The detection of the BRAF V600E mutation in melanoma samples is used to select patients who should respond to BRAF inhibitors. Different techniques are routinely used to determine BRAF status in ...clinical samples. However, low tumor cellularity and tumor heterogeneity can affect the sensitivity of somatic mutation detection. Digital PCR (dPCR) is a next-generation genotyping method that clonally amplifies nucleic acids and allows the detection and quantification of rare mutations. Our aim was to evaluate the clinical routine performance of a new dPCR-based test to detect and quantify BRAF mutation load in 47 paraffin-embedded cutaneous melanoma biopsies. We compared the results obtained by dPCR with high-resolution melting curve analysis and pyrosequencing or with one of the allele-specific PCR methods available on the market. dPCR showed the lowest limit of detection. dPCR and allele-specific amplification detected the highest number of mutated samples. For the BRAF mutation load quantification both dPCR and pyrosequencing gave similar results with strong disparities in allele frequencies in the 47 tumor samples under study (from 0.7% to 79% of BRAF V600E mutations/sample). In conclusion, the four methods showed a high degree of concordance. dPCR was the more-sensitive method to reliably and easily detect mutations. Both pyrosequencing and dPCR could quantify the mutation load in heterogeneous tumor samples.
It remains presently unclear whether disease progression in colorectal carcinoma (CRC), from early, to invasive and metastatic forms, is associated to a gradual increase in genetic instability and to ...a scheme of sequentially occurring Copy Number Alterations (CNAs).
In this work we set to determine the existence of such links between CRC progression and genetic instability and searched for associations with patient outcome. To this aim we analyzed a set of 162 Chromosomal Instable (CIN) CRCs comprising 131 primary carcinomas evenly distributed through stage 1 to 4, 31 metastases and 14 adenomas by array-CGH. CNA profiles were established according to disease stage and compared. We, also, asked whether the level of genomic instability was correlated to disease outcome in stage 2 and 3 CRCs. Two metrics of chromosomal instability were used; (i) Global Genomic Index (GGI), corresponding to the fraction of the genome involved in CNA, (ii) number of breakpoints (nbBP).
Stage 1, 2, 3 and 4 tumors did not differ significantly at the level of their CNA profiles precluding the conventional definition of a progression scheme based on increasing levels of genetic instability. Combining GGI and nbBP,we classified genomic profiles into 5 groups presenting distinct patterns of chromosomal instability and defined two risk classes of tumors, showing strong differences in outcome and hazard risk (RFS: p = 0.012, HR = 3; OS: p < 0.001, HR = 9.7). While tumors of the high risk group were characterized by frequent fractional CNAs, low risk tumors presented predominantly whole chromosomal arm CNAs. Searching for CNAs correlating with negative outcome we found that losses at 16p13.3 and 19q13.3 observed in 10% (7/72) of stage 2-3 tumors showed strong association with early relapse (p < 0.001) and death (p < 0.007, p < 0.016). Both events showed frequent co-occurrence (p < 1x10-8) and could, therefore, mark for stage 2-3 CRC susceptible to negative outcome.
Our data show that CRC disease progression from stage 1 to stage 4 is not paralleled by increased levels of genetic instability. However, they suggest that stage 2-3 CRC with elevated genetic instability and particularly profiles with fractional CNA represent a subset of aggressive tumors.
There is growing evidence that the immune system may prevent the occurrence, growth and metastatic diffusion of colorectal cancer (CRC). The role played by the adaptive immune response at the tumour ...site is critical in the balance between tumour invasion and defence against cancer. Recent data have shown that the evaluation of this immune response may help to define the prognosis and possibly the treatment of localised CRC as well as metastatic CRC. Tumour infiltrates with T cells (CD3+), cytotoxic T cells (CD8+) and memory T cells (CD45RO+) are the immune parameters most consistently and strongly associated with good clinical outcome in CRC. Several scoring systems have been developed, including the Immunoscore®, based on the immunohistochemical determination with a digital image analysis system of the density of CD3+ and CD8+ lymphocytes in the centre and the invasive margin of the tumour. This review will focus on the different immunoscoring systems developed in CRC, their performance, their limitations and their potential for improving patients' care in the future.
•The immune system helps preventing the occurrence, growth and diffusion of CRC.•Different scoring systems evaluating lymphocyte infiltrates in CRC have been developed.•The Immunoscore® system relies on immunohistochemistry and image analysis.•Its prognostic performance has been shown in primary and metastatic CRC.•Its predictive performance needs more prospective data.
In human carcinomas, especially breast cancer, chromosome arm 8p is frequently involved in complex chromosomal rearrangements that combine amplification at 8p11-12, break in the 8p12-21 region, and ...loss of 8p21-ter. Several studies have identified putative oncogenes in the 8p11-12 amplicon. However, discrepancies and the lack of knowledge on the structure of this amplification lead us to think that the actual identity of the oncogenes is not definitively established. We present here a comprehensive study combining genomic, expression, and chromosome break analyses of the 8p11-12 region in breast cell lines and primary breast tumors. We show the existence of four amplicons at 8p11-12 using array comparative genomic hybridization. Gene expression analysis of 123 samples using DNA microarrays identified 14 genes significantly overexpressed in relation to amplification. Using fluorescence in situ hybridization analysis on tissue microarrays, we show the existence of a cluster of breakpoints spanning a region just telomeric to and associated with the amplification. Finally, we show that 8p11-12 amplification has a pejorative effect on survival in breast cancer.
Two-stage hepatectomy of bilobar colorectal liver metastases is widely used and shows encouraging survival results. However, the risk of dropout after the first stage remains high and is associated ...with poor survival. The objective of our study was to evaluate the factors associated with long-term survival based on the pathologic response to preoperative systemic chemotherapy in colorectal liver metastases patients who underwent two-stage hepatectomy.
The pathologic response to preoperative chemotherapy and its effect on second-stage completion and survival were retrospectively evaluated in 67 patients treated between 2003 and 2013.
A total of 56 patients underwent two-stage hepatectomy for initially nonresectable colorectal liver metastases. Chemotherapy was combined with a biotherapy in 32 cases. The tumor regression grade, modified tumor regression grade, and Blazer grade were used to classify patients as responders (tumor regression grade and modified tumor regression grade 1–3, Blazer 0–1) or nonresponders (tumor regression grade and modified tumor regression grade 4–5, Blazer 2) after the first stage. Tumor response in the three classifications was associated with second-stage completion (tumor regression grade 1–3: OR = 4.01, 95% CI: 1.12–14.36, P = .033; modified tumor regression grade 1–3: OR = 3.8, 95% CI: 1.13–12.6, P = .03; Blazer 0–1: OR = 5.45, 95% CI: 1.66–17.85, P = .005). Triple chemotherapy was also associated with responders. The median overall survival of responders was significantly higher (Blazer 0–1: 42.9 months versus Blazer 2: 20.1 months, P = .018; tumor regression grade 1–3: 42.9 months versus tumor regression grade 4–5: 25.1 months, P = .04).
A pathologic response to chemotherapy is associated with second-stage completion and longer survival. Further studies are needed to achieve the early identification of patients for whom the benefit of the second surgical stage is less straightforward.
Rectal cancer is a common and serious disease in the Western hemisphere. Optimal treatment of rectal cancer involves a multidisciplinary approach, with collaboration required between radiologists, ...oncologists, surgeons, and pathologists to achieve local control and decrease the rate of recurrence. Several studies have been published that show the ability to accurately stage rectal cancer with magnetic resonance (MR) imaging. Moreover, advances in preoperative therapies require accurate preoperative staging with MR imaging to select those patients who may benefit from more intensive treatment, without subjecting those who will not benefit to unnecessary treatment. As we enter an era of individualized patient care, stratified according to the risk of both local and distant failure, imaging takes on the same importance as the tumor type and genetic susceptibility. MR imaging is now an essential tool to enable the oncology team to make appropriate treatment decisions. However, rectal cancer evaluation with MR imaging remains a challenge in the hands of nonexperts. This article describes a mnemonic device, "DISTANCE," to enable a systematic approach to the interpretation of MR images, thereby enabling all the clinically relevant features to be adequately assessed: DIS, for Distance from the Inferior part of the tumor to the transitional Skin; T, for T staging; A, for Anal complex; N, for Nodal staging; C, for Circumferential resection margin; and E, for Extramural vascular invasion.
To improve treatment efficacy, we decided to simultaneously target HER1 and HER2 with trastuzumab and cetuximab. Following promising preclinical results, we conducted a phase 1-2 trial in advanced ...pancreatic cancer patients after first-line gemcitabine-based chemotherapy failure. In this single-arm, non-randomized, multicenter trial, patients received weekly cetuximab (400mg/m², then 250mg/m²). They were sequentially included in two trastuzumab dose levels: 3.0 or 4.0mg/kg, then 1.5 or 2.0mg/kg/weekly. Endpoints were the objective response rate, safety, progression-free (PFS) and overall survival (OS). During phase 1 (n=10 patients), toxicities were evenly distributed except for skin toxicities that frequently caused compliance issues. The higher dose level was defined as the trastuzumab recommended dose. During phase 2 (n=39 patients), toxicities were mainly cutaneous reactions and asthenia. No objective response was observed. Nine patients were stabilized but arrested treatment due to toxicity. Median PFS was 1.8 months (95%CI: 1.7-2.0 months) and median OS was 4.6 months (95%CI: 2.7-6.6 months). Both were positively correlated with skin toxicity severity (P=0.027 and P=0.001, respectively). Conventional phase 1 dose-escalation schedules are unsuitable for targeted therapies because most cutaneous toxicities are not considered dose-limiting toxicities. The compliance issues caused by skin toxicities were particularly detrimental because of the toxicity-response correlation.
Triple-negative breast cancers (TNBCs) are characterised by lack of expression of hormone receptors and epidermal growth factor receptor 2 (HER-2). As they frequently express epidermal growth factor ...receptors (EGFRs), anti-EGFR therapies are currently assessed for this breast cancer subtype as an alternative to treatments that target HER-2 or hormone receptors. Recently, EGFR-activating mutations have been reported in TNBC specimens in an East Asian population. Because variations in the frequency of EGFR-activating mutations in East Asians and other patients with lung cancer have been described, we evaluated the EGFR mutational profile in tumour samples from European patients with TNBC.
We selected from a DNA tumour bank 229 DNA samples isolated from frozen, histologically proven and macrodissected invasive TNBC specimens from European patients. PCR and high-resolution melting (HRM) analyses were used to detect mutations in exons 19 and 21 of EGFR. The results were then confirmed by bidirectional sequencing of all samples.
HRM analysis allowed the detection of three EGFR exon 21 mutations, but no exon 19 mutations. There was 100% concordance between the HRM and sequencing results. The three patients with EGFR exon 21 abnormal HRM profiles harboured the rare R836R SNP, but no EGFR-activating mutation was identified.
This study highlights variations in the prevalence of EGFR mutations in TNBC. These variations have crucial implications for the design of clinical trials involving anti-EGFR treatments in TNBC and for identifying the potential target population.
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.
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