Assessment of KRAS status is mandatory in patients with metastatic colorectal cancer (mCRC) before applying targeted therapy. We describe here a blinded prospective study to compare KRAS and BRAF mutation status data obtained from the analysis of tumor tissue by routine gold-standard methods and of plasma DNA using a quantitative PCR-based method specifically designed to analyze circulating cell-free DNA (cfDNA). The mutation status was determined by both methods from 106 patient samples. cfDNA analysis showed 100% specificity and sensitivity for the BRAF V600E mutation. For the seven tested KRAS point mutations, the method exhibited 98% specificity and 92% sensitivity with a concordance value of 96%. Mutation load, expressed as the proportion of mutant alleles in cfDNA, was highly variable (0.5-64.1%, median 10.5%) among mutated samples. CfDNA was detected in 100% of patients with mCRC. This study shows that liquid biopsy through cfDNA analysis could advantageously replace tumor-section analysis and expand the scope of personalized medicine for patients with cancer.

To our knowledge, this is the first comprehensive study on the influence of several pre-analytical and demographic parameters that could be a source of variability in the quantification of nuclear and mitochondrial circulating DNA (NcirDNA and McirDNA). We report data from a total of 222 subjects, 104 healthy individuals and 118 metastatic colorectal cancer (mCRC) patients. Approximately 50,000 and 3,000-fold more mitochondrial than nuclear genome copies were found in the plasma of healthy individuals and mCRC patients, respectively. In healthy individuals, NcirDNA concentration was statistically influenced by age (p = 0.009) and gender (p = 0.048). Multivariate analysis with logistic regression specified that age over 47 years-old was predictive to have higher NcirDNA concentration (OR = 2.41; p = 0.033). McirDNA concentration was independent of age and gender in healthy individuals. In mCRC patients, NcirDNA and McirDNA levels were independent of age, gender, delay between food intake and blood collection, and plasma aspect, either with univariate or multivariate analysis. Nonetheless, ad hoc study suggested that menopause and blood collection time might have tendency to influence cirDNA quantification. In addition, high significant statistical differences were found between mCRC patients and healthy individuals for NcirDNA (p < 0.0001), McirDNA (p < 0.0001) and McirDNA/NcirDNA ratio (p < 0.0001). NcirDNA and McirDNA levels do not vary in the same way with regards to cancer vs healthy status, pre-analytical and demographic factors.

Evidence suggests that lean body mass (LBM) may be useful to normalize chemotherapy doses. Data from one prospective and one retrospective study were used to determine if the highest doses of oxaliplatin/kg LBM within FOLFOX regimens would be associated with dose‐limiting toxicity (DLT) in colon cancer patients. Toxicity over four cycles was graded according to NCI Common Toxicity Criteria V2 or V3 (Common Terminology Criteria for Adverse Events, National Cancer Institute, Bethesda, MD). Muscle tissue was measured by computerized tomography (CT) and used to evaluate the LBM compartment of the whole body. In prospective randomized clinical trials conducted in France (n = 58), for patients given FOLFOX‐based regimens according to body surface area, values of oxaliplatin/kg LBM were highly variable, ranging from 2.55 to 6.6 mg/kg LBM. A cut point of 3.09 mg oxaliplatin/kg LBM for developing toxicity was determined by Receiver Operating Characteristic (ROC) analysis, below this value 0/17 (0.0%) of patients experienced DLT; in contrast above this value 18/41 (44.0%) of patients were dose reduced or had treatment terminated owing to toxicity (≥Grade 3 or neuropathy ≥Grade 2); for 9/41 the DLT was sensory neuropathy. These findings were validated in an independent cohort of colon cancer patients (n = 80) receiving FOLFOX regimens as part of standard care, in Canada. Low LBM is a significant predictor of toxicity and neuropathy in patients administered FOLFOX‐based regimens using conventional body surface area (BSA) dosing.

The antiestrogen tamoxifen, a major endocrine therapy of estrogen receptor (ER)-positive breast cancer, is nevertheless inefficient in 30 to 40% of cases for unknown reasons. We retrospectively studied 50 ER-positive primary breast carcinomas. All of the patients had received tamoxifen as the only adjuvant therapy. They were divided into two groups depending on whether they relapsed within 5 years (16 tamoxifen-resistant cases) or did not relapse within 5 years (34 tamoxifen-sensitive cases). The expression of total ER␤ protein, and of ER␤cx protein, was estimated anonymously in formalin-fixed, paraffin-embedded tumor sections, by using specific antibodies and quantifiying nuclear immunostaining with a computer image analyzer. All of the tumors were found to be HER-2/neu-negative by immunohistochemistry.Univariate analysis showed that Scarff-Bloom-Richardsson grade modified by Elston (SBR grade; P < 0.001), tumor size (P ‫؍‬ 0.042), and MIB-1 proliferation index (P ‫؍‬ 0.02) were significantly higher in tamoxifen-resistant tumors. A low level of total ER␤, whether in percentage of positive cells or in quantitative immunocytochemical (QIC) score, was also associated with tamoxifen resistance (P ‫؍‬ 0.004). ER␤cx expression and lymph node status were similar between the two groups. The expression of ER␤ in the total population was positively correlated with ER␤cx (r ‫؍‬ 0.63, P < 0.001), and was independent of the other parameters. In a multivariate analysis, ER␤ expression was the most important variable (P ‫؍‬ 0.001), followed by SBR grade (I؉II versus III; P ‫؍‬ 0.008), and MIB-1 (P ‫؍‬ 0.016).To conclude, tamoxifen resistance is associated with classical variables of aggressive tumors (high SBR grade, proliferation index, and tumor size) but not with node invasiveness. Low ER␤ level is an additional independent marker, better than ER␣ level, to predict tamoxifen resistance.

The clinical management of metastatic colorectal cancer (mCRC) faces major challenges. Here, we show that nilotinib, a clinically approved drug for chronic myeloid leukaemia, strongly inhibits human CRC cell invasion in vitro and reduces their metastatic potential in intrasplenic tumour mouse models. Nilotinib acts by inhibiting the kinase activity of DDR1, a receptor tyrosine kinase for collagens, which we identified as a RAS‐independent inducer of CRC metastasis. Using quantitative phosphoproteomics, we identified BCR as a new DDR1 substrate and demonstrated that nilotinib prevents DDR1‐mediated BCR phosphorylation on Tyr177, which is important for maintaining β‐catenin transcriptional activity necessary for tumour cell invasion. DDR1 kinase inhibition also reduced the invasion of patient‐derived metastatic and circulating CRC cell lines. Collectively, our results indicate that the targeting DDR1 kinase activity with nilotinib may be beneficial for patients with mCRC.