Charles M. Perou

Transcriptomic Profiling of Mouse Mammary Tumors Enables Prognostic and Predictive Biomarker Discovery for Human Breast Cancer

Molecular evaluation of residual disease following neoadjuvant chemotherapy in triple negative breast cancer CALGB40603 (Alliance)

Kinase Plasticity with Vandetanib Treatment Enhances Sensitivity to Tamoxifen in Estrogen Receptor Positive Breast Cancer

Targeting Metabolic Vulnerabilities in Basal-Like Breast Cancer

BRD8 Regulates Anti-HER2 Response in HR+/HER2+ Breast Cancer

Integrated Multi-omic Profiling Identifies BRD8/EP400 as a Pivotal Chromatin Module Mediating Anti-HER2 Response in HR+/HER2+ Breast Cancer

Anti-CSF-1R therapy with combined immuno-chemotherapy coordinate an adaptive immune response to eliminate macrophage enriched triple negative breast cancers

Women diagnosed with metastatic triple negative breast cancer (mTNBC) have limited treatment options, are more prone to develop resistance and are associated with high mortality. A cold tumor immune microenvironment (TIME) characterized by low T cells and high tumor associated macrophages (TAMs) in mTNBC is associated with the failure of standard-of-care chemotherapy and immune checkpoint blockade (ICB) treatment. We demonstrate that the combination of immunomodulatory low-dose Cyclophosphamide (CTX) coupled with anti-CSF-1R antibody targeted therapy (SNDX-ms6352) and anti-PD-1 (ICB), was highly effective against aggressive metastatic Trp53 null TNBC transplantable syngeneic models that present with high macrophage infiltration. Mechanistically, CSF-1R inhibition along with CTX disrupted the M-CSF/CSF-1R axis which upregulated IL-17, IL-5 and type II interferon resulting in elevated B- and T cell infiltration. Addition of an anti-PD-1 maintenance dose helped overcome de novo PD-L1 intra-tumoral heterogeneity (ITH) associated recurrence in lung and liver mTNBC.

Exogenous Hormones, Tumor Intrinsic Subtypes, and Breast Cancer

HER2DX in older patients with HER2-positive early breast cancer: extended follow-up from the RESPECT trial of trastuzumab ± chemotherapy

Older adults with HER2-positive early breast cancer are underrepresented in clinical trials, and the benefit of chemotherapy in this population remains uncertain. We evaluated the HER2DX genomic assay within the randomized RESPECT trial (NCT01104935), which compared adjuvant trastuzumab with or without chemotherapy in patients aged 70-80 years. In this prespecified translational analysis (Trans-RESPECT), HER2DX scores were available for 154 patients. The HER2DX risk score classified 74.0% as low risk and 26.0% as high risk. Ten-year relapse-free and overall survival were higher in the low-risk group. HER2DX remained independently associated with overall survival in multivariable analysis. The HER2DX immune, luminal, and proliferation signatures that compose the risk score were also prognostic. While the HER2DX pCR score was not prognostic overall, exploratory subgroup analyses suggested a potential survival benefit from chemotherapy in the pCR-high group. HER2DX offers prognostic value and may guide chemotherapy use in older patients with HER2-positive early breast cancer. Clinical Trial Information NCT01104935.

Prognostic significance of CD8+ T cell Spatial Biomarkers in ER+ and ER− breast cancer: A retrospective cohort study

Real-World Validation of the Purity Independent Subtyping of Tumors Classifier for Informing Therapy Selection in Pancreatic Ductal Adenocarcinoma

Integration of Digital Histology and Gene Expression to Predict Treatment-Specific Responses in Breast Cancer

Integrative proteogenomics and forward genetics reveals a novel mitotic vulnerability in triple-negative breast cancer.

Overexpression of TSG101 causes the development of adenosquamous mammary carcinoma

BACKGROUND: The mammalian Tumor Susceptibility Gene 101 (TSG101) encodes a protein with diverse functions that control the proliferation and survival of cells, but its role in malignant transformation and cancer development has remained enigmatic. METHODS: To study the pro-tumorigenic functions of TSG101, we developed a bi-transgenic mouse model that expresses exogenous TSG101 along with a luciferase reporter in a ligand-controlled manner in the mammary gland epithelium. We performed a comprehensive histopathologic, biochemical, and molecular characterization of ductal hyperplasia and mammary tumors. Unsupervised hierarchical clustering based on 1,723 intrinsic genes of ten TSG101-overexpressing cancers alongside 251 tissue samples representing 31 reference mammary tumor models and normal mammary glands was conducted. RESULTS: Females overexpressing TSG101 develop ductal hyperplasia, adenomyoepitheliomas, and palpable adenosquamous carcinomas at an average latency of approximately ten months. These metaplastic mammary tumors are comprised of transforming basal and luminal epithelial cells. Using a GFP reporter strain to monitor the transgene activation at the single-cell level, we determined that the epithelial heterogeneity within transforming ducts and ensuing carcinomas originated from the luminal epithelium. At the molecular level, TSG101-induced mammary tumors are triple-negative and exhibit gene expression signatures of Wnt and inflammatory cytokine signaling, which are key regulators of epithelial cell fate. The ligand-controlled downregulation of exogenous TSG101 in established carcinomas led to tumor regression. We demonstrated that the TSG101-mediated activation of PI3K/AKT signaling, as well as upregulation of Cyclin D1 and MDM2, are dependent on the perpetual expression of the TSG101 oncoprotein. CONCLUSIONS: The collective findings of this study provide in vivo evidence that TSG101 possesses pro-tumorigenic properties that extend to cancer progression and maintenance, suggesting that this protein could be a rational molecular target to prevent and treat a subset of mammary tumors.

Exogenous Hormones, Tumor Intrinsic Subtypes, and Breast Cancer

Importance: Etiologic heterogeneity in breast carcinogenesis needs to be well characterized for targeted prevention. Associations between menopausal hormonal therapy (MHT) and oral contraceptive (OC) use and breast cancer intrinsic-like subtypes are not well understood. Objective: To examine whether exogenous hormone use is differentially associated with breast cancer subtypes and to evaluate heterogeneity by intrinsic-like subtypes. Design, Setting, and Participants: This study pooled data from 31 nested and population-based case-control studies involved in the Breast Cancer Association Consortium. The study population included individuals with breast cancer and control participants from 13 case-control studies nested in prospective cohorts (recruited between 1982 and 2011) and 18 population-based case-control studies (recruited between 1990 and 2013). Data analysis was performed in June 2024. Exposure: MHT use (estrogen-progestin therapy [EPT] or estrogen-only therapy [ET]) in postmenopausal women and OC use in premenopausal women (never, past use, or current use). Main Outcomes and Measures: Breast cancer intrinsic-like subtypes (luminal A-like, luminal B-like, luminal B-ERBB2 [formerly HER2 or HER2/neu]-like, ERBB2 enriched-like, or triple-negative) were determined by immunohistochemistry of tumor sections. Polytomous logistic regression was performed to estimate the association between exogenous hormones and risk of breast cancer by intrinsic-like subtypes. Analyses by subtypes were stratified by body mass index (BMI [calculated as weight in kilograms divided by height in meters squared]; healthy weight, 18.5-<25; overweight, 25-<30; or obesity, ≥30). Results: This study included 42 269 individuals with breast cancer (11 901 [28.2%] premenopausal and 30 368 [71.8%] postmenopausal; 23 353 [55.2%] had a known intrinsic-like subtype) and 71 072 control participants. The mean (SD) age of all participants was 57.9 (10.9) years. In postmenopausal women, associations between current MHT use (EPT or ET) and breast cancer differed by subtype. Current EPT users with healthy weight were more likely to be diagnosed with luminal A-like (odds ratio [OR], 2.51 [95% CI, 2.26-2.80]) or luminal B-ERBB2-like (OR, 1.95 [95% CI, 1.61-2.37]) subtypes. These associations were attenuated but remained for individuals with overweight (OR, 1.40 [95% CI, 1.02-1.92]) or obesity (OR, 1.68 [95% CI, 1.01-2.78]). EPT use increased the odds of being diagnosed with luminal B-like tumors solely in women with healthy weight (OR, 1.47 [95% CI, 1.17-1.86]). Current ET use was positively associated with luminal A-like disease in women with healthy weight only (OR, 1.16 [95% CI, 1.01-1.32]), showing inverse associations with higher BMI (obesity: OR, 0.65 [95% CI, 0.50-0.85]). In premenopausal women, recent OC use was associated with luminal B-ERBB2-like (OR, 1.50 [95% CI, 1.09-2.08]), ERBB2 enriched-like (OR, 2.33 [95% CI, 1.55-3.51]), and triple-negative (OR, 1.75 [95% CI, 1.33-2.29]; P < .04 for heterogeneity) tumors. Conclusions and Relevance: In this study, clear differences were observed in associations between current EPT use and luminal-like breast cancer subtypes and other subtypes. EPT users with healthy weight were more likely to be diagnosed with luminal-like breast cancer compared with nonusers. Subtype heterogeneity was less apparent in associations of OC and ET use. Future studies on contemporary formulations, patterns of use, and routes of administration of exogenous hormone usage are warranted.

Transcriptomic Predictors of Survival for Palbociclib + Endocrine Therapy Versus Capecitabine in Aromatase Inhibitor–Resistant Breast Cancer From the GEICAM/2013-02 PEARL Trial

PURPOSE: For hormone receptor-positive/human epidermal growth factor receptor 2-negative (HR+/HER2-) metastatic breast cancer (MBC), first-line cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) + endocrine therapy (ET) is the standard of care. They are also used after progression on first-line aromatase inhibitors (AIs), but some patients may respond better to chemotherapy-based options. We examined tumor features associated with survival from GEICAM/2013-02 PEARL, a phase III trial of palbociclib + ET versus capecitabine in AI-resistant HR+/HER2- MBC. METHODS: For 158 and 155 patients from each arm, 878 previously published gene expression signatures were derived using RNA sequencing on pretreatment tumor specimens, both primary and metastatic. Multivariable Cox models for progression-free survival (PFS) and overall survival (OS) were constructed with 16 preselected signatures related to proliferation, loss of retinoblastoma, and immune infiltration, and via Elastic Net using all signatures. RESULTS: Significant PFS difference by PAM50 intrinsic subtype was observed with palbociclib + ET. Comparing treatment arms, luminal A subtype trended toward longer PFS with palbociclib + ET, and luminal B and nonluminal subtypes had significantly longer PFS with capecitabine. Three B-cell (B-lymphocyte)-associated signatures correlated with shorter OS with palbociclib + ET. The immune-activated Immune1 TCGA breast cancer signature had significant treatment arm interaction for OS. Elastic Net iteratively selected B-cell-associated signatures independently associated with shorter OS with palbociclib + ET. CONCLUSION: PAM50 intrinsic subtype predicted PFS differences between palbociclib + ET and capecitabine. Lower B-cell-associated gene expression predicted longer OS with palbociclib + ET versus capecitabine. These features may help identify HR+/HER2- tumors resistant to further ET-based treatment with CDK4/6i.

Proteogenomic analysis of the CALGB 40601 (Alliance) HER2+ breast cancer neoadjuvant trial reveals resistance biomarkers

Proteogenomic analysis is applied to samples from the CALGB 40601 (Alliance) randomized neoadjuvant trial of trastuzumab, lapatinib, or the combination to identify biomarkers associated with pathological response status. Absence of ERBB2 gene amplification and human epidermal growth factor receptor 2 (HER2) protein overexpression by proteogenomics is associated with non-pathological compete response (pCR) (p < 0.05), highlighting potential false positives from standard diagnostics. Pathway analysis in proteogenomics-confirmed HER2+ samples identifies elevated epithelial-mesenchymal transition (EMT) and WNT-β-catenin signaling in non-pCR cases before treatment. Twenty-four pCR-associated proteins reproduce in a second proteomic dataset, and four (GPRC5A, TPBG, SP140L, and NEU1) are significant in a third. A meta-analysis of ten diverse neoadjuvant anti-HER2 treatment regimens from four independent studies confirms that non-pCR cases express higher levels of mRNA for G protein-coupled receptor class C group 5 member A (GPRC5A, p = 0.0002) and trophoblast glycoprotein (TPBG, p = 0.00008). Thus, proteogenomic analysis identifies negative biomarkers for pCR and alternative plasma membrane targets for treatment-resistant HER2+ breast cancer. This trial is registered at clinicaltrials.gov (NCT00770809).

Prognostic Significance of CD8 T-cell Spatial Biomarkers in ER+ and ER-Breast Cancer

Tumor Infiltrating Lymphocytes (TILs) have been shown to be prognostic in Triple-Negative Breast Cancer (TNBC), but are rarely considered in other subtypes, particularly estrogen receptor (ER) positive cancers (70-80% of breast cancers), due to lower TIL counts. However, the spatial proximity of lower abundance TILs has not been evaluated in relation to breast cancer prognosis. In this study, multiplex immunofluorescent-stained images were used to identify cell types based on cytokeratin (Ck), CD8, and FoxP3 expression. Using distance-based visual morphometry between epithelial and immune cells, we computed new metrics, proximity and consistency , which capture spatial relationships between Ck+ tumor cells and CD8+ T-cells. Prognostic significance of proximity and consistency were compared to lymphocyte counts using log-rank tests. Worse relapse-free survival (RFS) was observed for both ER+ and ER-breast cancers with low proximity and consistency of CD8+ cells. Among ER negative breast cancers, proximity had the highest RFS hazard ratio (HR 1.84, 95% CI [1.18,2.87]). Among ER positive participants, RFS hazard ratios for proximity and consistency were 2.04 (95% CI [1.39, 2.98]) and 1.82 (95% CI [1.23, 2.69]) respectively. These associations were stronger than those observed for lymphocyte count (HR 1.35, 95% CI [0.92,1.98], log-rank p-value = 0.13). These IF-derived spatial metrics were also associated with established TILs metrics and RNA-based expression-based measures of tumor adaptive immune response. The prognostic significance of proximity in ER+ breast cancer implies that spatial parameters may identify individuals who would benefit from immune therapy; up to 75% of breast cancers experience T-cell proximity suggestive of immune susceptibility.

Anti-CSF-1R therapy with combined immuno- chemotherapy coordinate an adaptive immune response to eliminate macrophage enriched Triple Negative Breast Cancers

Patients diagnosed with metastatic triple negative breast cancer (mTNBC) have limited treatment options, are more prone to develop resistance and are associated with high mortality. A cold tumor immune microenvironment (TIME) characterized by low T cells and high tumor associated macrophages (TAMs) in mTNBC is associated with the failure of standard-of-care chemotherapy and immune checkpoint blockade (ICB) treatment. We demonstrate that the combination of immunomodulatory metronomic Cyclophosphamide (CTX) coupled with anti-CSF-1R antibody targeted therapy (SNDX-ms6352) and anti-PD-1 (ICB), was highly effective against aggressive metastatic syngeneic Trp53 null TNBC genetically engineered mouse models (GEMMs) that present with high macrophage infiltration. Mechanistically, CSF-1R inhibition along with CTX disrupted the M-CSF/CSF-1R axis which upregulated IL-17, IL-15 and type II interferon resulting in elevated B- and T cell infiltration. Addition of an anti-PD-1 maintenance dose helped overcome de novo PD-L1 intra-tumoral heterogeneity (ITH) associated recurrence in lung and liver mTNBC.

B7-H3 and CSPG4 co-targeting as Pan-CAR-T cell treatment of triple-negative breast cancer

Purpose Chimeric antigen receptor T (CAR-T) cell therapy is under clinical investigation in patients with metastatic triple-negative breast cancer (TNBC). However, the identification of targetable antigens remains a high priority to avoid toxicity and prevent tumor escape. Experimental design Here we analyzed the gene expression of B7-H3 ( CD276 ) and chondroitin sulfate proteoglycan 4 ( CSPG4 ) in 98 TNBC samples identified in the AURORA US Network and Rapid Autopsy RNA sequencing data set at University of North Carolina (UNC). We then performed immunohistochemistry analysis for B7-H3 and CSPG4 protein expression in 151 TNBC samples collected at UNC. Finally, the validity of the proposed B7-H3 and CSGP4 co-targeting was tested in clinically relevant TNBC patient derived xenograft (PDX) models. Results We observed that CD276 and CSPG4 genes are broadly and comparably expressed in TNBC samples, and gene expression is generally conserved in tumor metastases. None of the TNBC analyzed met the criteria for simultaneous low expression of CSPG4 and CD276 genes. Immunohistochemistry analysis showed a median H-score of 138 (105–168, lower and upper quartile, respectively) for B7-H3 expression and a median H-score of 33 (14–78 lower and upper quartile, respectively) for CSPG4 expression. Notably, 49% of the TNBC cores with B7-H3 H-score ≤105 exhibited a CSPG4 H-score exceeding its median value, and 37% and 18% of the TNBC cores with low B7-H3 expression scored CSPG4 expression above its median H-score or exceeded its upper quartile, respectively, confirming that at least one of these two proteins is expressed in 94% of the analyzed tumors. Finally, optimized dual-specific B7-H3 and CSPG4 CAR-T cells eradicated tumors with mixed antigen expression in TNBC PDX models. Conclusions These data highlight the clinical potential of the proposed approach that could be applicable to the great majority of patients with TNBC as well as most of patients with breast cancer in general.