Michail Ignatiadis

The evaluation of tumor-infiltrating lymphocytes (TILs) in breast cancer: recommendations by an International TILs Working Group 2014

BACKGROUNDnThe morphological evaluation of tumor-infiltrating lymphocytes (TILs) in breast cancer (BC) is gaining momentum as evidence strengthens for the clinical relevance of this immunological biomarker. Accumulating evidence suggests that the extent of lymphocytic infiltration in tumor tissue can be assessed as a major parameter by evaluation of hematoxylin and eosin (H&E)-stained tumor sections. TILs have been shown to provide prognostic and potentially predictive value, particularly in triple-negative and human epidermal growth factor receptor 2-overexpressing BC.nnnDESIGNnA standardized methodology for evaluating TILs is now needed as a prerequisite for integrating this parameter in standard histopathological practice, in a research setting as well as in clinical trials. This article reviews current data on the clinical validity and utility of TILs in BC in an effort to foster better knowledge and insight in this rapidly evolving field, and to develop a standardized methodology for visual assessment on H&E sections, acknowledging the future potential of molecular/multiplexed approaches.nnnCONCLUSIONSnThe methodology provided is sufficiently detailed to offer a uniformly applied, pragmatic starting point and improve consistency and reproducibility in the measurement of TILs for future studies.

Cytokeratin-19 mRNA-Positive Circulating Tumor Cells After Adjuvant Chemotherapy in Patients With Early Breast Cancer

PURPOSEnTo evaluate the prognostic significance of cytokeratin-19 (CK-19) mRNA-positive circulating tumor cells (CTCs) in peripheral blood of women with early-stage breast cancer after the completion of adjuvant chemotherapy.nnnPATIENTS AND METHODSnBlood was obtained from 437 patients with early breast cancer before the start and after the completion of adjuvant chemotherapy, and the presence of CK-19 mRNA-positive CTCs was assessed by real-time reverse transcriptase polymerase chain reaction. Interaction with known prognostic factors and association of CTCs with clinical outcome were investigated.nnnRESULTSnCK-19 mRNA-positive CTCs were detected before chemotherapy in 179 patients (41.0%). After adjuvant chemotherapy, a significant change in CK-19 status was observed, as status for 51% of patients with initially CK-19 mRNA-positive disease turned negative, and status for 22% of patients with initially CK-19 mRNA-negative disease became positive (McNemar test P = .004). The detection of CK-19 mRNA-positive CTCs postchemotherapy was associated with involvement of more than three axillary lymph nodes (P = .026). Clinical relapses and disease-related deaths were significantly increased in patients with detectable postchemotherapy CK-19 mRNA-positive CTCs (both P < .001, respectively). Disease-free and overall survival were significantly reduced in patients with detectable CK-19 mRNA-positive CTCs postchemotherapy (P < .001 and P = .001, respectively). In multivariate analysis, the detection of CK-19 mRNA-positive CTCs before and after adjuvant chemotherapy was an independent factor associated with reduced disease-free survival (P < .001) and overall survival (P = .003).nnnCONCLUSIONnThe detection of CK-19 mRNA-positive CTCs in the blood after adjuvant chemotherapy is an independent risk factor indicating the presence of chemotherapy-resistant residual disease.

Different Prognostic Value of Cytokeratin-19 mRNA Positive Circulating Tumor Cells According to Estrogen Receptor and HER2 Status in Early-Stage Breast Cancer

PURPOSEnTo examine the prognostic value of cytokeratin-19 (CK-19) mRNA-positive circulating tumor cells (CTCs) in early-stage breast cancer patients focusing on clinically relevant subgroups based on estrogen receptor (ER) and HER2 expression.nnnPATIENTS AND METHODSnCK-19 mRNA-positive CTCs were detected by real-time reverse transcriptase polymerase chain reaction in the blood of 444 consecutive, stage I-III, breast cancer patients before initiation of adjuvant chemotherapy. The association between detection of CK-19 mRNA-positive CTCs and clinical outcome was analyzed for patients with ER-positive, ER-negative, triple-negative, HER2-positive, and ER-positive/HER2-negative tumors.nnnRESULTSnCK-19 mRNA-positive CTCs were detected in 181 (40.8%) of 444 patients; 109 (41.9%) of 260 patients with ER-positive tumors; 71 (40.6%) of 175 patients with ER-negative tumors; 27 (35%) of 77 patients with triple-negative tumors; 35 (39.8%) of 88 patients with HER2-positive tumors; and 82 (44.1%) of 186 patients with ER-positive/HER2-negative tumors. After a median follow-up of 53.5 months, patients with CK-19 mRNA-positive CTCs experienced reduced disease-free survival (DFS; P < .001) and overall survival (OS; P < .001); this was mainly observed in patients with ER-negative (P < .001 and P < .001, respectively) but not ER-positive tumors (P = .172 and P = .425, respectively) and in patients with triple-negative (P = .008 and P = .001, respectively) and HER2-positive (P = .023 and P = .040, respectively) but not ER-positive/HER2-negative tumors (P = .210 and P = .578, respectively). In multivariate analysis, the interaction between CK-19 mRNA-positive CTCs and ER status was the strongest independent prognostic factor for reduced DFS (hazard ratio [HR], 3.808; 95% CI, 2.415 to 6.003; P < .001) and OS (HR, 4.172; 95% CI, 2.477 to 9.161; P < .001).nnnCONCLUSIONnDetection of CK-19 mRNA-positive CTCs before adjuvant chemotherapy predicts poor clinical outcome mainly in patients with ER-negative, triple-negative, and HER2-positive early-stage breast cancer.

Clinical application of circulating tumor cells in breast cancer: overview of the current interventional trials

In 2004, circulating tumor cells (CTC) enumeration by the CellSearch® technique at baseline and during treatment was reported to be associated with prognosis in metastatic breast cancer patients. In 2008, the first evidence of the impact of CTC detection by this technique on survival of cM0(i+) patients were reported. These findings were confirmed by other non-interventional studies, whereas CTC were also investigated as a surrogate for tumor biology, mainly for HER2 expression/amplification. The aim of this report is to present the current prospective large interventional studies that have been specifically designed to demonstrate that CTC enumeration/characterization may improve the management of breast cancer patients: STIC CTC METABREAST (France) and Endocrine Therapy Index (USA) assess the CTC-guided hormone therapy vs chemotherapy decision in M1 patients; SWOG0500 (USA) and CirCe01 (France) assess the CTC count changes during treatment in metastatic patients; DETECT III (M1 patients, Germany) and Treat CTC (cM0(i+) patients, European Organization for Research and Treatment of Cancer/Breast International Group) assess the use of anti-HER2 treatments in HER2-negative breast cancer patients selected on the basis of CTC detection/characterization. These trials have different designs in various patient populations but are expected to be the pivotal trials for CTC implementation in the routine management of breast cancer patients.

Gene Modules and Response to Neoadjuvant Chemotherapy in Breast Cancer Subtypes: A Pooled Analysis

PURPOSEnTo investigate the association between chemotherapy response and gene expression modules describing important biologic processes and druggable oncogenic pathways in breast cancer (BC) subtypes.nnnPATIENTS AND METHODSnWe searched for publicly available gene expression studies evaluating anthracycline with or without taxane-based neoadjuvant chemotherapy and identified eight studies with 996 patients. We computed 17 gene modules and calculated odds ratios (ORs) for pathologic complete response (pCR) for one-unit increases in scaled modules with and without adjustment for clinicopathologic characteristics. Added predictive accuracy was evaluated using the area under the receiver operating characteristic curve (AUC) and integrated discrimination index (IDI). We used the false discovery rate (FDR) to adjust for multiple testing.nnnRESULTSnHigh immune module scores were associated with increased pCR probability in all BC subtypes. High module scores of chromosomal instability, phosphatase and tensin homolog (PTEN) loss, and E2F3 transcription factor were associated with increased pCR probability in estrogen receptor (ER) -negative/human epidermal growth factor receptor 2 (HER2) -negative and ER-positive/HER2-negative but not in HER2-positive tumors (interactions between HER2 and each of these modules for their association with pCR: P < .05; FDR, 0.17; trend for interaction between HER2 and PTEN). High values of insulin-like growth factor 1 activation module were associated with increased pCR probability only in ER-positive/HER2-negative tumors (interaction between insulin-like growth factor 1 and ER: P = .002; FDR, 0.03). When adding the immune module to clinicopathologic characteristics, we observed substantial increases in predictive accuracy for pCR in the HER2-positive subtype (IDI, 0.093; P = .004; increase in AUC from 0.760 to 0.836).nnnCONCLUSIONnDifferent processes and pathways are associated with pCR in different BC subtypes.

Global MicroRNA Expression Profiling Identifies MiR-210 Associated with Tumor Proliferation, Invasion and Poor Clinical Outcome in Breast Cancer

Purpose Aberrant microRNA (miRNA) expression is associated with cancer and has potential diagnostic and prognostic value in various malignancies. In this study, we investigated miRNA profiling as a complementary tool to improve our understanding of breast cancer (BC) biology and to assess whether miRNA expression could predict clinical outcome of BC patients. Experimental Design Global miRNA expression profiling using microarray technology was conducted in 56 systemically untreated BC patients who had corresponding mRNA expression profiles available. Results were further confirmed using qRT-PCR in an independent dataset of 89 ER-positive BC patients homogeneously treated with tamoxifen only. MiR-210 functional analyses were performed in MCF7 and MDA-MB-231 BC cell lines using lentiviral transduction. Results Estrogen receptor (ER) status, tumor grade and our previously developed gene expression grade index (GGI) were associated with distinct miRNA profiles. Several miRNAs were found to be clinically relevant, including miR-210, its expression being associated with tumor proliferation and differentiation. Furthermore, miR-210 was associated with poor clinical outcome in ER-positive, tamoxifen-treated BC patients. Interestingly, the prognostic performance of miR-210 was similar to several reported multi-gene signatures, highlighting its important role in BC differentiation and tumor progression. Functional analyses in BC cell lines revealed that miR-210 is involved in cell proliferation, migration and invasion. Conclusions This integrated analysis combining miRNA and mRNA expression demonstrates that miRNA expression provides additional biological information beyond mRNA expression. Expression of miR-210 is linked to tumor proliferation and appears to be a strong potential biomarker of clinical outcome in BC.

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) (pu200a=u200a0.03). 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.

External quality assurance of circulating tumor cell enumeration using the CellSearch(®) system: a feasibility study.

Circulating tumor cells (CTCs) are cells that have detached from solid tumors and entered the blood. CTCs can be detected, among others, by semi‐automated immunomagnetic enrichment and image cytometry using CellSearch® (Veridex, Raritan, NJ). We studied the feasibility of external quality assurance (EQA) of the entire CellSearch procedure from blood draw to interpretation of results in multiple laboratories.

Luminal breast cancer: from biology to treatment

Oestrogen receptor (ER)-positive—or luminal—tumours represent around two-thirds of all breast cancers. Luminal breast cancer is a highly heterogeneous disease comprising different histologies, gene-expression profiles and mutational patterns, with very varied clinical courses and responses to systemic treatment. Despite adjuvant endocrine therapy and chemotherapy treatment for patients at high risk of relapse, both early and late relapses still occur, a fact that highlights the unmet medical needs of these patients. Ongoing research aims to identify those patients who can be spared adjuvant chemotherapy and who will benefit from extended adjuvant hormone therapy. This research also aims to explore the role of adjuvant bisphosphonates, to interrogate new agents for targeting minimal residual disease, and to address endocrine resistance. Data from next-generation sequencing studies have given us new insight into the biology of luminal breast cancer and, together with advances in preclinical models and the availability of newer targeted agents, have led to the testing of rationally chosen combination treatments in clinical trials. However, a major challenge will be to make sense of the large amount of patient genomic data that is becoming increasingly available. This analysis will be critical to our understanding how intertumour and intratumour heterogeneity can influence treatment response and resistance.

Circulating Tumor Cells and Circulating tumor DNA for precision medicine: Dream or reality?

Next-generation sequencing studies have provided further evidence to support the notion that cancer is a disease characterized by Darwinian evolution. Today, we often fail to capture this evolution and treatment decisions, even in the metastatic setting, are often based on analysis of primary tumor diagnosed years ago. Currently, this is considered a major reason for treatment failures in cancer care. Recent technological advances in the detection and characterization of circulating tumor cells and circulating tumor DNA might address this and allow for treatment tailoring based on real-time monitoring of tumor evolution. In this review, we summarize the most important recent findings in the field, focusing on challenges and opportunities in moving these tools forward in clinical practice.