Farnesoid X receptor as marker of osteotropism of breast cancers through its role in the osteomimetism of tumor cells

peer reviewedBackground: The skeleton is the first and most common distant metastatic site for breast cancer. Such metastases complicate cancer management, inducing considerable morbidities and decreasing patient survival. Osteomimetism is part of the complex process of osteotropism of breast cancer cells. Recent data indicate that Farnesoid X Receptor (FXR) is involved in the transformation and progression of breast cancer. Methods: The expression of FXR, Runt-related transcription factor 2 (RUNX2) and bone proteins were evaluated on two tumor cell lines (MCF-7 and MDA-MB-231) by immunohistochemistry, immunofluorescence and western blotting and quantified. Results: In a series of 81 breast cancer patients who developed distant metastases, we found a strong correlation between FXR expression in primary breast tumors and the development of bone metastases, especially in patients with histological grade 3 tumors. In in vitro studies, FXR activation by Chenodeoxycholic acid (CDCA) increased the expression of numerous bone proteins. FXR inhibition by lithocholic acid and z-guggulsterone decreased bone protein expression. Short Hairpin RNA (ShRNA) against FXR validated the involvement of FXR in the osteomimetism of breast cancer cells. Conclusion: Our experimental results point to a relationship between the expression of FXR in breast cancer cells and the propensity of these tumor cells to develop bone metastases. FXR induces the expression of RUNX2 which itself causes the synthesis of bone proteins by tumor cells

Improvement of the clinical applicability of the Genomic Grade Index through a qRT-PCR test performed on frozen and formalin-fixed paraffin-embedded tissues

BACKGROUND: Proliferation and tumor differentiation captured by the genomic grade index (GGI) are important prognostic indicators in breast cancer (BC) especially for the estrogen receptor positive (ER+) disease. The aims of this study were to convert this microarray index to a qRT-PCR assay (PCR-GGI), which could be realized on formalin fixed paraffin embedded samples (FFPE), and to assess its prognostic performance and predictive value of clinical benefit in early and advanced ER+ BC patients treated with tamoxifen. METHODS: The accuracy and concordance of the PCR-GGI with the GGI was assessed using BC patients for which frozen and FFPE tissues as well as microarray data were available (n = 19). The evaluation of the prognostic value of the PCR-GGI was assessed on FFPE material using a consecutive series of 212 systemically treated early BC patients. The predictive performance for tamoxifen benefit was assessed using two ER+ BC populations treated either with adjuvant tamoxifen only (n = 77+139) or first-line tamoxifen for advanced disease (n = 270). RESULTS: The PCR-GGI is based on the expression of 8 genes (4 representative of the GGI and 4 reference genes). A significant correlation was observed between the microarray-derived GGI and the qRT-PCR assay using frozen (rho = 0.95, p < 10E-06) and FFPE material (rho = 0.89, p < 10E-06). The prognostic performance of the PCR-GGI was confirmed on FFPE samples (HRunivar. = 1.89; [95CI:1.01-3.54], p = 0.05). The PCR-GGI further identified two subgroups of patients with statistically different time to distant metastasis free survival (DMFS) across the two cohorts of ER+ BC patients treated with adjuvant tamoxifen. Additionally, the PCR-GGI was associated with response to tamoxifen in the advanced setting (HRunivar. = 1.98; [95CI:1.51-2.59], p = 6.9E-07). CONCLUSION: PCR-GGI recapitulates in an accurate and reproducible manner the performances of the GGI using frozen and FFPE samples

Results of a worldwide survey on the currently used histopathological diagnostic criteria for invasive lobular breast cancer

Invasive lobular carcinoma (ILC) represents the second most common subtype of breast cancer (BC), accounting for up to 15% of all invasive BC. Loss of cell adhesion due to functional inactivation of E-cadherin is the hallmark of ILC. Although the current world health organization (WHO) classification for diagnosing ILC requires the recognition of the dispersed or linear non-cohesive growth pattern, it is not mandatory to demonstrate E-cadherin loss by immunohistochemistry (IHC). Recent results of central pathology review of two large randomized clinical trials have demonstrated relative overdiagnosis of ILC, as only similar to 60% of the locally diagnosed ILCs were confirmed by central pathology. To understand the possible underlying reasons of this discrepancy, we undertook a worldwide survey on the current practice of diagnosing BC as ILC. A survey was drafted by a panel of pathologists and researchers from the European lobular breast cancer consortium (ELBCC) using the online tool SurveyMonkey (R). Various parameters such as indications for IHC staining, IHC clones, and IHC staining procedures were questioned. Finally, systematic reporting of non-classical ILC variants were also interrogated. This survey was sent out to pathologists worldwide and circulated from December 14, 2020 until July, 1 2021. The results demonstrate that approximately half of the institutions use E-cadherin expression loss by IHC as an ancillary test to diagnose ILC and that there is a great variability in immunostaining protocols. This might cause different staining results and discordant interpretations. As ILC-specific therapeutic and diagnostic avenues are currently explored in the context of clinical trials, it is of importance to improve standardization of histopathologic diagnosis of ILC diagnosis

Uncovering the genomic heterogeneity of multifocal breast cancer.

Multifocal breast cancer (MFBC), defined as multiple synchronous unilateral lesions of invasive breast cancer, is relatively frequent and has been associated with more aggressive features than unifocal cancer. Here, we aimed to investigate the genomic heterogeneity between MFBC lesions sharing similar histopathological parameters. Characterization of different lesions from 36 patients with ductal MFBC involved the identification of non-silent coding mutations in 360 protein-coding genes (171 tumour and 36 matched normal samples). We selected only patients with lesions presenting the same grade, ER, and HER2 status. Mutations were classified as 'oncogenic' in the case of recurrent substitutions reported in COSMIC or truncating mutations affecting tumour suppressor genes. All mutations identified in a given patient were further interrogated in all samples from that patient through deep resequencing using an orthogonal platform. Whole-genome rearrangement screen was further conducted in 8/36 patients. Twenty-four patients (67%) had substitutions/indels shared by all their lesions, of which 11 carried the same mutations in all lesions, and 13 had lesions with both common and private mutations. Three-quarters of those 24 patients shared oncogenic variants. The remaining 12 patients (33%) did not share any substitution/indels, with inter-lesion heterogeneity observed for oncogenic mutation(s) in genes such as PIK3CA, TP53, GATA3, and PTEN. Genomically heterogeneous lesions tended to be further apart in the mammary gland than homogeneous lesions. Genome-wide analyses of a limited number of patients identified a common somatic background in all studied MFBCs, including those with no mutation in common between the lesions. To conclude, as the number of molecular targeted therapies increases and trials driven by genomic screening are ongoing, our findings highlight the presence of genomic inter-lesion heterogeneity in one-third, despite similar pathological features. This implies that deeper molecular characterization of all MFBC lesions is warranted for the adequate management of those cancers

Comprehensive evaluation of methods to assess overall and cell-specific immune infiltrates in breast cancer

Background: Breast cancer (BC) immune infiltrates play a critical role in tumor progression and response to treatment. Besides stromal tumor infiltrating lymphocytes (sTILs) which have recently reached level 1B evidence as a prognostic marker in triple negative BC, a plethora of methods to assess immune infiltration exists, and it is unclear how these compare to each other and if they can be used interchangeably. Methods: Two experienced pathologists scored sTIL, intra-tumoral TIL (itTIL), and 6 immune cell types (CD3+, CD4+, CD8+, CD20+, CD68+, FOXP3+) in the International Cancer Genomics Consortium breast cancer cohort using hematoxylin and eosin-stained (n = 243) and immunohistochemistry-stained tissue microarrays (n = 254) and whole slides (n = 82). The same traits were evaluated using transcriptomic- and methylomic-based deconvolution methods or signatures. Results: The concordance correlation coefficient (CCC) between pathologists for sTIL was very good (0.84) and for cell-specific immune infiltrates slightly lower (0.63-0.66). Comparison between tissue microarray and whole slide pathology scores revealed systematically higher values in whole slides (ratio 2.60-5.98). The Spearman correlations between microscopic sTIL and transcriptomic- or methylomic-based assessment of immune infilt

Low CD10 mRNA Expression Identifies High-Risk Ductal Carcinoma In Situ (DCIS)

PURPOSE: Optimal management of breast ductal carcinoma in situ (DCIS) is controversial, and many patients are still overtreated. The local death of myoepithelial cells (MECs) is believed to be a pre-requisite to tumor invasion. We thus hypothesized that loss of CD10 expression, a MEC surface peptidase, would signify basement membrane disruption and confer increased risk of relapse in DCIS. The aim of our study was to retrospectively evaluate the prognostic value of CD10 in DCIS. EXPERIMENTAL DESIGN: CD10 expression was evaluated by quantitative RT-PCR and immunohistochemistry using paraffin-embedded samples of normal breast tissue (n = 11); of morphologically normal ducts associated with DCIS (n = 10); and of DCIS without an invasive component (n = 154). RESULTS: CD10 immunostaining was only observed in MECs in normal tissue and in DCIS. Normal tissue showed high mRNA expression levels of CD10, whereas DCIS showed a variable range. After a median follow-up of 6 years, DCIS with CD10 expression below the levels observed in normal tissue (71%) demonstrated a higher risk of local relapse (HR = 1.88; [95CI:1.30-2.70], p = 0.001) in univariate analysis. No relapse was observed in patients expressing high CD10 mRNA levels (29%) similar to the ones observed in normal tissue. In multivariate analysis including known prognostic factors, low CD10 mRNA expression remained significant (HR = 2.25; [95%CI:1.24-4.09], p = 0.008), as did the recently revised Van Nuys Prognostic Index (VNPI) score (HR = 2.03; [95%CI:1.23-3.35], p = 0.006). CONCLUSION: The decrease of CD10 expression in MECs is associated with a higher risk of relapse in DCIS; this knowledge has the potential to improve DCIS management

The role of the pathologist in tissue banking: European Consensus Expert Group Report

Human tissue biobanking encompasses a wide range of activities and study designs and is critical for application of a wide range of new technologies (-“omics”) to the discovery of molecular patterns of disease and for implementation of novel biomarkers into clinical trials. Pathology is the cornerstone of hospital-based tissue biobanking. Pathologists not only provide essential information identifying the specimen but also make decisions on what should be biobanked, making sure that the timing of all operations is consistent with both the requirements of clinical diagnosis and the optimal preservation of biological products. This document summarizes the conclusions of a Pathology Expert Group Meeting within the European Biological and Biomolecular Research Infrastructure (BBMRI) Program. These recommendations are aimed at providing guidance for pathologists as well as for institutions hosting biobanks on how to better integrate and support pathological activities within the framework of biobanks that fulfill international standards

Molecular apocrine tumours in EORTC 10994/BIG 1-00 phase III study: pathological response after neoadjuvant chemotherapy and clinical outcomes

Background: We explored, within the EORTC10994 study, the outcomes for patients with molecular apocrine (MA) breast cancer, and defined immunohistochemistry (IHC) as androgen-receptor (AR) positive, oestrogen (ER) and progesterone (PR) negative. We also assessed the concordance between IHC and gene expression arrays (GEA) in the identification of MA cancers. Methods: Centrally assessed biopsies for AR, ER, PR, HER2 and Ki67 by IHC were classified into six subtypes: MA, triple-negative (TN) basal-like, luminal A, luminal B HER2 negative, luminal B HER2 positive and “other”. The two main objectives were the pCR rates and survival outcomes in the overall MA subtype (and further divided by HER2 status) and the remaining five subtypes. Results: IHC subtyping was obtained in 846 eligible patients. Ninety-three (11%) tumours were classified as the MA subtype. Both IHC and GEA data were available for 64 patients. In this subset, IHC concordance was 88.3% in identifying MA tumours compared with GEA. Within the MA subtype, pCR was observed in 33.3% of the patients (95% CI: 29.4–43.9) and the 5-year recurrence-free interval was 59.2% (95% CI: 48.2–68.6). Patients with MA and TN basal-like tumours have lower survival outcomes. Conclusions: Irrespective of their HER2 status, the prognosis for MA tumours remains poor and adjuvant trials evaluating anti-androgens should be considered.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Processed pseudogenes acquired somatically during cancer development

Cancer evolves by mutation, with somatic reactivation of retrotransposons being one such mutational process. Germline retrotransposition can cause processed pseudogenes, but whether this occurs somatically has not been evaluated. Here we screen sequencing data from 660 cancer samples for somatically acquired pseudogenes. We find 42 events in 17 samples, especially non-small cell lung cancer (5/27) and colorectal cancer (2/11). Genomic features mirror those of germline LINE element retrotranspositions, with frequent target-site duplications (67%), consensus TTTTAA sites at insertion points, inverted rearrangements (21%), 5′ truncation (74%) and polyA tails (88%). Transcriptional consequences include expression of pseudogenes from UTRs or introns of target genes. In addition, a somatic pseudogene that integrated into the promoter and first exon of the tumour suppressor gene, MGA, abrogated expression from that allele. Thus, formation of processed pseudogenes represents a new class of mutation occurring during cancer development, with potentially diverse functional consequences depending on genomic context