Isabelle Veys

CD4+ follicular helper T cell infiltration predicts breast cancer survival

CD4⁺ T cells are critical regulators of immune responses, but their functional role in human breast cancer is relatively unknown. The goal of this study was to produce an image of CD4⁺ T cells infiltrating breast tumors using limited ex vivo manipulation to better understand the in vivo differences associated with patient prognosis. We performed comprehensive molecular profiling of infiltrating CD4⁺ T cells isolated from untreated invasive primary tumors and found that the infiltrating T cell subpopulations included follicular helper T (Tfh) cells, which have not previously been found in solid tumors, as well as Th1, Th2, and Th17 effector memory cells and Tregs. T cell signaling pathway alterations included a mixture of activation and suppression characterized by restricted cytokine/chemokine production, which inversely paralleled lymphoid infiltration levels and could be reproduced in activated donor CD4⁺ T cells treated with primary tumor supernatant. A comparison of extensively versus minimally infiltrated tumors showed that CXCL13-producing CD4⁺ Tfh cells distinguish extensive immune infiltrates, principally located in tertiary lymphoid structure germinal centers. An 8-gene Tfh signature, signifying organized antitumor immunity, robustly predicted survival or preoperative response to chemotherapy. Our identification of CD4⁺ Tfh cells in breast cancer suggests that they are an important immune element whose presence in the tumor is a prognostic factor.

HER2-positive circulating tumor cells in breast cancer.

Purpose Circulating Tumor Cells (CTCs) detection and phenotyping are currently evaluated in Breast Cancer (BC). Tumor cell dissemination has been suggested to occur early in BC progression. To interrogate dissemination in BC, we studied CTCs and HER2 expression on CTCs across the spectrum of BC staging. Methods Spiking experiments with 6 BC cell lines were performed and blood samples from healthy women and women with BC were analyzed for HER2-positive CTCs using the CellSearch®. Results Based on BC cell lines experiments, HER2-positive CTCs were defined as CTCs with HER2 immunofluoresence intensity that was at least 2.5 times higher than the background. No HER2-positive CTC was detected in 42 women without BC (95% confidence interval (CI) 0–8.4%) whereas 4.1% (95%CI 1.4–11.4%) of 73 patients with ductal/lobular carcinoma in situ (DCIS/LCIS) had 1 HER2-positive CTC/22.5 mL, 7.9%, (95%CI 4.1–14.9%) of 101 women with non metastatic (M0) BC had ≥1 HER2-positive CTC/22.5 mL (median 1 cell, range 1–3 cells) and 35.9% (95%CI 22.7–51.9%) of 39 patients with metastatic BC had ≥1 HER2-positive CTC/7.5 mL (median 1.5 cells, range 1–42 cells). In CTC-positive women with DCIS/LCIS or M0 BC, HER2-positive CTCs were more commonly detected in HER2-positive (5 of 5 women) than HER2-negative BC (5 of 12 women) (p = 0.03). Conclusion HER2-positive CTCs were detected in DCIS/LCIS or M0 BC irrespective of the primary tumor HER2 status. Nevertheless, their presence was more common in women with HER2-positive disease. Monitoring of HER2 expression on CTCs might be useful in trials with anti-HER2 therapies.

Polysomy 17 in HER-2/neu Status Elaboration in Breast Cancer: Effect on Daily Practice

Purpose: To assess the effect of chromosome 17 copy number on HER-2/neu status determination in breast cancers. Experimental Design: HER-2/neu gene copy and chromosome 17 centromere numbers were evaluated on 893 breast carcinomas using double color fluorescence in situ hybridization (FISH). The net and chromosome 17 corrected (ratio) HER-2/neu copy numbers were compared and related to immunohistochemistry done according to the Food and Drug Administration (FDA)–approved scoring system (0, 1+, 2+, and 3+) as a first screening step in 584 cases. Results: When a ratio ≥2 was considered as criterion for FISH positivity, 49.3% (440 of 893) of cases showed amplification versus 56.2% (502 of 893) by using a net HER-2/neu gene copy number >4 as a alternative criterion; 14.8% (67 of 453) of cases having a ratio <2 had a net HER-2/neu gene copy number >4 and 1.1% (5 of 440) with a ratio ≥2 had a net HER-2/neu gene copy number <4. Among discordant cases, 88.8% (64 of 72) were polysomic (>2.25 chromosomes 17/cell) and among polysomic cases, 12.8% (40 of 312) of the low polysomic (2.26-3.75 chromosomes 17/cell) and 36.9% (24 of 65) of the highly polysomic (>3.75 chromosomes 17/cell) cases showed discordance. In cases with a ratio <2, polysomy 17 incidences were 85.7% (6 of 7) in IHC 3+, 42.4% (79 of 186) in IHC 2+, 33.3% (15 of 45) in IHC 1+, and 29.1% (16 of 55) in IHC 0. Conclusion: A net increase in HER-2/neu gene copy number consecutive to polysomy 17 in the absence of specific gene amplification might lead to a strong protein overexpression in a small subset of breast carcinomas. HER-2/neu status determination by FISH is dependent on the criterion considered for positivity in clinical practice.

Correction for chromosome-17 is critical for the determination of true Her-2/neu gene amplification status in breast cancer.

Purpose: Trastuzumab is the cornerstone for treatment of women with HER2-overexpressing breast cancer, both in the adjuvant and in the metastatic settings. The accurate assessment of HER2 is, therefore, critical to identifying patients who may benefit from trastuzumab-based therapy. This project aimed to determine the optimal scoring method for fluorescence in situ hybridization (FISH) assay. Methods: FISH assay was done on 893 samples of breast cancer. Three scoring methods were evaluated: Her2/CEP17≥2, Her2>4, or Her2>6. Protein and gene expression were evaluated by immunohistochemistry (n = 584) and mRNA/assay/nucleic acid sequence–based amplification (NASBA; n = 90). Results: Samples were divided into five groups based on FISH results: disomic amplified and nonamplified, polysomic amplified, nonamplified, and discordant (10.8% of cases, mostly positive with Her2>4 scoring, but negative with the others). Her2/CEP17≥2 and Her2>6 scoring methods showed the best association (a) with regard to FISH scoring (κ = 0.906, P < 10−6) and (b) between FISH and immunohistochemistry (3+ as positive; κ > 0.650, P < 10−6) or NASBA (κ > 0.536, P < 10−6). Polysomy had an effect on Her2 copy number (P < 10−6), but had no effect on protein and mRNA content. Therefore, within the discordant subgroup, for which additive Her-2 gene copies are due to high polysomy, protein and mRNA levels were similar to those of the nonamplified samples. For this subgroup, the best concordance between FISH/immunohistochemistry/NASBA was observed with the Her2/CEP17 ratio and Her-2>6 scoring (68% and 58% perfect matches, respectively). No perfect matches were observed using the Her2>4 scoring method. Conclusion: Correction for chromosome-17 is the method of choice for clinical practice; Her-2>6, but not Her-2>4, could be used as an alternative. [Mol Cancer Ther 2006;5(10):2572–9]

Safety and usefulness of cryopreservation of ovarian tissue to preserve fertility: a 12-year retrospective analysis

STUDY QUESTION Do the benefits of ovarian tissue cryopreservation outweigh the risks for patients seeking to preserve fertility before gonadotoxic treatment in various indications? SUMMARY ANSWER In >90% of the patients undergoing cryopreservation of ovarian tissue, oncological treatment was associated with a reduced ovarian reserve and in 30% of patients, premature ovarian failure (POF) occurred within 5 years. WHAT IS KNOWN ALREADY Ovarian tissue cryopreservation is an effective fertility preservation option, especially for pre-pubertal patients and patients who have a short time between diagnosis of a disease and gonadotoxic treatment. STUDY DESIGN, SETTING, DURATION This study retrospectively analysed ovarian function and fertility recovery rates, as well as ovarian tissue characteristics, of patients who underwent ovarian tissue cryopreservation at Erasme Hospital between 1999 and 2011. PARTICIPANTS/MATERIALS, SETTINGS, METHODS A total of 225 patients referred from 15 Belgian oncological units underwent cryopreservation of ovarian tissue before gonadotoxic therapy for malignant or benign diseases. There were 28 patients (12.4%) who died during follow-up due to recurrence of disease. One severe adverse event occurred during anaesthesia for ovarian tissue collection, leading to the death of the patient. Ovarian function and fertility outcomes were available for 114 patients including 13 girls who were pre-pubertal at the time of the procedure. Eight patients had undergone ovarian tissue transplantation in order to restore their fertility after remission of the disease. MAIN RESULTS AND THE ROLE OF CHANCE Breast cancer and haematological disease were the most frequent indications for ovarian tissue cryopreservation. Overall, 90% of post-pubertal patients were diagnosed with poor ovarian reserve (AMH < 0.5 ng/ml) after a mean of 50 months of follow-up (11-125 months), including 30% with POF (FSH > 40 IU/ml). Breast cancer patients had a lower rate of POF than did post-pubertal patients with haematological diseases (11 versus 34.5%, respectively), despite the older age (mean 31 versus 23.5 years old, respectively) of the breast cancer patients. Ovarian function returned in 71 post-pubertal patients without the need for grafts of cryopreserved tissue. Spontaneous pregnancies were reported for 33 of them, leading to 34 live births. Among the 13 pre-pubertal patients who reached pubertal age during the follow-up, 10 had POF. Eight patients received cryopreserved ovarian grafts to reverse POF and three of them have already become pregnant. LIMITATIONS, REASONS FOR CAUTION This study is a retrospective analysis. The cohort was not compared with a control group of patients who did not undergo the procedure. WIDER IMPLICATIONS OF THE FINDINGS After careful evaluation of the surgical risks, ovarian tissue cryopreservation can be proposed as an efficient option to preserve the fertility of children and young adults facing gonadotoxic therapies. However, alternative procedures such as oocyte or embryo cryopreservation should be considered as first options especially for older patients or if there is high risk of neoplastic cells within the ovaries. STUDY FUNDING/COMPETING INTEREST This study was supported by the Télévie, FNRS-FRSM and Fondation Belge contre le cancer. There are no competing interests to report.

Tumor-infiltrating lymphocyte composition, organization and PD-1/ PD-L1 expression are linked in breast cancer

ABSTRACT The clinical relevance of tumor-infiltrating lymphocytes (TIL) in breast cancer (BC) has been clearly established by their demonstrated correlation with long-term positive outcomes. Nevertheless, the relationship between protective immunity, observed in some patients, and critical features of the infiltrate remains unresolved. This study examined TIL density, composition and organization together with PD-1 and PD-L1 expression in freshly collected and paraffin-embedded tissues from 125 patients with invasive primary BC. Tumor and normal breast tissues were analyzed using both flow cytometry and immunohistochemistry. TIL density distribution is a continuum with 25% of tumors identified as TIL-negative at a TIL density equivalent to normal breast tissues. TIL-positive tumors (75%) were equally divided into TIL-intermediate and TIL-high. Tumors had higher mean frequencies of CD4+ T cells and CD19+ B cells and a lower mean frequency of CD8+ T cells compare with normal tissues, increasing the CD4+/CD8+ T-cell ratio. Tertiary lymphoid structures (TLS), principally located in the peri-tumoral stroma, were detected in 60% of tumors and correlated with higher TIL infiltration. PD-1 and PD-L1 expression were also associated with higher TIL densities and TLS. TIL density, TLS and PD-L1 expression were correlated with more aggressive tumor characteristics, including higher proliferation and hormone receptor negativity. Our findings reveal an important relationship between PD-1/PD-L1 expression, increased CD4+ T and B-cell infiltration, TIL density and TLS, suggesting that evaluating not only the extent but also the nature and location of the immune infiltrate should be considered when evaluating antitumor immunity and the potential for benefit from immunotherapies.

Eighteen months clinical experience with the GeneSearch breast lymph node assay

BACKGROUND The accuracy of a molecular reverse transcriptase-polymerase chain reaction (RT-PCR)-based assay for metastases detection in axillary sentinel lymph nodes (SLNs) has recently been validated in our institution and adopted as an intraoperative test for breast cancer patient management. METHODS Molecular assay performance was compared to standard postoperative histology in 253 consecutive patients with clinically node-negative T1 early breast cancer (<2 cm). RESULTS The molecular assay correctly identified 26/27 macrometastases and 11/15 micrometastases. Overall concordance with histopathology was 93%, with 87% sensitivity, 94% specificity, and 75% positive and 97% negative predictive values. The molecular assay was positive in 13/14 patients with SLNs and nonsentinel lymph node (axillary lymph node [ALN])-positive histology. Notably, 2/12 patients with assay-positive/histology-negative SLNs exhibited ALN positivity. CONCLUSIONS This molecular assay can raise the standard of care for patient management as its accuracy is similar to that of standard postoperative histology with the advantage of being standardized, objective, and fast enough for intraoperative use.

Association between SPARC mRNA expression, prognosis and response to neoadjuvant chemotherapy in early breast cancer: a pooled in-silico analysis.

Introduction SPARC is an important regulator of the extracellular matrix and has been suggested to improve delivery of albumin-bound cytotoxics. However, little is known regarding its role in breast cancer (BC). Methods We conducted a pooled analysis of publically available datasets, in which BC patients who received no systemic therapy or received neoadjuvant chemotherapy were eligible. Patients were assigned to molecular subtypes using PAM-50. We computed a SPARC module (SPARC7), composed of genes with an absolute correlation with SPARC >0.7. In the systemically untreated cohort, we evaluated 1) expression of SPARC/SPARC7 according to breast cancer subtype, 2) association between SPARC/SPARC7 and biological processes related to proliferation, immune and stroma, and 3) association between SPARC/SPARC7 and relapse-free survival in a Cox model in all patients and in the different molecular subtypes adjusted for tumor size, nodal status, histological grade, and age. In the neoadjuvant cohort, we evaluated the association between SPARC and pCR in a logistic regression model, adjusted for the same clinicopathologic factors. Results 948 (10 datasets), and 791 (8 datasets) patients were included in the systemically untreated and neoadjuvant cohorts, respectively. High SPARC expression was associated with small tumor size, low histological grade and luminal-A tumors (all p<0.0001). There was a positive correlation between SPARC and stroma-related modules but negative correlation with proliferation modules. High SPARC expression was associated with poor prognosis in patients with basal and HER2+ breast cancer even after adjusting for clinicopathologic parameters. In the neoadjuvant cohort, a subgroup analysis suggested that high SPARC is associated with low rates of pCR in the HER2 subtype. Same results were observed on replacing SPARC by SPARC7. Conclusion This analysis suggests a potential role of SPARC in determining prognosis and response to primary chemotherapy in early BC. This information could guide further development of albumin-bound cytotoxics in BC.

Genomic grade adds prognostic value in invasive lobular carcinoma

BACKGROUND The prognostic value of histologic grade (HG) in invasive lobular carcinoma (ILC) remains uncertain, and most ILC tumors are graded as HG2. Genomic grade (GG) is a 97-gene signature that improves the prognostic value of HG. This study evaluates whether GG may overcome the limitations of HG in ILC. METHODS Gene expression data were generated from frozen tumor samples, and GG calculated according to the expression of 97 genes. The prognostic value of GG was assessed in a stratified Cox regression model for invasive disease-free survival (IDFS) and overall survival (OS). RESULTS A total of 166 patients were classified by GG. HG classified 33 (20%) tumors as HG1, 120 (73%) as HG2 and 12 (7%) as HG3. GG classified 106 (64%) tumors as GG low (GG1), 29 (17%) as GG high (GG3) and 31 (19%) as equivocal (cases not classified as GG1 or GG3). The median follow-up time was 6.5 years. In multivariate analyses, GG was associated with IDFS [HRGG3 vs GG1 5.6 (2.1-15.3); P < 0.001] and OS [HRGG3 vs GG1 7.2, 95% CI (1.6-32.2); P = 0.01]. CONCLUSIONS GG outperformed HG in ILC and added prognostic value to classic clinicopathologic variables, including nodal status.BACKGROUND The prognostic value of histologic grade (HG) in invasive lobular carcinoma (ILC) remains uncertain, and most ILC tumors are graded as HG2. Genomic grade (GG) is a 97-gene signature that improves the prognostic value of HG. This study evaluates whether GG may overcome the limitations of HG in ILC. METHODS Gene expression data were generated from frozen tumor samples, and GG calculated according to the expression of 97 genes. The prognostic value of GG was assessed in a stratified Cox regression model for invasive disease-free survival (IDFS) and overall survival (OS). RESULTS A total of 166 patients were classified by GG. HG classified 33 (20%) tumors as HG1, 120 (73%) as HG2 and 12 (7%) as HG3. GG classified 106 (64%) tumors as GG low (GG1), 29 (17%) as GG high (GG3) and 31 (19%) as equivocal (cases not classified as GG1 or GG3). The median follow-up time was 6.5 years. In multivariate analyses, GG was associated with IDFS [HR(GG3 vs GG1) 5.6 (2.1-15.3); P < 0.001] and OS [HR(GG3 vs GG1) 7.2, 95% CI (1.6-32.2); P = 0.01]. CONCLUSIONS GG outperformed HG in ILC and added prognostic value to classic clinicopathologic variables, including nodal status.

The Genomic Grade Assay Compared With Ki67 to Determine Risk of Distant Breast Cancer Recurrence

IMPORTANCE The Genomic Grade Index (GGI) was previously developed, evaluated on frozen tissue, and shown to be prognostic in early breast cancer. To test the GGI in formalin-fixed, paraffin-embedded breast cancer tumors, a quantitative reverse transcriptase polymerase chain reaction assay was developed and named the Genomic Grade (GG). The GG assay has the potential to increase the clinical application of the GGI, but robust demonstration of the clinical validity of the GG assay is required. OBJECTIVE To evaluate the prognostic ability of the GG assay to detect breast cancer recurrence compared with centrally reviewed immunohistochemical testing of Ki67 antigen proliferation. DESIGN, SETTING, AND PARTICIPANTS This is an internationally collaborative substudy of a large phase 3 4-arm adjuvant trial. Patients had endocrine receptor-positive, node-positive, or node-negative nonmetastatic primary breast cancer. Patients included in this study had available formalin-fixed, paraffin-embedded samples of their primary tumors and were randomized to either a 5-year tamoxifen monotherapy arm or a 5-year letrozole monotherapy arm. Associations between either GG assay results or log2-transformed Ki67 data and survival end points were evaluated using Cox regression models stratified for chemotherapy use; the 2 vs 4 arm randomization option; and endocrine therapy assignment with and without adjustment for clinicopathological parameters, including centrally reviewed histological grade, hormone receptors, and ERBB2 (formerly HER2 or HER2/neu). The likelihood ratio statistic was used to assess the added prognostic value. INTERVENTIONS Central evaluation and comparison, blinded for clinical information, of the GG assay, breast cancer histological grade, and Ki67. MAIN OUTCOMES AND MEASURES Distant recurrence-free interval (DRFI). RESULTS Genomic Grade assay data were obtained in 883 breast cancer samples (62%). At a median follow-up of 8.1 years, 84 (10%) had distant recurrences. Increasing GG or Ki67 were both significantly associated with lower DRFI and added independent prognostic information to the clinicopathological prognostic factors. In patients with early node-negative breast cancer who were endocrine-only treated, 38% were GG1 with a 10-year DRFI of 99% (95% CI, 97%-100%), and 18% were histological grade 1 with a 10-year DRFI of 100% (95% CI, 100%-100%). For GG equivocal patients, the 10-year DRFI was 94% (95% CI, 90%-98%), and for GG3 patients, the 10-year DRFI was 87% (95% CI, 80%-94%). CONCLUSIONS AND RELEVANCE Either the GG assay or centrally reviewed Ki67 significantly improves clinicopathological models to determine distant recurrence of breast cancer. Compared with the histological grade, the GG assay can identify a higher proportion of endocrine-only treated patients with very low risk of distant recurrence at 10 years. TRIAL REGISTRATION clinicaltrials.gov Identifiers: NCT00004205 and NCT00004205.