Co-expression of mesothelin and CA125/MUC16 is a prognostic factor for breast cancer, especially in luminal-type breast cancer patients

The expression of mesothelin correlates with a poor prognosis in patients with breast cancer. Since mesothelin plays a role in cancer metastasis in association with CA125, we herein examined the expression of mesothelin and CA125, and the clinicopathological meaning and prognosis of the co-expression of mesothelin and CA125 in breast cancer. Our results showed that among 478 patients, mesothelin and CA125 were co-expressed in 48 (10 %), mesothelin only in 75 (16 %), CA125 only in 217 (45 %), and neither in 234 (49 %). A high correlation was observed between the expression of mesothelin and CA125 (P =0.0004). The co-expression of mesothelin and CA125 correlated with poor patient relapse-free survival (RFS) (P = 0.0001) and was identified as an independent predictor of RFS by Cox’s multivariate analysis. In conclusion, this is the first to report the prognostic significance of the co-expression of mesothelin and CA125 in breast cancer. The co-expression of mesothelin and CA125 may be clinically useful for prognostication after surgical therapy in patients with breast cancer.

A ferroptosis-associated gene signature for the prediction of prognosis and therapeutic response in luminal-type breast carcinoma

Ferroptosis is a new form of regulated cell death (RCD), and its emergence has provided a new approach to the progression and drug resistance of breast cancer (BRCA). However, there is still a great gap in the study of ferroptosis-related genes in BRCA, especially luminal-type BRCA patients. We downloaded the mRNA expression profiles and corresponding clinical data of BRCA patients from the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) and The Cancer Genome Atlas (TCGA) databases. Then, we built a prognostic multigene signature with ferroptosis-related differentially expressed genes (DEGs) in the METABRIC cohort and validated it in the TCGA cohort. The predictive value of this signature was investigated in terms of the immune microenvironment and the probability of a response to immunotherapy and chemotherapy. The patients were divided into a high-risk group and a low-risk group according to the ferroptosis-associated gene signature, and the high-risk group had a worse overall survival (OS). The risk score based on the 10 ferroptosis-related gene-based signature was determined to be an independent prognostic predictor in both the METABRIC and TCGA cohorts (HR, 1.41, 95% CI, 1.14–1.76, P = 0.002; HR, 2.19, 95% CI, 1.13–4.26, P = 0.02). Gene set enrichment analysis indicated that the term “cytokine-cytokine receptor interaction” was enriched in the high-risk score subgroup. Moreover, the immune infiltration scores of most immune cells were significantly different between the two groups, the low-risk group was much more sensitive to immunotherapy, and six drugs might have potential therapeutic implications in the high-risk group. Finally, a nomogram incorporating a classifier based on the 10 ferroptosis-related genes, tumor stage, age and histologic grade was established. This nomogram showed favorable discriminative ability and could help guide clinical decision-making for luminal-type breast carcinoma.

Automated immunostaining-free prediction of breast carcinoma ER and PR status, Ki67 count, patient therapy stratification index and quantification of prognostic quiescence burden in TNBC from pretreatment H&E stained histopathology images in breast cancer.

e12535 Background: Categorical combinations of ER, PR, HER2, and Ki67 levels are traditionally used to classify patients into luminal A and B-like subtypes in order to inform treatment choice. Accounting for nearly 70% of all breast malignancies, luminal cancer is heterogeneous, harboring subtypes with distinct molecular profiles and clinical outcomes. Although most patients with luminal-type disease respond well to endocrine therapy alone, some develop recurrences benefiting from additional cytotoxic therapy. Identifying such cases a priori remains a challenge but would enable patients to be spared the debilitating side-effects of ineffective chemotherapy. In this regard, the efficacy of chemotherapy and disease recurrence relate to (i) ER driven G1/S perturbations and/or (ii) quiescent cell populations arrested in the G0/G1 phase of cell cycle. This study aimed to develop a histopathology whole slide image (WSI)-based, low cost, rapid and automated approach to: (i) predict ER/PR/Ki67 status, (ii) quantify quiescence burden, (iii) develop a G1/S-based patient stratification system for luminal A/B patients, and (iv) achieve a quiescence burden-based stratification of TNBC patients. Methods: This investigation centered on the initial clinical validation of a novel, immunostaining-free technology which uses information extracted from pre-treatment hematoxylin and eosin (H&E) stained slide WSIs alone to achieve these aims. Unlike conventional artificial intelligence-based approaches, the underlying proprietary algorithm and its prediction criteria are based on deterministic, hard-coded observational relationships of continuous scales drawn from WSI morphological features. In this instance, these represent tumor-related biological pathway disruptions and mitotic checkpoint perturbations, where G1/S perturbations enable luminal subtype stratification, and G0/G1 perturbations reflect quiescence burden. Back projecting the algorithm’s quiescence burden output on to the original WSIs enables morphological patterns to be mapped to quiescence burden.

Molecular profiles of genomically high risk ER+ HER2- breast cancer tumors classified as functionally basal or luminal B by the 80-gene signature.

563 Background: The 80-gene signature (BluePrint/BP) classifies early-stage breast cancers based on functional molecular pathways as luminal, HER2, or Basal-type. In the NBRST study, 13% of immunochemistry (IHC) defined ER+ HER2- cancers reclassified as Basal-type by the BP assay (ER+ Basal), and these had worse prognosis but responded better to neoadjuvant chemotherapy than ER+ HER2- cancers classified as genomically luminal-type. The 70-gene risk of recurrence signature (MammaPrint/MP) further stratifies luminal-type cancers into low risk luminal A or high risk (HR) luminal B. HR cancers can be further stratified into High 1 (H1) or High 2 (H2), and the I-SPY2 trial has shown higher pCR rates in ER+ cancers classified as H2. Here, we investigated biological differences among ER+ Basal, ER- Basal, H1 luminal B, and H2 luminal B cancers by full transcriptome analysis. Methods: From the FLEX Study (NCT03053193), 1501 breast cancers with known IHC ER status were classified by MP and BP: 103 ER+ Basal, 210 ER- Basal and 1188 luminal B (H1 n=1034, H2 n=154). Clinical factors were assessed by either the Chi-square or Fisher’s exact tests; ANOVA or t test were used to analyze age. Differentially expressed genes (DEGs) were detected using Limma and pathway analyses were performed with GSEA. DEGs with a fold change >2 and FDR < 0.05 were considered significant. Results: Basal-type cancers (ER+/ER-) were larger and higher grade than luminal B cancers. Clustering analysis showed similar transcriptional profiles between ER+ Basal and ER- Basal cancers, distinct from luminal B cancers. Few DEGs were detected between ER+ Basal and ER- Basal cancers, and significantly more DEGs were found between ER+ Basal and luminal B cancers. Only three upregulated genes were detected in ER+ Basal compared to ER- Basal cancers: ESR1 and two immune-related genes ( FDCSP and LTF). Enrichment analysis of DEGs indicated increased immune activation and cell proliferation in ER+ Basal and ER- Basal cancers, and decreased estrogen response between ER+ Basal and luminal B cancers. Enrichment analysis between luminal B H1 and H2 cancers showed H2 cancers had higher immune activation and cell proliferation and lower estrogen response. Conclusions: Reclassification by BP of IHC defined ER+ HER2- cancers identified a subgroup of ER+ cancers that are biologically closer to ER- Basal than luminal-type cancers. Significant differences in response to neoadjuvant chemotherapy that have been seen between ER+ Basal and luminal B breast cancers lend support to the clinical importance of these findings. These data explain the poor prognosis observed in patients with ER+ Basal cancers and suggest that optimized chemotherapy, such as that for triple negative cancer, might be of benefit. BP provides clinically actionable information beyond pathological subtyping, which may guide neoadjuvant treatment recommendations. Clinical trial information: NCT03053193.

Concordance between 21-gene score (Oncotype DX) and clinical-pathologic prognostic models in early-breast cancer in Medellín, Colombia.

e12521 Background: The genomic-based 21-gene recurrence-score assay (Oncotype DX, Genomic Health)(ODx) is used to decide on the use of adjuvant chemotherapy (ACT) in luminal-type Early-Breast Cancer (LT-EBC). Patients with low RS can safely avoid ACT. Other predictive models based on standard clinical and histopathological (C&H) variables also have been developed. These include, Magee equations (ME), Predict model (PM), and Tennessee nomogram score (TNS). This study aims to establish the concordance between ODx and ME, PM and TNS models in a set of patients with EBC in Medellín, Colombia. Methods: Patients with unifocal, stage I and IIA, LT-EBC (HR+/Her2-) with results for ODx were included. For inclusion, key standard C&H variables needed to be available as to allow accurate assessment of ME, PM and TNS predictive models. ODx was used as the reference test and the predictive models as index tests. Low-risk (LR) was defined < 18 in all three ME; < 3% in the PM; and a probability calculated for LR > 90% in the TNS. A second analysis was performed in the > 50 years-old cohort (+50C), using standard and a modified criteria. In the last one, intermediate-risk (IR) patients were either excluded or grouped with high-risk scores for analysis. Concordance between the models and the ODx was evaluated using Cohen's kappa index (K). The degree of concordance was classified according to the categories established by Landis and Koch. AUROC (area under receiver operating characteristics) was estimated. Statistical analyses were performed with Stata v16. Results: 122 patients were included. Median age: 58 (IQR 49-66). Main histology was ductal carcinoma (85.2%), and median size was 15 mm (IQR 10-20). LR was adjudicated in 80.3%, 57.4%, 89.3% and 69.7% with ODx, ME, PM and TNS, respectively. Concordance between the ODx and ME and PM in the all patient population was fair, with K of 0.35 (95% CI: 0.18-0.50; p < 0.001), and 0.24 (95% CI: 0, 04 - 0.45; p < 0.001), respectively. Concordance of ODx and TNS was inferior with a K of 0.16 (95% CI: 0.03-0.34, p = 0.04). AUC for ME, PM, and TNS was 0.61 (95% CI: 0.49-0.73), 0.61 (95% CI: 0.52-0.70) and 0.59 (95% CI: 0.48-0.70), respectively. 85 patients were included in the +50C (69.7%). For one analysis, 41 patients with IR with either ME or PM were excluded. Concordance between the ME and the ODx was fair. LR in both ME and ODx was found 41/43 patients (95.5%, 95% CI 87.9-100). As for PM and TNS, the degree of concordance with ODx were low, and non-significant, respectively. Concordance between ODx LR was found in 36/37 patients with LR in all three ME, PM and TNS (97.3%, 95% CI: 90.7-100). Discrimination capacity: 79%. Conclusions: The high cost of ODx can be safely avoided in stage I/IIA luminal-type EBC and > 50 year-old with low-risk scores in all three ME, PM, and TNS.

Optima: Optimal personalised treatment of early breast cancer using multi-parameter analysis, an international randomized trial of tumor gene expression test-directed chemotherapy treatment in a largely node-positive population.

TPS599 Background: Multi-parameter tumor gene expression assays (MPAs) are validated tools to assist adjuvant chemotherapy decisions for post-menopausal women with luminal-type node-negative breast cancer. Currently there is less certainty for women with 1-3 involved axillary lymph nodes and no information on MPA use for patients with higher level nodal involvement. Three RCTs with available data report chemotherapy benefit for premenopausal women; with limited use of ovarian function suppression (OFS) for non-chemotherapy treated participants, chemotherapy-induced menopause may explain these results. Methods: OPTIMA is an international academic, partially-blinded RCT of test-directed chemotherapy treatment with an adaptive design. Women and men aged 40 or older with resected luminal-type breast cancer may participate if they fulfil one of the following stage criteria: pN1-2; pN1mi with pT ≥20mm; pN0 with pT ≥30mm. Consenting patients are randomized between standard treatment with chemotherapy followed by endocrine therapy or to undergo Prosigna testing; those with high-Prosigna Score ( > 60) tumors receive standard treatment whilst those with low-score tumors are treated with endocrine therapy alone. Patients are informed only of their treatment; test details, and randomization for chemotherapy-treated patients are masked. Clinical choice of chemotherapy is declared at randomization from a menu of standard regimens. Endocrine therapy must be for at least 5 years. Women postmenopausal at trial entry should receive an AI; men, tamoxifen; and premenopausal women, either an AI or tamoxifen, and OFS for 3 or more years; OFS initiation may be deferred because of post-chemotherapy amenorrhea. OPTIMA aims to randomize 2250 patients in each arm to demonstrate non-inferiority of test directed treatment, defined as not more than 3% below the estimated 85% 5-year IDFS for the control arm with a one sided 5% significance level. Power is 81% assuming recruitment over 96-months from January 2017 and 12 months minimum follow-up. OPTIMA also has at least 80% power to demonstrate 3.5% non-inferiority of IDFS for patients with low Prosigna Score tumors (estimated 65% of participants). Cox proportional hazards models will be used to explore important prognostic factors including menopausal status. Additional secondary endpoints include DRFI. A cost-effectiveness analysis of protocol specified MPA driven treatment against standard clinical practice will be conducted. At 31/01/2021, 2004 patients had been randomized. The DMC reviewed the trial in December 2020 with knowledge of related trial results and suggested that the trial continues as planned. OPTIMA is registered as ISRCTN42400492 and funded by the UK NIHR Health Technology Assessment Programme, award number 10/34/501. Clinical trial information: ISRCTN42400492.

Sensitization of MCF7 Cells with High Notch1 Activity by Cisplatin and Histone Deacetylase Inhibitors Applied Together

Histone deacetylase inhibitors (HDIs) are promising anti-cancer agents that inhibit proliferation of many types of cancer cells including breast carcinoma (BC) cells. In the present study, we investigated the influence of the Notch1 activity level on the pharmacological interaction between cisplatin (CDDP) and two HDIs, valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA, vorinostat), in luminal-like BC cells. The type of drug–drug interaction between CDDP and HDIs was determined by isobolographic analysis. MCF7 cells were genetically modified to express differential levels of Notch1 activity. The cytotoxic effect of SAHA or VPA was higher on cells with decreased Notch1 activity and lower for cells with increased Notch1 activity than native BC cells. The isobolographic analysis demonstrated that combinations of CDDP with SAHA or VPA at a fixed ratio of 1:1 exerted additive or additive with tendency toward synergism interactions. Therefore, treatment of CDDP with HDIs could be used to optimize a combined therapy based on CDDP against Notch1-altered luminal BC. In conclusion, the combined therapy of HDIs and CDDP may be a promising therapeutic tool in the treatment of luminal-type BC with altered Notch1 activity.