Clinical utility of circulating DNA analysis for rapid detection of actionable mutations to select metastatic colorectal patients for anti-EGFR treatment.

BACKGROUND: While tumor-tissue remains the 'gold standard' for genetic analysis in cancer patients, it is challenged with the advent of circulating cell-free tumor DNA (ctDNA) analysis from blood samples. Here, we broaden our previous study on the clinical validation of plasma DNA in metastatic colorectal cancer patients, by evaluating its clinical utility under standard management care. PATIENTS AND METHODS: Concordance and data turnaround-time of ctDNA when compared with tumor-tissue analysis were studied in a real-time blinded prospective multicenter clinical study (n = 140 metastatic colorectal patients). Results are presented according to STARD criteria and were discussed in regard with clinical outcomes of patients. RESULTS: Much more mutations were found by ctDNA analysis: 59%, 11.8% and 14.4% of the patients were found KRAS, NRAS and BRAF mutant by ctDNA analysis instead of 44%, 8.8% and 7.2% by tumor-tissue analysis. Median tumor-tissue data turnaround-time was 16 days while 2 days for ctDNA analysis. Discordant samples analysis revealed that use of biopsy, long delay between tumor-tissue and blood collection and resection of the tumor at time of blood draw, tumor site, or type of tissue analyzed seem to affect concordance. Altogether, the clinical data with respect to the anti-epidermal growth factor receptor response (RAS status) and the prognosis (BRAF status) of those discordant patients do not appear contradictory to the mutational status as determined by plasma analysis. Lastly, we present the first distribution profile of the RAS and BRAF hotspot mutations as determined by ctDNA analysis (n = 119), revealing a high proportion of patients with multiple mutations (45% of the population and up to 5 mutations) and only 24% of WT scored patients for both genes. Mutation profile as determined from ctDNA analysis with using various detection thresholds highlights the importance of the test sensitivity. CONCLUSION: Our study showed that ctDNA could replace tumor-tissue analysis, and also clinical utility of ctDNA analysis by considerably reducing data turnaround time.

Sorafenib and irinotecan (NEXIRI) as second- or later-line treatment for patients with metastatic colorectal cancer and KRAS-mutated tumours: a multicentre Phase I/II trial.

BACKGROUND: This trial evaluated the feasibility and efficacy of combined sorafenib and irinotecan (NEXIRI) as second- or later-line treatment of patients with KRAS-mutated metastatic colorectal cancer (mCRC), who had progressed after irinotecan-based chemotherapy. METHODS: In Phase I, in a 3+3 dose escalation schedule, patients received irinotecan (125, 150 or 180 mg m(-2) every 2 weeks), in combination with 400 mg sorafenib b.d. The primary end point was the maximum-tolerated dose of irinotecan. In Phase II, the primary end point was disease control rate (DCR). Secondary end points were progression-free survival (PFS), overall survival (OS) and toxicity. RESULTS: Phase I included 10 patients (median age 63 (49-73)); no dose-limiting toxicity was seen. In Phase II, 54 patients (median age 60 (43-80) years) received irinotecan 180 mg m(-)(2) every 2 weeks with sorafenib 400 mg b.d. Nine patients (17%) remained on full-dose sorafenib. The DCR was 64.9% (95% CI, 51-77). Median PFS and OS were 3.7 (95% CI, 3.2-4.7) and 8.0 (95% CI, 4.8-9.7) months, respectively. Toxicities included Grade 3 diarrhoea (37%), neutropenia (18%), hand-foot syndrome (13%) and Grade 4 neutropenia (17%). CONCLUSION: The NEXIRI regimen showed promising activity as second- or later-line treatment in this heavily pretreated mCRC population (ClinicalTrials.gov NCT00989469).

A large-scale candidate gene approach identifies SNPs in SOD2 and IL13 as predictive markers of response to preoperative chemoradiation in rectal cancer.

Neoadjuvant radiochemotherapy followed by total mesorectal excision is now the standard treatment for locally advanced rectal cancer. However, tumor response to chemoradiation varies widely among individuals and cannot be determined before the final pathologic evaluation. The aim of this study was to identify germline genetic markers that could predict sensitivity or resistance to preoperative radiochemotherapy (RT-CT) in rectal cancer. We evaluated the predictive value of 128 single-nucleotide polymorphisms (SNPs) in 71 patients preoperatively treated by RT-CT. The selected SNPs were distributed over 76 genes that are involved in various cellular processes such as DNA repair, apoptosis, proliferation or immune response. The SNPs superoxide dismutase 2 (SOD2) rs4880 (P=0.005) and interleukin-13 (IL13) rs1800925 (P=0.0008) were significantly associated with tumor response to chemoradiation. These results reinforce the idea of using germline polymorphisms for personalized treatment.

[Considerations for normalisation of RT-qPCR in oncology]. / La RT-qPCR en oncologie: considérations pour la normalisation.

Gene expression analysis has many applications in the management of cancer, including diagnosis, prognosis, and therapeutic care. In this context, the reverse transcription quantitative polymerase chain reaction (RT-qPCR) has become the "gold standard" for mRNA quantification. However, this technique involves several critical steps such as RNA extraction, cDNA synthesis, quantitative PCR, and analysis, which all can be source of variation. To obtain biologically meaningful results, data normalisation is required to correct sample-to-sample variations that may be introduced during this multistage process. Normalisation can be carried out against a housekeeping gene, total RNA mass, or cell number. Careful choice of the normalization method is crucial, as any variation in the reference will introduce errors in the quantification of mRNA transcripts. By reviewing the different methods available and their related problems, the aim of this article is to provide recommendations for the set up of an appropriate normalisation strategy for RT-qPCR data in oncology.

Functional categories of TP53 mutation in colorectal cancer: results of an International Collaborative Study.

BACKGROUND: Loss of TP53 function through gene mutation is a critical event in the development and progression of many tumour types including colorectal cancer (CRC). In vitro studies have found considerable heterogeneity amongst different TP53 mutants in terms of their transactivating abilities. The aim of this work was to evaluate whether TP53 mutations classified as functionally inactive (< or=20% of wildtype transactivation ability) had different prognostic and predictive values in CRC compared with mutations that retained significant activity. MATERIALS AND METHODS: TP53 mutations within a large, international database of CRC (n = 3583) were classified according to functional status for transactivation. RESULTS: Inactive TP53 mutations were found in 29% of all CRCs and were more frequent in rectal (32%) than proximal colon (22%) tumours (P < 0.001). Higher frequencies of inactive TP53 mutations were also seen in advanced stage tumours (P = 0.0003) and in tumours with the poor prognostic features of vascular (P = 0.006) and lymphatic invasion (P = 0.002). Inactive TP53 mutations were associated with significantly worse outcome only in patients with Dukes' stage D tumours (RR = 1.71, 95%CI 1.25-2.33, P < 0.001). Patients with Dukes' C stage tumours appeared to gain a survival benefit from 5-fluorouracil-based chemotherapy regardless of TP53 functional status for transactivation ability. CONCLUSIONS: Mutations that inactivate the transactivational ability of TP53 are more frequent in advanced CRC and are associated with worse prognosis in this stage of disease.

p53 status and response to radiotherapy in rectal cancer: a prospective multilevel analysis.

The aim of this study was to evaluate, in a prospective study, the predictive role of p53 status analysed at four different levels in identifying the response to preoperative radiotherapy in rectal adenocarcinoma. Before treatment, 70 patients were staged and endoscopic forceps biopsies from the tumour area were taken. p53 status was assessed by total cDNA sequencing, allelic loss analysis, immunohistochemistry, and p53 antibodies. Neoadjuvant treatment was based on preoperative radiotherapy or radiochemotherapy. Response to therapy was evaluated after surgery by both pathologic downstaging and histologic tumour regression grade. In all, 35 patients (50.0%) had p53 gene mutations; 44.4% of patients had an allelic loss; nuclear p53 overexpression was observed in 39 patients (55.7%); and p53 antibodies were detected in 11 patients (16.7%). In the multilevel analysis of p53 status, gene mutations correlated with both nuclear protein overexpression (P < 0.0001) and loss of heterozygosity (P = 0.013). In all, 29 patients (41.4%) were downstaged by pathologic analysis, and 19 patients (29.2%) were classified as tumour regression grade 1. Whatever the method of evaluation of treatment response, no correlation between p53 alterations and response to radiotherapy was observed. Our results do not support the use of p53 alterations alone as a predictive marker for response to radiotherapy in rectal carcinoma.

A homogeneous europium cryptate-based assay for the diagnosis of mutations by time-resolved fluorescence resonance energy transfer.

Oligonucleotide ligation assay (OLA) is considered to be a very useful methodology for the detection and characterization of mutations, particularly for clinical purposes. The fluorescence resonance energy transfer between a fluorescent donor and a suitable fluorophore as acceptor has been applied in the past to several scientific fields. This technique is well adapted to nucleic acid analysis such as DNA sequencing, DNA hybridization and polymerase chain reaction. We describe here a homogeneous format based on the use of a rare earth cryptate label as donor: tris-bipyridine-Eu(3+). The long-lived fluorescence of this label makes it possible to reach a high sensitivity by using a time-resolved detection mode. A non-radiative energy transfer technology, known as time-resolved amplification of cryptate emission (TRACE((R))) characterized by a temporal and spectral selectivity has been developed. The TRACE((R)) detection of characterized single nucleotide polymorphism using the OLA for allelic discrimination is proposed. We demonstrate the potentialities of this OLA-TRACE((R)) methodology through the analysis of K-ras oncogene point mutations.

K-ras mutation detection by hybridization to a polypyrrole DNA chip.

BACKGROUND: Detection of mutations in cancer-related genes is of major importance for both basic knowledge and clinical practice. Several strategies have been developed to diagnose these alterations. We describe a method based on polypyrrole DNA chip technology to detect K-ras gene mutations in tumors. METHODS: An oligodeoxynucleotide array was constructed on a silicon device by copolymerization of 5'-pyrrole-labeled oligodeoxynucleotides and pyrrole. The samples to be analyzed were then amplified by PCR, and the single-stranded biotin-labeled amplified DNA was specifically hybridized to the addressed probes. Perfectly matched duplexes were detected by fluorescence microscopy using R-phycoerythrin as the detection label. The developed methodology was applied to genotype assignment of K-ras in human samples. The genotypes of 75 DNA genomic samples from colorectal cancer patients were analyzed side by side using direct DNA sequencing and a polypyrrole DNA chip. RESULTS: The chip method unequivocally defined all of the genotypes. Mutations present at <10% of the wild-type DNA concentration could be distinguished. CONCLUSIONS: This probe array assay is a rapid and reliable procedure that may be used to detect mutations.

Deregulation of the cytoplasmic tyrosine kinase cSrc in the absence of a truncating mutation at codon 531 in human bladder carcinoma.

The involvement of the cytoplasmic tyrosine kinase cSrc was investigated in human bladder carcinogenesis. Kinase activity was determined in tissue lysates from bladder transitional cell carcinoma (TCC) relative to normal epithelia. Strong kinase activation was observed at all stages of carcinogenesis with a peak at the stage pT1, where tumor cells disrupt the basement membrane and invade the submucosa. In agreement with a role for cSrc in cell invasion, immunocytochemistry analysis showed a strong staining of invading cells. An increase in cSrc protein level were also found in most tumor samples, however, it did not correlate with an increase in activity (r = 0.44) suggesting that cSrc is deregulated in these tumors. Indeed, high Src activity was affinity-purified from a column (IRSVSSDGHE(p)YIYVDP-Affigel 10) that specifically retains active Src. Enzymatic regulation involves the C-terminus, recently found mutated at codon 531 in a subset of advanced human colon cancers. However, no such mutations were detected in TCC, suggesting the existence of other mechanisms for kinase activation.

Rapid and large-scale method to detect K-ras gene mutations in tumor samples.

We have developed a rapid and large-scale method for the detection of K-ras gene mutations in tumors. First, DNA is amplified by an asymmetric PCR; second, the single-strand dinitrophenyl (DNP)-labeled amplified DNA is hybridized specifically to oligonucleotide probes affixed on a tube. Finally, perfectly matched duplexes are easily detected by a monoclonal anti-DNP antibody bearing 125I. The usefulness of this technique is illustrated by analyzing K-ras codon 12 mutations in human colorectal samples. This reliable assay procedure can be applied to the rapid screening of virtually any genetic disease caused by previously described point mutations.

Detection of loss of heterozygosity by a tube-based assay.

Here we report a tube format for detecting loss of heterozygosity in human genomic DNA. This method is based on the detection of a polymorphic restriction site in the selected gene by tube support analysis. After a PCR, amplification products were hybridized to an 125I-labeled oligonucleotide probe and affinity-collected on a tube. The captured hybrids were then subjected to an enzymatic digestion, and the remaining bound radioactivity was measured. The usefulness of this technique is illustrated by analyzing the p53 gene LOH in human colorectal samples. This development, similar to immunoanalysis tests, eliminates the electrophoresis step and makes the results easy to study.