p=0.026). Among women with high BMI, those with low European ancestry had increased levels of basophils infiltration R2=0.66; p=0.17) and those with high European ancestry had reduced infiltration of naïve CD8+ T cells (R2= -0.53; p=0.008) and reduced plasmacytoid dendritic cells (pDC, R2=-0.51; p=0.01). Conclusions: We found that luminal B tumors have infiltration of immune cells that may contribute to differences in the response to treatment and outcome. To the best of our knowledge, the role of infiltrating basophils into breast cancer tissues has not been described. However, circulating basophils and predicted infiltration of basophils into ovarian and colorectal cancer tissues have been correlated with survival. Our findings have to be confirmed in a larger set of samples and by additional methods, including immunohistochemistry. Citation Format: Jone Garai, Julie Dutil, Douglas W. Cress, Victoria Seewaldt, Jamie K. Teer, Laura Fejerman, Lucio Miele, Jovanny Zabaleta. Cell infiltration prediction of breast cancer tissues identifies basophils as potentially important immune cells in the tumor microenvironment [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 2729.

Abstract 1188: Reclassification of ER+ (luminal A/luminal B1 minus ER low)-like and ER- like breast tumors based on proteomic/gene and clinical outcome signatures

Abstract Introduction: Classification of breast cancer can incorporate immunohistochemical (IHC) detection of ER/PR/HER2/KI67 to stratify the subtypes. High throughput proteomics analysis allows for the expansion of biomarker discovery within the subtypes. We evaluated a cohort of 109 tumors characterized as ER+ (Luminal A and Luminal B1; HER2+ and ER low (1-10%) cases were excluded) compared to ER-/HER2- tumors. Utilizing an integrated bioinformatics approach, we developed a proteomic marker signature to reclassify tumors into ER+(like) and ER-(like) tumors. CPTAC (Proteomic)/TCGA (RNAseq) datasets and larger METBRIC and GSE96058 cohorts were used to validate this marker signature. The selected biomarkers demonstrated significant differences impacting survival outcome. Methods: Clinical IHC subtyping of core biopsies was used to select a cohort of patients with ER+/HER2- and ER-/HER2- primary tumors from flash-frozen surgical samples. The positive/negative status of ER/PR/HER2 was defined using updated ASCO 2020 guidelines. Ki-67 status was determined using the 2011 St. Gallen's International Expert Consensus recommendations. Proteomic analysis was performed using Thermo Q-Exactive+ LC MS/MS analysis. Differential analysis was applied to select the significantly altered proteins between ER+ and ER- cases, Univariate survival analysis was engaged to filter informative protein/genes using TCGA RNA-Seq data. Nearest centroid analysis was deployed to define the classifier to predict novel molecular subtypes. Results/Conclusions: We selected 34 proteins/genes from 164 significantly differentially expressed proteins for further analysis. The centroid model constructed with the 34 proteins defined 2 groups: ER+(like) and ER-(like). An additional 4 groups were defined across subtypes: luminal tumors classified both by IHC and marker signature (LL), luminal tumors classified by IHC but marker signature more like triple negative (LT), triple negative tumors classified by IHC but marker signature more like luminal (TL), and triple negative classified by both IHC and marker signature (TT). This marker signature segregated close to 5000 tumors across CPTAC, TCGA, METABRIC and GSE96058 cohorts. Survival analysis in these groups of patients revealed differences in radiation, hormone/radiation, hormone therapy, and hormone/radiation/chemotherapy treatments. In summary using proteomics data we identified a 34 gene/protein marker signature, validated in large external cohorts and exhibited impact on survival and response to therapy. Further, this signature was enriched in metabolism and microenvironmental associated factors that could represent novel targets or development combination strategies based on this signature. Citation Format: Guisong Wang, Punit Shah, Rick Searfoss, Leigh Fantacone-Campbell, Jeffrey A. Hooke, Brenda Deyarmin, Rebecca N. Zingmark, Stella Somiari, Jianfang Liu, Leonid Kvecher, Bradley Mostoller, Lori A. Sturtz, Praven-Kumar Raj-Kumar, Elder Granger, Linda Vahdat, Mary L. Cutler, Chas Bountra, Rangaprasad Sarangarajan, Hai Hu, Michael A. Kiebish, Albert J. Kovatich, Niven R. Narain, Craig D. Shriver. Reclassification of ER+ (luminal A/luminal B1 minus ER low)-like and ER- like breast tumors based on proteomic/gene and clinical outcome signatures [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 1188.

Ki-67 proliferative index correlation to the immunohistochemistry profile in early female breast cancer: a review of 515 cases

Introduction: Many biological markers are used as prognostic and predictive indicators in invasive breast cancers management. Among them, tumour size, grade, patho-morphological subtype, hormone receptors status and HER2 receptor expression in addition to Ki-67 proliferative index. Also, they play a key role in adjuvant treatment decision making. Our aim was to evaluate the association between Ki-67 proliferative index and breast cancer immunological subtype. Material and methods: A total of 515 early invasive patients were enrolled, tumour biological characteristics as histopathological subtype, immune-histo-chemistry (ER,PR,HER2) status and Ki-67 proliferation index values have been collected. The Ki-67 index level of 20%, was used as the cut-off point to differentiate between low and high Ki-67 expression levels. Statistical analysis has been performed using the Chi square test online tool. Results: In this cohort, about 42%, 33%, 7%, and 18% of the cases were grouped as luminal A-like, luminal B-like, HER2 enriched subtype, and triple-negative, respectively. All luminal A-like patients had Ki-67 level less than 20%. About 3% of the cohort, are luminal B-like tumours with Ki-67 level less than 20%, where 30.3% of the patients were luminal B-like tumours with Ki-67 level ≥ 20%. In HER2 enriched subtype, Ki- 67 of < 20% level seen in 1.9% of cases, and Ki-67 levels ≥ 20% was observed in 5.2% of the cases. In the triple-negative group, Ki-67 was 20% or higher in 16% of cases, and only 1.7% of patients had Ki-67 level less than 20%. Conclusion: Luminal A-like tumours were the most frequently encountered subtype, they have low Ki-67 levels and are known to be of a low histological grade tumour, and usually associated with a good prognosis. Also, data indicates that high Ki-67 levels are seen more often in Luminal B-Like breast cancers as well as in triple-negative breast cancers and HER2 enriched tumours.

Biological and clinical features of triple negative Invasive Lobular Carcinomas of the breast. Clinical outcome and actionable molecular alterations

BackgroundWe report here for the first time, a comprehensive characterization of biological and clinical features of early-stage triple negative Invasive Lobular Carcinomas(TN-ILCs) MethodsWe analyzed all consecutive patients with early-stage TN-ILC operated at two reference cancer-centers between 1994 and 2012.Primary objective was to assess the invasive disease-free survival(iDFS).Co-primary objective was to assess biological features of TN-ILCs, including molecular intrinsic subtypes based on PAM-50 assay, expression of androgen receptor (AR) and mutational status of ERBB2-gene.Additionally, DNA mutational status of an independent cohort of 45 TN-ILCs from three databases were analyzed, to confirm mutations in ERBB2-gene and to identify other recurrently mutated genes. ResultsAmong 4152 ILCs, 74(1.8%) were TN and were analyzed.The iDFS at 5 and 10 years of FUP were 50.4%(95%CI,38.0–61.6) and 37.2%(95%CI,25.5–48.8), respectively.The molecular subtype was defined through PAM50-classifier for 31 out of 74 TN-ILCs: 48% were Luminal-A(15/31), 3% luminal-B(1/31), 32% HER2-enriched (10/31), and only 16% basal-like(5/31).Luminal tumors expressed AR more frequently than non-luminal tumors (AR≥1% in 94% of luminal tumors versus 53% in non-luminal tumors; p-value = 0.001).20% of TN-ILCs analyzed(7/35), harbored a pathogenetic and actionable mutation in the ERBB2-gene. Analysis of the independent cohort of 45 TN-ILCs from three different databases, confirmed similar percentage of pathogenetic and actionable mutations in ERBB2-gene(20%; 9/45).Among the top 10 molecular pathways significantly enriched for recurrently mutated genes in TN-ILCs(FDR<0.05), there were ErbB-signaling and DNA-damage-response pathways. ConclusionsTN-ILCs are rare tumors with poor prognosis. Their specific biological features require newly defined targeted therapeutic strategies

Molecular characterization of luminal breast tumors in African American women.

550 Background: Racial disparities in breast cancer (BC) mortality are attributed to later stage diagnoses and a higher incidence of triple-negative BC among African American (AA) women. In previous work, we showed that AA women with ER+ BC are more likely to develop biologically aggressive disease and are more likely to die from early stage, ER+ BC than non-Hispanic White women (Hoskins et al, JAMA Oncol, 2021). The underlying molecular drivers of this disparity are unknown. Here we report the molecular characterization of a series of luminal BC from AA women. Methods: Consecutive breast tumor specimens received in the Pathology Department underwent next generation sequencing (NGS). Unstained FFPE tissue sections were macro-dissected to isolate tumor cells, and nucleic acids were extracted using commercially available kits. DNA and RNA sequencing libraries were prepared with the Oncomine Comprehensive Assay v3 (OCAv3) (Thermo Fisher), which includes 161 driver genes and detects SNVs, CNVs, INDELs and gene fusions. Sequencing was performed on the Ion S5XL sequencer. Sequencing reads were mapped to the UCSC human genome build GRCh37/hg19 using Torrent Suite™ software (version 5.10; Thermo Fisher). Data analysis and variant calling was performed using the Ion Reporter analysis tool. Results: We identified 60 somatic driver gene alterations in luminal tumors from 35 AA patients (primary tumors, n = 26; metastatic tumors, n = 9). Recurrently altered genes identified in &gt; 5% of tumors are listed in the Table. The most frequently altered gene was PIK3CA (42% of tumors). ESR1 gene fusions were seen in 25% of tumors. Interestingly, an equal frequency of ESR1 fusions were detected in primary (27%) and metastatic (22%) tumors, in contrast to activating mutations which are found in recurrent tumors following treatment with aromatase inhibitors. ARID1A alterations were identified in 17% of primary tumors. ARID1A encodes a subunit of the SWI/SNF chromatin remodeling complex. Alterations in ARID1A confer endocrine resistance, and are enriched in recurrent tumors in the literature. We also found a high number of CNVs in members of the FGF gene family (36% of tumors), which are also associated with resistance to endocrine therapy. An in silico analysis comparing our findings with publicly available datasets will be presented. Conclusions: This study of somatic driver gene alterations in a consecutive series of luminal breast tumors from AA patients found a higher than expected frequency of alterations in genes associated with endocrine resistance in untreated primary tumors, suggesting a partial explanation for racial disparities in survival.[Table: see text]

Disparities within luminal breast cancer: Clinical and molecular features of African American and non-Hispanic white patients.

1009 Background: African American breast cancer patients (AA) are diagnosed younger, have more high-risk features, and poorer clinical outcomes than non-Hispanic White patients (NHW), despite similar treatments. Although comorbidities such as obesity and metabolic syndrome may contribute to differences, ancestry-specific factors and effects of structural violence that disproportionately afflict AA individuals may influence tumor biology and outcomes. We previously reported differentially expressed genes (DEGs) associated with tumor aggressiveness in Basal tumors from AA compared with NHW (Sharma et al., 2020). Here, we compare DEGs in luminal tumors between AA and NHW. Methods: The prospective, observational FLEX study (NCT03053193) includes stage I-III breast cancer patients who receive 70-gene signature (MammaPrint/MP)/80-gene signature (BluePrint/BP) testing and consent to full transcriptome and clinical data collection. AA (n=364) and NHW (n=400, random selection) with BP luminal tumors, enrolled from 2017 to present, were included. Race/ethnicity was self-reported. AA were younger than NHW (mean, 59 vs. 62 years, p=0.001); thus, an age-matched subset (n= 360 AA, NHW) was compared. Differential gene expression analysis was performed with R limma package. Comparisons were made between AA and age-matched or randomly selected NHW in: (1) all, (2) luminal A, (3) luminal B, and (4) luminal B, obese. DEGs with FDR&lt;0.05 were significant. Different fold change (FC) thresholds were evaluated. Results: Compared with age-matched NHW, AA were similar in menopausal status, T stage, grade, and tumor type; obesity, T2DM status, and nodal stage were significantly different ( p&lt;0.01). Tumors from AA were more often MP high risk ( p&lt;0.001), regardless of age matching. Luminal B AA vs. age-matched NHW comparison resulted in more DEGs (n=1070) than other comparisons; however, most were FC&lt;2. Notably, 5/6 DEGs ( PSPH, NOTCH2NL, POLR1A, MAP1LC3P and RPS26P10) in basal tumors (Nunes et al. 2019) were also identified here. Of 9 DEGs (FC&gt;1.7) in the luminal B age-matched comparison, 2 ( PSPH and LINC01139) were also found in the luminal B, obese subset. Consistently upregulated DEGs in AA were associated with metabolism, translation, and cellular stress response pathways. Conclusions: We found significant transcriptomic differences between luminal tumors from AA and NHW, when controlling for age, obesity, and genomic classification. A subset of DEGs in luminal B tumors were consistent with those in Basal tumors, suggesting that similar race-associated factors drive DEGs regardless of tumor subtype. DEGs that may be unique to AA luminal tumors were also found. This study suggests that some biological differences in breast tumors may result from patient ancestry or shared adverse socioeconomic exposures and underscores the need for inclusion of diverse patient groups in clinical trials. Clinical trial information: NCT03053193.

ER and immune-related signatures define benefit to palbociclib, trastuzumab, pertuzumab +/- fulvestrant in ER+/HER2+ breast cancer patients in the NA-PHER2 trial.

555 Background: In the NA-PHER2 study we assessed the association between biological pathways with pathological complete response (pCR) and Ki67 down-regulation Methods: Patients with centrally confirmed ER+ ( &gt; 10%) HER2+ breast cancer (BC) were treated in two independent, non-randomized cohorts with neoadjuvant trastuzumab, pertuzumab, palbocilib with (Fulv, n = 30) or without (NoFulv, n = 28) fulvestrant (+/-LHRH analogues). We assessed RNA-seq on core-biopsies obtained pre-treatment [n = 53/58 (91.4%)], at day 14 [n = 49/58 (84.5%)], and on residual disease at surgery [n = 42/45 (93.3%)]. We investigated biomarker dynamics and association with pCR or Ki67 down-regulation (centrally evaluated) at day 14 and at surgery. In the overall population and in each cohort, we primarily assessed three pre-defined biomarkers (ER-metagene [from OncotypeDX], a CD8-metagene and ERBB2 expression), and secondarily we explored a pre-defined list of genesets. Continuous and categorical (median cut-point) variables were evaluated. Results: In the biomarker population, pCR rate was 22.5% (28.6% and 16.0% in Fulv and NoFulv cohorts). At baseline, continuous CD8-metagene (OR 1.85 [1.12-3.06], p = 0.016) and ER-metagene (OR 0.56 [0.34-0.90], p = 0.016) associated with higher and lower pCR rate, respectively. High ERBB2 (above median) was marginally associated with pCR (OR 3.83 [0.90-16.3], p = 0.068). Only ER- and CD8-metagenes retained significance in multivariate analysis and were similarly predictive in both cohorts. Combining categorical variables, the groups with high-CD8/low-ER and low-CD8/high-ER had 61.5% and 0% pCR rate respectively, whereas low-CD8/low-ER and high-CD8/high-ER had similar 15% pCR (p = 0.001). The association was significant in both cohorts (p = 0.019 Fulv; p = 0.028 NoFulv). Dynamic assessment of the same biomarkers at day 14 did not improve prediction. Higher ER-metagene at baseline, but not CD8 and ERBB2, was associated with robust down-regulation of Ki67 at day 14 (Ki67 &lt; 2.7%, complete cell cycle arrest) only in Fulv cohort (p = 0.016). ER-metagene also associated with retained Ki67 down-regulation (Ki67 &lt; 10%) at surgery (p = 0.002). Alternative ER- and immune-related signatures provided very similar results. The comprehensive landscape of complex molecular dynamics and exploratory association with outcome will be presented. Conclusions: In ER+/HER2+ BC, low expression of ER-related and high expression of immune-related genes identified patients with very high likelihood of achieving pCR with a chemo-free regimen. In the fulvestrant cohort, the group with high ER-metagene, despite a lower pCR rate, had higher Ki67 down-regulation at day 14, which has been associated with long-term benefit in luminal tumors. These findings provide a potential tool for tailored de-escalation strategies.