Circulating tumor cells (CTC) in blood are promising new biomarkers potentially useful for prognostic prediction and monitoring of therapies in patients with solid tumors including colon cancer. Moreover, CTC research opens a new avenue for understanding the biology of metastasis in patients with cancer. However, an in-depth investigation of CTCs is hampered by the very low number of these cells, especially in the blood of patients with colorectal cancer. Thus, the establishment of cell cultures and permanent cell lines from CTCs has become the most challenging task over the past year. Here, we describe, for the first time, the establishment of cell cultures and a permanent cell line from CTCs of one patient with colon cancer. The cell line designated CTC-MCC-41 has been cultured for more than one year, and the cells Ó2015 AACR.

Newly detected KRAS and/or EGFR mutations in plasma ctDNA from patients refractory to anti-EGFR treatment appear to derive from rare, pre-existing clones in the primary tumors. These rare clones were associated with shorter PFS in patients receiving anti-EGFR treatment. Multiple simultaneous mutations in KRAS and EGFR in the ctDNA and the decline in allele frequency after discontinuation of anti-EGFR therapy in a subset of patients suggest that several resistance mechanisms can co-exist and that relative clonal burdens may change over time. Monitoring treatment-induced genetic alterations by sequencing ctDNA could identify biomarkers for treatment screening in anti-EGFR-refractory patients.

Mitochondria are considered as the power‐generating units of the cell due to their key role in energy metabolism and cell signaling. However, mitochondrial components could be found in the extracellular space, as fragments or encapsulated in vesicles. In addition, this intact organelle has been recently reported to be released by platelets exclusively in specific conditions. Here, we demonstrate for the first time, that blood preparation with resting platelets, contains whole functional mitochondria in normal physiological state. Likewise, we show, that normal and tumor cultured cells are able to secrete their mitochondria. Using serial centrifugation or filtration followed by polymerase chain reaction‐based methods, and Whole Genome Sequencing, we detect extracellular full‐length mitochondrial DNA in particles over 0.22 µm holding specific mitochondrial membrane proteins. We identify these particles as intact cell‐free mitochondria using fluorescence‐activated cell sorting analysis, fluorescence microscopy, and transmission electron microscopy. Oxygen consumption analysis revealed that these mitochondria are respiratory competent. In view of previously described mitochondrial potential in intercellular transfer, this discovery could greatly widen the scope of cell‐cell communication biology. Further steps should be developed to investigate the potential role of mitochondria as a signaling organelle outside the cell and to determine whether these circulating units could be relevant for early detection and prognosis of various diseases.

Purpose: Circulating cell-free DNA (ccfDNA) is a valuable source of tumor material obtained from a simple blood sampling that enables noninvasive analysis of the tumor genome. Our goal was to carry out a multiparametric analysis of ccfDNA and evaluate its prognostic value by investigating the overall survival (OS) of 97 metastatic colorectal cancer patients (mCRC).Experimental Design: Qualitative parameters (determination of the main KRAS exon2 and BRAF V600E mutations) and quantitative parameters (total ccfDNA concentration, mutant ccfDNA concentration, the proportion of mutant ccfDNA, and ccfDNA integrity index) were determined simultaneously in a single run using a unique Q-PCR multimarker approach (100% success rate).Results: The median follow-up time was 36 months and median OS was 22 months. Patients showing high ccfDNA levels had significantly shorter OS (18.07 months vs. 28.5 months, P ¼ 0.0087). Moreover, multivariate analysis revealed that a high ccfDNA level is an independent prognostic factor (P ¼ 0.034). All ccfDNA parameters were of prognostic interest: patients with higher levels of mutant ccfDNA and higher mutation loads for the detected mutations had shorter OS (P ¼ 0.0089 and P ¼ 0.05, respectively). In addition, the level of ccfDNA fragmentation correlated positively with decreased OS in the exclusive KRAS/ BRAF-mutant cohort of patients (P ¼ 0.0052) and appeared as a strong independent prognostic factor (P ¼ 0.0072), whereas it was not significant in the exclusive KRAS/BRAF WT cohort of patients (P ¼ 0.67).Conclusions: Our data provide for the first time qualitative and quantitative evidence in favor of multiparametric ccfDNA analysis in mCRC patients for prognostic assessment.

The management of patients with colorectal cancer (CRC) and potentially resectable liver metastases (LM) requires quick assessment of mutational status and of response to pre-operative systemic therapy. In a prospective phase II trial (NCT01442935), we investigated the clinical validity of circulating tumor cell (CTC) and circulating tumor DNA (ctDNA) detection. CRC patients with potentially resectable LM were treated with first-line triplet or doublet chemotherapy combined with targeted therapy. CTC (Cellsearch®) and Kirsten RAt Sarcoma (KRAS) ctDNA (droplet digital polymerase chain reaction (PCR)) levels were assessed at inclusion, after 4 weeks of therapy and before LM surgery. 153 patients were enrolled. The proportion of patients with high CTC counts (≥3 CTC/7.5mL) decreased during therapy: 19% (25/132) at baseline, 3% (3/108) at week 4 and 0/57 before surgery. ctDNA detection sensitivity at baseline was 91% (N=42/46) and also decreased during treatment. Interestingly, persistently detectable KRAS ctDNA (p = 0.01) at 4 weeks was associated with a lower R0/R1 LM resection rate. Among patients who had a R0/R1 LM resection, those with detectable ctDNA levels before liver surgery had a shorter overall survival (p < 0.001). In CRC patients with limited metastatic spread, ctDNA could be used as liquid biopsy tool. Therefore, ctDNA detection could help to select patients eligible for LM resection.

Circulating tumor cells (CTCs) are important clinical indicators for prognosis and treatment efficacy. However, CTC investigation is hampered by their low number, making the establishment of permanent CTC lines very challenging. We derived and characterized nine CTC lines using blood samples from a patient with metastatic colorectal cancer collected before and after chemotherapy and targeted therapy, and during cancer progression. These cell lines displayed an intermediate epithelial/mesenchymal phenotype, stem-cell like characteristics, angiogenesis potential, an osteomimetic signature and the capacity to escape from the immune system. Moreover, they showed changes in mRNA and protein expression (e.g., DEFA6, ABCB1 and GAL), whereas analysis of chromosomal copy number aberrations revealed no significant variation over time. These data indicate that although CTC lines derived from sequential blood samples during therapy have common traits, treatment-resistant CTC clones with distinct phenotypic characteristics are selected over time.

(1) Background: Oxaliplatin is among the most neurotoxic anticancer drugs. Little data are available on the long-term prevalence and consequences of chemotherapy-induced peripheral neuropathy (CIPN), even though the third largest population of cancer survivors is made up of survivors of colorectal cancer. (2) Methods: A multicenter, cross-sectional study was conducted in 16 French centers to assess the prevalence of CIPN, as well as its consequences (neuropathic pain, anxiety, depression, and quality of life) in cancer survivors during the 5 years after the end of adjuvant oxaliplatin chemotherapy. (3) Results: Out of 406 patients, the prevalence of CIPN was 31.3% (95% confidence interval: 26.8–36.0). Little improvement in CIPN was found over the 5 years, and 36.5% of patients with CIPN also had neuropathic pain. CIPN was associated with anxiety, depression, and deterioration of quality of life. None of the patients with CIPN were treated with duloxetine (recommendation from American Society of Clinical Oncology), and only 3.2%, 1.6%, and 1.6% were treated with pregabalin, gabapentin, and amitriptyline, respectively. (4) Conclusions: Five years after the end of chemotherapy, a quarter of patients suffered from CIPN. The present study showed marked psychological distress and uncovered a failure in management in these patients.

Purpose: Circulating tumor DNA (ctDNA) has been suggested as a major prognostic factor in resected stage-III colon cancer. We analyzed ctDNA of patients randomized in the phase III IDEA-France trial. Experimental Design: ctDNA was tested for WIF1 and NPY by droplet digital PCR with method developed and validated for colorectal cancer. Disease-free survival (DFS) and overall survival (OS) were analyzed via multivariable analysis in patients with ctDNA samples and in sub-groups according to treatment duration (3/6 months) and disease stage (high/low-risk stage III). Results: Of 2,010 randomized patients, 1,345 had available ctDNA samples (1,017 collected both post-surgery and pre-chemotherapy). More Eastern Cooperative Oncology Group performance status (ECOG PS) of 0 (78% versus 69%) and T4 and/or N2 (40% versus 36%) were observed in patients studied (n = 1017) versus not analyzed (n = 993). There were 877 ctDNA-negative (86.2%) and 140 ctDNA-positive (13.8%) patients; their baseline characteristics were similar. With a median follow-up of 6.6 years, the 3-year DFS rate was 66.39% for ctDNA-positive patients and 76.71% for ctDNA-negative patients (P = 0.015). ctDNA was confirmed as an independent prognostic marker for DFS (adjusted HR = 1.55, 95% CI 1.13–2.12, P = 0.006) and OS (HR = 1.65, 95% CI 1.12–2.43, P = 0.011). ctDNA was prognostic in patients treated for 3 months and with T4 and/or N2 tumors, but not in those treated for 6 months and with T1–3/N1 tumors. Conclusions: In this first ctDNA assessment of a large series of patients with stage III colon cancer enrolled in phase III trial, post-surgery ctDNA was found in 13.8% of them and was confirmed as an independent prognostic marker. See related commentary by Bent and Kopetz, p. 5449