EarlyR: A Robust Gene Expression Signature for Predicting Outcomes of Estrogen Receptor–Positive Breast Cancer

Introduction Early stage estrogen receptor (ER)-positive breast cancer may be treated with chemotherapy in addition to hormone therapy. Currently available molecular signatures assess the risk of recurrence and the benefit of chemotherapy; however, these tests may have large intermediate risk groups, limiting their usefulness. Methods The EarlyR prognostic score was developed using integrative analysis of microarray data sets and formalin-fixed, paraffin-embedded–based quantitative real-time PCR assay and validated in Affymetrix data sets and METABRIC cohort using Cox proportional hazards models and Kaplan-Meier survival analysis. Concordance index was used to measure the probability of prognostic score agreement with outcome. Results The EarlyR score and categorical risk strata (EarlyR-Low, EarlyR-Int, EarlyR-High) derived from expression of ESPL1, MKI67, SPAG5, PLK1 and PGR was prognostic of 8-year distant recurrence-free interval in Affymetrix (categorical P = 3.5 × 10−14; continuous P = 8.8 × 10−15) and METABRIC (categorical P < 2.2 × 10−16; continuous P < 10−16) data sets of ER+ breast cancer. Similar results were observed for the breast cancer–free interval end point. At most 13% of patients were intermediate risk and at least 66% patients were low risk in both ER+ cohorts. The EarlyR score was significantly prognostic (distant recurrence-free interval; P < .001) in both lymph node–negative and lymph node–positive patients and was independent from clinical factors. EarlyR and surrogates of current molecular signatures were comparable in prognostic significance by concordance index. Conclusion The 5-gene EarlyR score is a robust prognostic assay that identified significantly fewer patients as intermediate risk and more as low risk than currently available assays. Further validation of the assay in clinical trial–derived cohorts is ongoing

Upregulation of HSF1 in estrogen receptor positive breast cancer

Heat shock transcription factor 1 (HSF1), a key regulator of the heat-shock response, is deregulated in many cancers. HSF1 can mediate cancer cell survival and metastasis. High levels of HSF1 have been associated with poor prognosis in breast cancer. The nature of HSF1 upregulation needs to be validated in different cohorts to further validate its prognostic utility in breast cancer. We first evaluated its expression in a cohort of breast cancer tissue microarrays with Oncotype DX recurrence scores available using immunohistochemistry. To further confirm the clinical relevance and prognostic impact, mutational and methylation status of the gene were also assessed in The Cancer Genome Atlas and publically available microarray datasets. Immunohistochemical analysis showed that HSF1 expression is independent of Oncotype DX high recurrence score in ER-positive node-negative patients. Analysis of The Cancer Genome Atlas data revealed upregulation of HSF1 is not due to methylation or mutation. HSF1 copy number variations and amplifications (15%) were not associated with survival. In publicly available microarray datasets, a prognostic impact was observed in ER-positive tumors, but not in ER-negative tumors. Patients with ER-positive tumors with high HSF1 levels were associated with shorter overall survival (P = 0.00045) and relapse-free survival (P = 0.0057). In multivariable analysis, HSF1 remained a significant prognostic parameter. The mRNA expression levels of HSF1 in ER-positive breast cancer are associated with both shorter relapse-free and overall survival. This prognostic impact is specific to mRNA expression, but stayed insignificant by protein expression or by analyzing amplification events

CIBERSORT analysis of TCGA and METABRIC identifies subgroups with better outcomes in triple negative breast cancer

Studies have shown that the presence of tumor infiltrating lymphocytes (TILs) in Triple Negative Breast Cancer (TNBC) is associated with better prognosis. However, the molecular mechanisms underlying these immune cell differences are not well delineated. In this study, analysis of hematoxylin and eosin images from The Cancer Genome Atlas (TCGA) breast cancer cohort failed to show a prognostic benefit of TILs in TNBC, whereas CIBERSORT analysis, which quantifies the proportion of each immune cell type, demonstrated improved overall survival in TCGA TNBC samples with increased CD8 T cells or CD8 plus CD4 memory activated T cells and in Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) TNBC samples with increased gamma delta T cells. Twenty-five genes showed mutational frequency differences between the TCGA high and low T cell groups, and many play important roles in inflammation or immune evasion (ATG2B, HIST1H2BC, PKD1, PIKFYVE, TLR3, NOTCH3, GOLGB1, CREBBP). Identification of these mutations suggests novel mechanisms by which the cancer cells attract immune cells and by which they evade or dampen the immune system during the cancer immunoediting process. This study suggests that integration of mutations with CIBERSORT analysis could provide better prediction of outcomes and novel therapeutic targets in TNBC cases

The AKT inhibitor triciribine in combination with paclitaxel has order-specific efficacy against Zfp217-induced breast cancer chemoresistance

We previously identified the transcription factor ZNF217 (human) / Zfp217 (mouse) as an oncogene and prognostic indicator of reduced survival, increased metastasis, and reduced response to therapy in breast cancer patients. Here we investigated the role of Zfp217 in chemotherapy resistance. Preclinical animal models of Zfp217 overexpression were treated with a combination therapy of the microtubule inhibitor epothilone B, doxorubicin (Adriamycin), and cyclophosphamide (EAC). Tumors overexpressing Zfp217 increased their tumor burden compared to control tumors after treatment and accumulated a mammary gland progenitor cell population (K8+K14+). To overcome this chemoresistance after ZNF217 overexpression, we treated tumors ± Zfp217 overexpression with paclitaxel and triciribine, a nucleoside analog and AKT inhibitor that kills cells that overexpress ZNF217. Treatment order critically impacted the efficacy of the therapy. Combination treatment of triciribine followed by paclitaxel (TCN→PAC) inhibited tumor burden and increased survival in tumors that overexpressed Zfp217, whereas single agent or combination treatment in the reverse order (PAC→TCN) did not improve response. Analysis of these tumors and patient-derived tumor xenograft tumors treated with the same therapies suggested that Zfp217 overexpression in tumors contributes both to decreased microvessel density and vessel maturity, while TCN→PAC tumors overexpressing Zfp217 showed improved vessel maturity

Splicing factor ESRP1 controls ER-positive breast cancer by altering metabolic pathways

The epithelial splicing regulatory proteins 1 and 2 (ESRP1 and ESRP2) control the epithelial-to-mesenchymal transition (EMT) splicing program in cancer. However, their role in breast cancer recurrence is unclear. In this study, we report that high levels of ESRP1, but not ESRP2, are associated with poor prognosis in estrogen receptor positive (ER+) breast tumors. Knockdown of ESRP1 in endocrine-resistant breast cancer models decreases growth significantly and alters the EMT splicing signature, which we confirm using TCGA SpliceSeq data of ER+ BRCA tumors. However, these changes are not accompanied by the development of a mesenchymal phenotype or a change in key EMT-transcription factors. In tamoxifen-resistant cells, knockdown of ESRP1 affects lipid metabolism and oxidoreductase processes, resulting in the decreased expression of fatty acid synthase (FASN), stearoyl-CoA desaturase 1 (SCD1), and phosphoglycerate dehydrogenase (PHGDH) at both the mRNA and protein levels. Furthermore, ESRP1 knockdown increases the basal respiration and spare respiration capacity. This study reports a novel role for ESRP1 that could form the basis for the prevention of tamoxifen resistance in ER+ breast cancer

Association of Stromal Tumor-Infiltrating Lymphocytes With Recurrence-Free Survival in the N9831 Adjuvant Trial in Patients With Early-Stage HER2-Positive Breast Cancer

Importance The presence of tumor-infiltrating lymphocytes at diagnosis is reported to be prognostic in triple-negative breast cancer. Objective To evaluate the association of stromal tumor-infiltrating lymphocytes (STILs) with recurrence-free survival (RFS) in women with human epidermal growth factor receptor 2 (HER2)–positive breast cancer treated with chemotherapy or chemotherapy plus trastuzumab in the N9831 trial. Design, Setting, and Participants Hematoxylin-eosin–stained tumor slides from patients with early-stage HER2-positive breast cancer in 2 of the 3 arms of the N9831 trial were assessed for STILs at an academic medical center. The amounts of STILs were quantitated in deciles, and a level of at least 60% STILs was used for the prespecified categorical cutoff. The association between STILs and RFS was evaluated with Cox models. Exposure Standard chemotherapy consisting of doxorubicin-cyclophosphamide followed by weekly paclitaxel (arm A) or doxorubicin-cyclophosphamide followed by weekly paclitaxel plus trastuzumab followed by trastuzumab alone (arm C). Main Outcomes and Measures Stromal tumor-infiltrating lymphocytes and their association with RFS. Results A total of 489 patients from arm A and 456 patients from arm C were assessed with a median (range) follow-up of 4.4 (0-13.6) years. The 10-year Kaplan-Meier estimates for RFS in arm A were 90.9% and 64.5% for patients with high and low levels of STILs, respectively (hazard ratio [HR], 0.23 [95% CI, 0.07-0.73]; P = .01). The 10-year estimates for RFS in arm C were 80.0% and 80.1% for patients with high and low levels of STILs, respectively (HR, 1.26 [95% CI, 0.50-3.17]; P = .63). The test for interaction between trastuzumab treatment and STIL status was statistically significant (P = .03). In a multivariable analysis, STIL status remained significantly associated with RFS in arm A and not significantly associated in arm C (HR, 1.01 [95% CI, 0.89-1.15]; interaction P = .04). Conclusions and Relevance This analysis of participants in the N9831 trial found that the presence of STILs was prognostically associated with RFS in patients treated with chemotherapy alone but not in patients treated with chemotherapy plus trastuzumab. High levels of STILs were associated with lack of trastuzumab therapy benefit, in contrast to a previously reported association between increased levels of STILs and increased trastuzumab benefit in HER2-positive patients

Thymic Carcinomas and Second Malignancies: A Single-Center Review

Thymic carcinomas account for less than 0.01% of new cancer diagnoses annually and are more aggressive than thymomas. Autoimmune disorders have been associated with thymomas and only recently with thymic carcinomas. Second malignancies are well described after thymomas. The aim of this study was to analyze the incidence of second malignancies in patients with thymic carcinomas. All cases of thymic carcinomas were identified from the pathology archives of Indiana University. Histological materials were reviewed and further correlated with clinical data to identify incidence of second cancers in patients with thymic carcinomas. Histological material was available for review in 92 cases of thymic carcinoma. Clinical data were available for 85 patients. Fourteen of these (16.5%) patients had a second malignancy; these included small cell lung carcinoma, "testicular cancer", embryonal carcinoma, seminoma, breast carcinoma (two cases), prostatic adenocarcinoma, Hodgkin's lymphoma, thyroid carcinoma, bladder carcinoma (two cases), renal cell carcinoma, and melanoma. The latter could precede, be concurrent with, or follow the diagnosis thymic carcinoma. The incidence of second cancers in patients with thymic carcinomas is similar to that reported for thymomas. Abnormalities in immunological surveillance may be responsible for this high incidence of second malignancies in thymic tumors

The Birth of an Adenoid Cystic Carcinoma

Adenoid cystic carcinoma is a rare malignancy of exocrine glands defined by the presence of a dual population of cells (epithelial and myoepithelial cells) organized in varying combinations of cribriform, tubular, and solid patterns.1,2 This neoplasm most frequently originates in the salivary glands; however, it can also occur in other anatomical sites, including the breast.1,3 More than 90% of adenoid cystic carcinomas of the breast harbor the recurrent translocation t(6;9), resulting in the MYB-NFIB fusion gene, which leads to MYB overexpression.4 Adenoid cystic carcinoma in situ has been described in the breast; however, its identifica-tion is not trivial.3 Here, we illustrate an in situ adenoid cystic carcinoma partially involving a mammary duct in a 68-year-old woman with primary adenoid cystic carci-noma of the right breast. The double population of cells composing this intraductal lesion can be appreciated by its immunohistochemical profile (Figure 1). Given that the neoplastic cells of the in situ lesion already express MYB, our findings are consistent with the notion that MYB overexpression is an early event in the tumorigen-esis of adenoid cystic carcinomas.4,

Quantification of spatial pharmacogene expression heterogeneity in breast tumors.

BACKGROUND: Chemotherapeutic drug concentrations vary across different regions of tumors and this is thought to be involved in development of chemotherapy resistance. Insufficient drug delivery to some regions of the tumor may be due to spatial differences in expression of genes involved in the disposition, transport, and detoxification of drugs (pharmacogenes). Therefore, in this study, we analyzed the spatial expression of 286 pharmacogenes in six breast cancer tissues using the recently developed Visium spatial transcriptomics platform to (1) determine if these pharmacogenes are expressed heterogeneously across tumor tissue and (2) to determine which pharmacogenes have the most spatial expression heterogeneity. METHODS AND RESULTS: The spatial transcriptomics technology sequences the transcriptome of 55 um diameter barcoded sections (spots) across a tissue sample. We analyzed spatial gene expression profiles of four biobank-sourced breast tumor samples in addition to two breast tumor sample datasets from 10× Genomics. We define heterogeneity as the interquartile range of read counts. Collectively, we identified 8887 spots in tumor regions, 3814 in stroma, 44 in lymphocytes, and 116 in normal regions based on pathologist annotation of the tissues. We showed statistically significant differences in expression of pharmacogenes in tumor regions compared to surrounding non-tumor regions. We also observed that the most heterogeneously expressed genes within tumor regions were involved in reactive oxygen species (ROS) handling and detoxification mechanisms. GPX4, GSTP1, MGST3, SOD1, CYP4Z1, CYB5R3, GSTK1, and NAT1 showed the most heterogeneous expression within tumor regions. CONCLUSIONS: The heterogeneous expression of these pharmacogenes may have important implications for cancer therapy due to their ability to impact drug distribution and efficacy throughout the tumor. Our results suggest that chemoresistance caused by expression of GPX4, GSTP1, MGST3, and SOD1 may be intrinsic, not acquired, since the heterogeneity is not specific to chemotherapy-treated samples or cell type. Additionally, we identified candidate chemoresistance pharmacogenes that can be further tested through focused follow-up studies