Expression of insulin‐like growth factor‐1 receptor in circulating tumor cells of patients with breast cancer is associated with patient outcomes

In patients with breast cancer, markers of aggressiveness such as dysregulation of the insulin‐like growth factor receptor (IGF1R) system and E‐cadherin loss are commonly observed. Reduced IGF1R expression is correlated with decreased E‐cadherin levels and increased cell motility. We assessed IGF1R and E‐cadherin expression in circulating tumor cells (CTCs) in patients with breast cancer. Peripheral blood mononuclear cells of early (n = 87)‐ and metastatic (n = 126)‐stage breast cancer patients (obtained prior to adjuvant and first‐line chemotherapy) were evaluated using double immunofluorescence (IF) staining for cytokeratin (CK) and IGF1R. Triple IF using CK, IGF1R, and E‐cadherin antibodies was performed in selected CTC(+) patients. IGF1R(+) CTCs were more frequently observed in early disease than in metastatic disease (86% vs 68% of CTCs, P = 0.04) stage, whereas IGF1R(−) CTCs were more common in metastatic than in early disease (32% vs 14% of CTCs, P = 0.002). 100% of CTC(+) patients with early disease, compared to 79% of those with metastatic disease, harbored IGF1R(+) CTCs (P = 0.007). Patients with early disease and exclusively IGF1R(+) CTCs had longer disease‐free (P = 0.02) and overall survival (P = 0.001) compared to patients with both IGF1R(+) and IGF1R(−) CTC populations. 67% of early‐stage CTC(+) patients evaluated had exclusively IGF1R(+)/E‐cadherin(+) CTCs, 33% also had IGF1R(−)/E‐cadherin(−) CTCs, and none had exclusively IGF1R(−)/E‐cadherin(−) CTCs compared to 17%, 75%, and 8% of metastatic patients, respectively (P = 0.027). Similarly, in paired samples of patients with early disease that progressed to metastatic disease, the proportion of IGF1R(+)/E‐cadherin(+) CTCs was reduced and IGF1R(−)/E‐cadherin(−) CTCs were increased in the metastatic stage compared to early disease stage. IGF1R(+) CTCs are commonly detected in breast cancer, and their frequency decreases in the metastatic disease stage. IGF1R(+)/E‐cadherin(+) CTCs also decrease in metastatic patients. IGF1R(+) CTCs are associated with favorable outcomes in early disease stage, suggesting that IGF1R expression is correlated with reduced metastatic potential in breast cancer

Circulating microRNAs in the early prediction of disease recurrence in primary breast cancer

Abstract Background In primary breast cancer metastases frequently arise from a state of dormancy that may persist for extended periods of time. We investigated the efficacy of plasma micro-RNA (miR)-21, miR-23b, miR-190, miR-200b and miR-200c, related to dormancy and metastasis, to predict the outcome of patients with early breast cancer. Methods miRNAs were evaluated by RT-qPCR in plasma obtained before adjuvant chemotherapy. miRNA expression, classified as high or low according to median values, correlated with relapse and survival. Receiver operating characteristic (ROC) curves were constructed to determine miRNA sensitivity and specificity. Results miR-21 (p < 0.001), miR-23b (p = 0.028) and miR-200c (p < 0.001) expression were higher and miR-190 was lower (p = 0.013) in relapsed (n = 49), compared to non-relapsed patients (n = 84). Interestingly, miR-190 was lower (p = 0.0032) in patients with early relapse (at < 3 years; n = 23) compared to those without early relapse (n = 110). On the other hand, miR-21 and miR-200c were higher (p = 0.015 and p < 0.001, respectively) in patients with late relapse (relapse at ≥ 5 years; n = 20) as compared to non-relapsed patients. High miR-200c was associated with shorter disease-free survival (DFS) (p = 0.005) and high miR-21 with both shorter DFS and overall survival (OS) (p < 0.001 and p = 0.033, respectively) compared to low expression. ROC curve analysis revealed that miR-21, miR-23b, miR-190 and miR-200c discriminated relapsed from non-relapsed patients. A combination of of miR-21, miR-23b and miR-190 showed higher sensitivity and specificity in ROC analyses compared to each miRNA alone; accuracy was further improved by adding lymph node infiltration and tumor grade to the panel of three miRs (AUC 0.873). Furthermore, the combination of miR-200c, lymph node infiltration, tumor grade and estrogen receptor predicted late relapse (AUC 0.890). Conclusions Circulating miRNAs are differentially expressed among relapsed and non-relapsed patients with early breast cancer and predict recurrence many years before its clinical detection. Our results suggest that miRNAs represent potential circulating biomarkers in early breast cancer

Dynamic monitoring of PD‐L1 and Ki67 in circulating tumor cells of metastatic non‐small cell lung cancer patients treated with pembrolizumab

Programmed cell death protein ligand‐1 (PD‐L1) expression in non‐small cell lung cancer (NSCLC) tumors guides treatment selection. PD‐L1 expression in circulating tumor cells (CTCs) may provide further information. We have explored PD‐L1 and marker of proliferation Ki‐67 (Ki67; also known as MKI67) in CTCs in longitudinal samples of 47 advanced NSCLC patients receiving pembrolizumab. A triple immunofluorescence, against cytokeratin, PD‐L1, and Ki67, was performed on peripheral blood mononuclear cells, at baseline, post‐first cycle, post‐third, and primary resistance (PMR). Patients displaying PMR (progression at first evaluation) were classified as progressive disease (PD) and those with clinical benefit as disease control (DC). CTCs were categorized as PD‐L1high/low/medium/negative and Ki67+ or Ki67−. CTC evaluation revealed a significant increase in the PD‐L1low CTC rate at PMR compared to baseline (2.5% at baseline vs. 36.5% at PMR), whereas a reduction in the PD‐L1high CTC rate was observed (31.5% vs. 0%, respectively). Investigation of CTC status between PD and DC patients showed that PD patients more frequently increased total and PD‐L1low CTCs after first cycle compared to DC (83% of PD vs. 37% of DC and 67% of PD vs. 8% of DC, respectively). Progression‐free survival (PFS) was longer in patients with decreased total and PD‐L1low CTCs after first cycle compared to those with increased CTCs (median PFS: not reached vs. 2 months). PD‐L1+ patients presenting a high Ki67 index (% Ki67+ CTCs > 30%) before treatment had a shorter PFS compared to those with a low Ki67 (≤ 30%), and overall survival (OS) was shorter in PD‐L1+ patients harboring Ki67+ CTCs compared to those not presenting (median OS: 11.8 months vs. 33.1 months, respectively). In sequential samples of patients with a durable benefit, a low Ki67 index was observed. Our results suggest that monitoring PD‐L1 and Ki67 expression in CTCs of NSCLC patients treated with pembrolizumab may be predictive for pembrolizumab efficacy

A Comparison of Three Methods for the Detection of Circulating Tumor Cells in Patients with Early and Metastatic Breast Cancer

Background: We directly compared CTC detection rates and prognostic significance, using three different methods in patients with breast cancer (BC). Methods: Early (n=200) and metastatic (n=164) patients were evaluated before initiating adjuvant or first-line chemotherapy, using the CellSearchTM System, an RT-qPCR for CK-19 mRNA detection and by double immunofluorescence (IF) microscopy using A45-B/B3 and CD45 antibodies. Results: Using the CellSearchTM System, 37% and 16.5% of early BC patients were CTC-positive (at ≥1 and ≥2 CTCs/23 ml of blood), 18.0% by RT-qPCR and 16.9% by IF; no agreement was observed between methods. By the CellSearchTM 34.8% and 53.7% (at≥ 5 and ≥ 2 CTCs/7.5 ml) of metastatic patients were CTC-positive, 37.8% by RT-qPCR and 28.5% by IF. A significant agreement existed only between the CellSearchTM and RT-qPCR. In 60.8% of cases, differential EpCAM and CK-19 expression on CTCs by IF could explain the discrepancies between the CellSearchTM and RT-qPCR. CTC-positivity by either method was associated with decreased overall survival in metastatic patients. Conclusion: A significant concordance was observed between the CellSearchTM and RT-qPCR in metastatic but not in early BC. Discordant results could be explained in part by CTC heterogeneity. CTC detection by all methods evaluated had prognostic relevance in metastatic patients

Additional file 1: of Circulating microRNAs in the early prediction of disease recurrence in primary breast cancer

Table S1. Tenfold cross-validation results of nine different sets of combinations of predictor variables. (DOCX 13 kb