Monitoring in metastatic breast cancer: Is imaging outdated in the era of circulating tumor cells?

In clinical practice imaging technologies such as computed tomography (CT), positron emission tomography (PET)/CT and magnetic resonance imaging (MRI) are well-established methods for monitoring metastatic breast cancer (MBC) patients and for assessing therapeutic efficacy. However, several weeks of treatment are required before these technologies can offer any reliable information on effective disease regression, and, in the meanwhile, the patients are exposed to potentially unnecessary therapy. Circulating tumor cells (CTCs) have been shown to be powerful prognostic and predictive markers and provide clinicians with valuable information. However, in one clinical trial, an early change of chemotherapy based on CTC detection did not result in improved survival. Currently, CTC detection outside clinical trials should be limited to selected clinical situations, i.e. increased treatment toxicity or as risk estimation

Comparison of HER2 Expression in Primary Tumor and Disseminated Tumor Cells in the Bone Marrow of Breast Cancer Patients

Objective: The aim of this study was to measure the human epidermal growth factor receptor 2 (HER2) status of disseminated tumor cells (DTCs) from bone marrow (BM) aspirates and to assess correspondence or discrepancy with the primary tumor. Methods: DTCs were isolated from the BM of 156 breast cancer patients. Cytokeratin-positive DTCs were further analyzed by the chromogenic in situ hybridization method to detect HER2 gene amplification. Results: A significant correlation (p = 0.021) was found between the HER2 status of DTCs and the primary tumors. Sixty-one (68.5%) patients had a corresponding status. However, a shift of phenotype between primary tumor and DTCs was found in the remaining patients. Conclusion: This study showed a significant grade of discordance of the HER2 status between primary tumors and DTCs in the BM of a relevant subgroup of patients. Detection of HER2 amplification on DTCs could therefore help to better stratify patients for a more tailored therapy, since they would benefit from a HER2-targeted therapy. (C) 2016 S. Karger AG, Base

Circulating DNA as prognostic biomarker in patients with advanced hepatocellular carcinoma: a translational exploratory study from the SORAMIC trial

Abstract: Background: Liquid biopsy based on cell-free DNA circulating in plasma has shown solid results as a non-invasive biomarker. In the present study we evaluated the utility of circulating free DNA (cfDNA) and the sub-type tumor DNA (ctDNA) in hepatocellular cancer (HCC) patients to assess therapy response and clinical outcome. Methods: A cohort of 13 patients recruited in the context of the SORAMIC trial with unresectable, advanced HCC and different etiological and clinicopathological characteristics was included in this exploratory study. Plasma samples were collected between liver micro-intervention and beginning of sorafenib-based systemic therapy and then in correspondence of three additional follow-ups. DNA was isolated from plasma and next generation sequencing (NGS) was performed on a panel of 597 selected cancer-relevant genes. Results: cfDNA levels showed a significant correlation with the presence of metastases and survival. In addition cfDNA kinetic over time revealed a trend with the clinical history of the patients, supporting its use as a biomarker to monitor therapy. NGS-based analysis on ctDNA identified 28 variants, detectable in different combinations at the different time points. Among the variants, HNF1A, BAX and CYP2B6 genes showed the highest mutation frequency and a significant association with the patients’ clinicopathological characteristics, suggesting a possible role as driver genes in this specific clinical setting. Conclusions: Taken together, the results support the prognostic value of cfDNA/ctDNA in advanced HCC patients with the potential to predict therapy response. These findings support the clinical utility of liquid biopsy in advanced HCC improving individualized therapy and possible earlier identification of treatment responders

Early monocyte response following local ablation in hepatocellular carcinoma

Local ablative therapies are established treatment modalities in the treatment of early- and intermediate-stage hepatocellular carcinoma (HCC). Systemic effects of local ablation on circulating immune cells may contribute to patients’ response. Depending on their activation, myeloid cells are able to trigger HCC progression as well as to support anti-tumor immunity. Certain priming of monocytes may already occur while still in the circulation. By using flow cytometry, we analyzed peripheral blood monocyte cell populations from a prospective clinical trial cohort of 21 HCC patients following interstitial brachytherapy (IBT) or radiofrequency ablation (RFA) and investigated alterations in the composition of monocyte subpopulations and monocytic myeloid-derived suppressor cells (mMDSCs) as well as receptors involved in orchestrating monocyte function. We discovered that mMDSC levels increased following both IBT and RFA in virtually all patients. Furthermore, we identified varying alterations in the level of monocyte subpopulations following radiation compared to RFA. (A) Liquid biopsy liquid biopsy of circulating monocytes in the future may provide information on the inflammatory response towards local ablation as part of an orchestrated immune response

Whole blood microRNAs as potential biomarkers in post-operative early breast cancer patients

Background: microRNAs (miRNAs) are considered promising cancer biomarkers, showing high reliability, sensitivity and stability. Our study aimed to identify associations between whole blood miRNA profiles, presence of circulating tumor cells (CTCs) and clinical outcome in post-operative early breast cancer patients (EBC) to assess the utility of miRNAs as prognostic markers in this setting. Method: A total of 48 post-operative patients, recruited in frame of the SUCCESS A trial, were included in this retrospective study and tested with a panel of 8 miRNAs (miR-10b, −19a, − 21, − 22, −20a, − 127, − 155, −200b). Additional 17 female healthy donors with no previous history of cancer were included in the study as negative controls. Blood samples were collected at different time points (pre-adjuvant therapy, post-adjuvant therapy, 2 years follow up), total RNA was extracted and the relative concentration of each miRNA was measured by quantitative PCR and compared in patients stratified on blood collection time or CTC detection. Furthermore, we compared miRNA profiles of patients, for each time point separately, and healthy donors. CTCs were visualized and quantified with immunocytochemistry analysis. Data were analyzed using non-parametric statistical tests. Results: In our experimental system, miR-19a, miR-22 and miR-127 showed the most promising results, differentiating patients at different time points and from healthy controls, while miR-20a, miR-21 and miR-200b did not show any difference among the different groups. miR-10b and miR-155 were never detectable in our experimental system. With respect to patients’ clinical characteristics, we found a significant correlation between miR-200b and lymph node status and between miR-20a and tumor type. Furthermore, miR-127 correlated with the presence of CTCs. Finally, we found a borderline significance between Progression Free Survival and miR-19a levels. Conclusions_ This pilot study suggests that profiling whole blood miRNAs could help to better stratify post-operative EBC patients without any sign of metastasis to prevent later relapse or metastatic events

Quantifying HER-2 expression on circulating tumor cells by ACCEPT

Circulating tumor cells (CTCs) isolated from blood can be probed for the expression of treatment targets. Immunofluorescence is often used for both the enumeration of CTC and the determination of protein expression levels related to treatment targets. Accurate and reproducible assessment of such treatment target expression levels is essential for their use in the clinic. To enable this, an open source image analysis program named ACCEPT was developed in the EU-FP7 CTCTrap and CANCER-ID programs. Here its application is shown on a retrospective cohort of 132 metastatic breast cancer patients from which blood samples were processed by CellSearch (R) and stained for HER-2 expression as additional marker. Images were digitally stored and reviewers identified a total of 4084 CTCs. CTC's HER-2 expression was determined in the thumbnail images by ACCEPT. 150 of these images were selected and sent to six independent investigators to score the HER-2 expression with and without ACCEPT. Concordance rate of the operators' scoring results for HER2 on CTCs was 30% and could be increased using the ACCEPT tool to 51%. Automated assessment of HER-2 expression by ACCEPT on 4084 CTCs of 132 patients showed 8 (6.1%) patients with all CTCs expressing HER-2, 14 (10.6%) patients with no CTC expressing HER-2 and 110 (83.3%) patients with CTCs showing a varying HER-2 expression level. In total 1576 CTCs were determined HER-2 positive. We conclude that the use of image analysis enables a more reproducible quantification of treatment targets on CTCs and leads the way to fully automated and reproducible approaches

Detection of circulating tumor cells using manually performed immunocytochemistry (MICC) does not correlate with outcome in patients with early breast cancer – Results of the German SUCCESS-A- trial

Background: Recently, the prognostic significance of circulating tumor cells (CTCs) in primary breast cancer as assessed using the Food-and-Drug-Administration-approved CellSearch® system has been demonstrated. Here, we evaluated the prognostic relevance of CTCs, as determined using manually performed immunocytochemistry (MICC) in peripheral blood at primary diagnosis, in patients from the prospectively randomized multicenter SUCCESS-A trial (EudraCT2005000490-21). Methods: We analyzed 23 ml of blood from 1221 patients with node-positive or high risk node-negative breast cancer before adjuvant taxane-based chemotherapy. Cells were separated using a density gradient followed by epithelial cell labeling with the anti-cytokeratin-antibody A45-B/B3, immunohistochemical staining with new fuchsin, and cytospin preparation. All cytospins were screened for CTCs, and the cutoff for positivity was at least one CTC. The prognostic value of CTCs with regard to disease-free survival (DFS), distant disease-free survival (DDFS), breast-cancer-specific survival (BCSS), and overall survival (OS) was assessed using both univariate analyses applying the Kaplan–Meier method and log-rank tests, and using multivariate Cox regressions adjusted for other predictive factors. Results: In 20.6% of all patients (n = 251) a median of 1 (range, 1–256) CTC was detected, while 79.4% of the patients (n = 970) were negative for CTCs before adjuvant chemotherapy. A pT1 tumor was present in 40.% of patients, 4.8% had G1 grading and 34.6% were node-negative. There was no association between CTC positivity and tumor stage, nodal status, grading, histological type, hormone receptor status, Her2 status, menopausal status or treatment. Univariate survival analyses based on a median follow-up of 64 months revealed no significant differences between CTC-positive and CTC-negative patients with regard to DFS, DDFS, BCSS, or OS. This was confirmed by fully adjusted multivariate Cox regressions, showing that the presence of CTCs (yes/no) as assessed by MICC did not predict DFS, DDFS, BCSS or OS. Conclusions: We could not demonstrate prognostic relevance regarding CTCs that were quantified using the MICC method at the time of primary diagnosis in our cohort of early breast cancer patients. Further studies are necessary to evaluate if the presence of CTCs assessed using MICC has prognostic relevance, or can be used for risk stratification and treatment monitoring in adjuvant breast cancer. Trial registration The ClinicalTrial.gov registration ID of this prospectively randomized trial is NCT02181101; the (retrospective) registration date was June 2014 (study start date September 2005)