Final Report Summary - DISSECT (Disseminating tumor cells as novel biomarkers: Dissecting the metastatic cascade in cancer patients)
Current tumour staging is mainly based on determining local extension, lymph node involvement and distant metastasis. However, even high resolution imaging procedures are not sensitive enough to detect early tumour cell dissemination. Therefore, we have developed ultrasensitive methods not only to trace but also to further characterize disseminated and circulating tumour cells (DTC, CTC). Within the DISSECT project our team has addressed (a) whether there is increased tumour cell release during therapeutic interventions and what happens with these cells during chemo- and radiotherapy and (b) how new approaches to enrich, detect, isolate and characterize CTCs, circulating DNA, circulating mRNS and micro-RNA can be developed. Besides the development of a comprehensive platform for circulating “liquid biopsy” biomarkers, the major goal of the DISSECT project was to investigate the influence of current diagnostic procedures and therapeutic interventions on tumour cell dissemination in order to improve the clinical management of cancer patients.
We performed studies to monitor tumour cell spread during diagnostic biopsies, surgical intervention and radiotherapy. Our key finding was the significant release of CTCs after diagnostic needle biopsies of the prostate in patients with histologically confirmed prostate cancer. Follow up studies on the potential clinical relevance of this finding are ongoing.
In addition, the influence of chemotherapy on CTC release was studied in breast cancer patients who received neoadjuvant chemotherapy within multi-center clinical trials. Here the key finding was that high CTC counts predicted an unfavorable clinical outcome independently from the pathological complete response (pCR) of the primary tumour which is currently the “gold standard” to assess response to neoadjuvant therapy. This finding underlines the importance of blood-borne tumour cell dissemination for metastatic progression and demonstrates that the limitation to focus solely on the primary tumour response.
Besides chemotherapy targeted therapies have broadened the spectrum of systemic therapies in cancer patients. In DISSECT, HER2 expression of CTC was analyzed as precondition for anti-HER therapies. Moreover, to investigated the relevance of mutations of the PIK3CA gene on CTCs as resistance mechanisms against HER2-targeting therapies. Besides breast cancer, we extended our HER2-studies to patients suffering from urinary bladder cancer, indicating a marked heterogeneity of HER2 expression on CTCs.
To obtain insights into the molecular biology of CTCs, we performed an in-depth characterization of CTCs at the genomic, transcriptomic and protein level. To assess the genome, we established and applied protocols for WGA of DNA isolated from single CTC followed by investigation of copy number aberrations, gene amplification (HER2, EGFR) and mutations (K-RAS, B-RAF and PIK3CA) including array-CGH and next generation sequencing. Comparative evaluation of primary tumour areas and CTCs obtained at the time of metastasis revealed evidence that heterogeneous pathways of molecular evolution can lead to metastatic progression. At the mRNA level, we could show an 84-gene signature of individual CTCs in breast and prostate cancer and identified pathways relevant to survival and selection by systemic therapies, such as the expression of the androgen receptor variant 7 in prostate cancer. At the protein level, we established sensitive and specific immunostaining procedures to analyse the expression of the estrogen receptor and the immune checkpoint inhibitor ligand PD-L1 in breast cancer patients or PSMA in prostate cancer, and showed a marked discrepancy between protein expression in the primary tumour and CTCs at the time of metastatic relapse, demonstrating the need for re-staging of metastatic disease for improved stratification of cancer therapies tailored to the actual characteristics of the tumour burden.
In depth characterization is limited by the low concentration and yield of CTCs. To further improve CTC detection, we developed the first in vivo capture system for CTCs and validated it in patients with lung cancer. Moreover, CTCs that undergo an epithelial-mesenchymal transition (EMT) usually are missed by current detection methods focusing on epithelial markers. We therefore explored new proteins such as plastin-3 that are not downregulated during EMT in order to detect also EMT-like CTC.
Before the DISSECT project, the characterization of CTCs was restricted to descriptive studies. For functional testing of CTC, we developed the first xenograft mouse model in collaboration with Dr. Andreas Trumpp (Heidelberg) and showed that CTC isolated from breast cancer patients contain subfractions of metastasis-initiating cells characterized by EpCAM, CD44 and cMET expression. As unique models for functional CTC studies, we further established permanent cell lines from CTCs of patients with colorectal and breast cancer. Interestingly, these cell lines expressed epithelial markers such as EpCAM or keratins, suggesting that the epithelial or intermediate (E/M) phenotype might be important for cell growth.
Besides CTCs we established and validated also methods for the improved detection, characterization and quantification of circulating tumour DNA, mRNA and miRNA in blood plasma and serum collected mainly from patients with breast, colorectal and prostate cancer. More recently, circulating tumour-derived exosomes came into the focus of our liquid biopsy research. For example, in clinical studies on patients with breast and ovarian cancer we could show that exosomal microRNAs have diagnostic and prognostic relevance. Moreover, in collaboration with David Lyden (New York) and other colleagues we demonstrated, for the first time, that tumour-derived exosomes that are taken up by organ-specific cells might help to prepare the pre-metastatic niche in various types of solid tumours.
In conclusion, the DISSECT project has shown that in depth characterization of CTCs/DTCs and tumour-cell derived products in cancer patients can provide unique insights into human cancer biology with important implications for the improvement of existing diagnostic and therapeutic procedures as well as for the development of new approaches to reduce tumour spread and metastasis as primary cause of cancer-related death.
We performed studies to monitor tumour cell spread during diagnostic biopsies, surgical intervention and radiotherapy. Our key finding was the significant release of CTCs after diagnostic needle biopsies of the prostate in patients with histologically confirmed prostate cancer. Follow up studies on the potential clinical relevance of this finding are ongoing.
In addition, the influence of chemotherapy on CTC release was studied in breast cancer patients who received neoadjuvant chemotherapy within multi-center clinical trials. Here the key finding was that high CTC counts predicted an unfavorable clinical outcome independently from the pathological complete response (pCR) of the primary tumour which is currently the “gold standard” to assess response to neoadjuvant therapy. This finding underlines the importance of blood-borne tumour cell dissemination for metastatic progression and demonstrates that the limitation to focus solely on the primary tumour response.
Besides chemotherapy targeted therapies have broadened the spectrum of systemic therapies in cancer patients. In DISSECT, HER2 expression of CTC was analyzed as precondition for anti-HER therapies. Moreover, to investigated the relevance of mutations of the PIK3CA gene on CTCs as resistance mechanisms against HER2-targeting therapies. Besides breast cancer, we extended our HER2-studies to patients suffering from urinary bladder cancer, indicating a marked heterogeneity of HER2 expression on CTCs.
To obtain insights into the molecular biology of CTCs, we performed an in-depth characterization of CTCs at the genomic, transcriptomic and protein level. To assess the genome, we established and applied protocols for WGA of DNA isolated from single CTC followed by investigation of copy number aberrations, gene amplification (HER2, EGFR) and mutations (K-RAS, B-RAF and PIK3CA) including array-CGH and next generation sequencing. Comparative evaluation of primary tumour areas and CTCs obtained at the time of metastasis revealed evidence that heterogeneous pathways of molecular evolution can lead to metastatic progression. At the mRNA level, we could show an 84-gene signature of individual CTCs in breast and prostate cancer and identified pathways relevant to survival and selection by systemic therapies, such as the expression of the androgen receptor variant 7 in prostate cancer. At the protein level, we established sensitive and specific immunostaining procedures to analyse the expression of the estrogen receptor and the immune checkpoint inhibitor ligand PD-L1 in breast cancer patients or PSMA in prostate cancer, and showed a marked discrepancy between protein expression in the primary tumour and CTCs at the time of metastatic relapse, demonstrating the need for re-staging of metastatic disease for improved stratification of cancer therapies tailored to the actual characteristics of the tumour burden.
In depth characterization is limited by the low concentration and yield of CTCs. To further improve CTC detection, we developed the first in vivo capture system for CTCs and validated it in patients with lung cancer. Moreover, CTCs that undergo an epithelial-mesenchymal transition (EMT) usually are missed by current detection methods focusing on epithelial markers. We therefore explored new proteins such as plastin-3 that are not downregulated during EMT in order to detect also EMT-like CTC.
Before the DISSECT project, the characterization of CTCs was restricted to descriptive studies. For functional testing of CTC, we developed the first xenograft mouse model in collaboration with Dr. Andreas Trumpp (Heidelberg) and showed that CTC isolated from breast cancer patients contain subfractions of metastasis-initiating cells characterized by EpCAM, CD44 and cMET expression. As unique models for functional CTC studies, we further established permanent cell lines from CTCs of patients with colorectal and breast cancer. Interestingly, these cell lines expressed epithelial markers such as EpCAM or keratins, suggesting that the epithelial or intermediate (E/M) phenotype might be important for cell growth.
Besides CTCs we established and validated also methods for the improved detection, characterization and quantification of circulating tumour DNA, mRNA and miRNA in blood plasma and serum collected mainly from patients with breast, colorectal and prostate cancer. More recently, circulating tumour-derived exosomes came into the focus of our liquid biopsy research. For example, in clinical studies on patients with breast and ovarian cancer we could show that exosomal microRNAs have diagnostic and prognostic relevance. Moreover, in collaboration with David Lyden (New York) and other colleagues we demonstrated, for the first time, that tumour-derived exosomes that are taken up by organ-specific cells might help to prepare the pre-metastatic niche in various types of solid tumours.
In conclusion, the DISSECT project has shown that in depth characterization of CTCs/DTCs and tumour-cell derived products in cancer patients can provide unique insights into human cancer biology with important implications for the improvement of existing diagnostic and therapeutic procedures as well as for the development of new approaches to reduce tumour spread and metastasis as primary cause of cancer-related death.