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

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

International study on inter-reader variability for circulating tumor cells in breast cancer

Introduction Circulating tumor cells (CTCs) have been studied in breast cancer with the CellSearch® system. Given the low CTC counts in non-metastatic breast cancer, it is important to evaluate the inter-reader agreement. Methods CellSearch® images (N = 272) of either CTCs or white blood cells or artifacts from 109 non-metastatic (M0) and 22 metastatic (M1) breast cancer patients from reported studies were sent to 22 readers from 15 academic laboratories and 8 readers from two Veridex laboratories. Each image was scored as No CTC vs CTC HER2- vs CTC HER2+. The 8 Veridex readers were summarized to a Veridex Consensus (VC) to compare each academic reader using % agreement and kappa (κ) statistics. Agreement was compared according to disease stage and CTC counts using the Wilcoxon signed rank test. Results For CTC definition (No CTC vs CTC), the median agreement between academic readers and VC was 92% (range 69 to 97%) with a median κ of 0.83 (range 0.37 to 0.93). Lower agreement was observed in images from M0 (median 91%, range 70 to 96%) compared to M1 (median 98%, range 64 to 100%) patients (P < 0.001) and from M0 and <3CTCs (median 87%, range 66 to 95%) compared to M0 and ≥3CTCs samples (median 95%, range 77 to 99%), (P < 0.001). For CTC HER2 expression (HER2- vs HER2+), the median agreement was 87% (range 51 to 95%) with a median κ of 0.74 (range 0.25 to 0.90). Conclusions The inter-reader agreement for CTC definition was high. Reduced agreement was observed in M0 patients with low CTC counts. Continuous training and independent image review are require

International study on inter-reader variability for circulating tumor cells in breast cancer

Introduction: Circulating tumor cells (CTCs) have been studied in breast cancer with the CellSearch® system. Given the low CTC counts in non-metastatic breast cancer, it is important to evaluate the inter-reader agreement.Methods: CellSearch® images (N = 272) of either CTCs or white blood cells or artifacts from 109 non-metastatic (M0) and 22 metastatic (M1) breast cancer patients from reported studies were sent to 22 readers from 15 academic laboratories and 8 readers from two Veridex laboratories. Each image was scored as No CTC vs CTC HER2- vs CTC HER2+. The 8 Veridex readers were summarized to a Veridex Consensus (VC) to compare each academic reader using % agreement and kappa (κ) statistics. Agreement was compared according to disease stage and CTC counts using the Wilcoxon signed rank test.Results: For CTC definition (No CTC vs CTC), the median agreement between academic readers and VC was 92% (range 69 to 97%) with a median κ of 0.83 (range 0.37 to 0.93). Lower agreement was observed in images from M0 (median 91%, range 70 to 96%) compared to M1 (median 98%, range 64 to 100%) patients (P < 0.001) and from M0 and <3CTCs (median 87%, range 66 to 95%) compared to M0 and ≥3CTCs samples (median 95%, range 77 to 99%), (P < 0.001). For CTC HER2 expression (HER2- vs HER2+), the median agreement was 87% (range 51 to 95%) with a median κ of 0.74 (range 0.25 to 0.90).Conclusions: The inter-reader agreement for CTC definition was high. Reduced agreement was observed in M0 patients with low CTC counts. Continuous training and independent image review are required

Immune infiltration in invasive lobular breast cancer

Background: Invasive lobular breast cancer (ILC) is the second most common histological subtype of breast cancer after invasive ductal cancer (IDC). Here, we aimed at evaluating the prevalence, levels and composition of tumor infiltrating lymphocytes (TIL) and their association with clinico-pathological, and outcome variables in ILC, and to compare it with IDC. Methods: We considered two patient series with TIL data: a multi-centric retrospective series (n=614) and the BIG 02-98 study (n=149 ILC and 807 IDC). We compared immune subsets identified by immuno-histochemistry in the ILC (n=159) and IDC (n=468) patients from the Nottingham series, as well as the CIBERSORT immune profiling of the ILC (n=98) and IDC (n=388) METABRIC and TCGA patients. All ILC/IDC comparisons were done in ER-positive/HER2-negative tumors. All statistical tests were two-sided. Results: TIL levels were statistically significantly lower in ILC compared to IDC (fold change =0.79; 95%CI: 0.70-0.88, P<.001). In ILC, high TIL levels were associated with young age, lymph node involvement, and high proliferative tumors. In the univariable analysis, high TIL levels were associated with worse prognosis in the retrospective and BIG 02-98 lobular series, although it did not reach statistical significance in the latter. The Nottingham series revealed that the levels of intra-tumoral but not total CD8+ were statistically significantly lower in ILC compared to IDC. Comparison of the CIBERSORT profiles highlighted statistically significant differences in terms of immune composition. Conclusion: This study shows differences between the immune infiltrates of ER-positive/HER2-negative ILC and IDC in terms of prevalence, levels, localization, composition, and clinical associations

Infrared (IR) imaging: A new tool to refine breast cancer prognosis

abstract 89Pinfo:eu-repo/semantics/publishe

Discrimination between healthy and tumor tissues on formalin-fixed paraffin-embedded breast cancer samples using IR imaging

This work presents a pilot study to illustrate the potential of Fourier transform infrared (FT-IR) imaging in breast cancer research. Using this technique, we have acquired infrared (IR) microspectroscopic images from healthy and cancerous breast tissue section from one patient. First of all, a Student t-test was applied, showing DNA/RNA spectral region (1400-1000 cm-1) as the most discriminant for the differentiation between healthy and tumor samples. Afterwards, a supervised pattern recognition method, Partial Least Squares (PLS) was used to develop an automated classifier to discriminate the two classes of data. Infrared spectra of independent IR measurements were used to test the classifier. The class identity was correlated with information obtained by histopathologic gold standard. The results showed that more than 95% of the training and validation spectra were correctly identified. We demonstrate that combination between IR microspectroscopic imaging and multivariate data analysis can be used as a complement to present diagnostic tools for breast cancer. © 2010 - IOS Press and the authors. All rights reserved.SCOPUS: cp.jinfo:eu-repo/semantics/publishe

CD10 expression and risk of relapse in DCIS

Abstract: e22053info:eu-repo/semantics/publishe

Chromogenic in situ hybridization: a practical alternative for fluorescence in situ hybridization to detect HER-2/neu oncogene amplification in archival breast cancer samples.

Determination of HER-2/neu oncogene amplification has become necessary for selection of breast cancer patients for trastuzumab (Herceptin) therapy. Fluorescence in situ hybridization (FISH) is currently regarded as a gold standard method for detecting HER-2/neu amplification, but it is not very practical for routine histopathological laboratories. We evaluated a new modification of in situ hybridization, the chromogenic in situ hybridization (CISH), which enables detection of HER-2/neu gene copies with conventional peroxidase reaction. Archival formalin-fixed paraffin-embedded tumor tissue sections were pretreated (by heating in a microwave oven and using enzyme digestion) and hybridized with a digoxigenin-labeled DNA probe. The probe was detected with anti-digoxigenin fluorescein, anti-fluorescein peroxidase, and diaminobenzidine. Gene copies visualized by CISH could be easily distinguished with a x40 objective in hematoxylin-stained tissue sections. HER-2/neu amplification typically appeared as large peroxidase-positive intranuclear gene copy clusters. CISH and FISH (according to Vysis, made from frozen pulverized tumor samples) correlated well in a series of 157 breast cancers (kappa coefficient, 0.81). The few different classifications were mostly because of low-level amplifications by FISH that were negative by CISH and immunohistochemistry with monoclonal antibody CB-11. We conclude that CISH, using conventional bright-field microscopy in evaluation, is a useful alternative for determination of HER-2/neu amplification in paraffin-embedded tumor samples, especially for confirming the immunohistochemical staining results.Comparative StudyJournal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe