Frequent detection of K-ras mutation in stool samples of colorectal carcinoma patients after improved DNA extraction: comparison with tissue samples

Fecal occult blood testing is widely used in the clinical screening of colorectal tumors. However, this method has so frequent false-positive results that more accurate screening-strategy should be established. Although the molecular screening using K-ras gene mutation in stools has been attempted to improve the results, the low rate of DNA extraction from stools leaves this measurement under utility value. In this study, we investigated whether or not our applied DNA extraction method from stools could produce enough DNA for the molecular screening of colorectal tumors by K-ras gene mutations in stools. We applied cetyltrimethylammonium bromide (CTAB) solution to improve human DNA extraction from stools and a mutant-allele-sensitive amplification (MASA) method to detect K-ras mutation within codon 12. We were able to confirm the stool DNA by identifying K-ras fragments in all the 20 patients. Tissue K-ras mutation was identified in 4 (2 cancers and 2 adenomas) of 20 patients. Stool K-ras mutations were found in 6 patients, 3 tissue K-ras mutation positive patients (2 cancers and an adenoma) and 3 tissue K-ras mutation negative patients. These results indicate that it is possible to extract enough DNA from human stool samples of all patients with colorectal tumors for K-ras mutation studies. K-ras mutations are more frequently detected in stools than in resected colorectal tumors. This study indicates that K-ras mutation screening in stools for colorectal cancer may include not only a primary colorectal cancer but also precancerous lesions in all parts of a gastrointestinal tract.     

Novel Methylation Panel for the Early Detection of Colorectal Tumors in Stool DNA

BackgroundPrevious studies showed that the assessment of promoter hypermethylation of a limited number of genes in tumor biopsies may identify the majority of colorectal tumors. This study aimed to assess the clinical usefulness of a panel of methylation biomarkers in stool DNA in the identification of colorectal tumors, using methylation-specific melting curve analysis (MS-MCA), a technique that simultaneously analyzes all cytosine-phosphate-guanine (CpG) residues within a promoter.Materials and MethodsThe promoter methylation status of 4 tumor-related genes (RARB2, p16^INK4a, MGMT, and APC) was analyzed in DNA stool samples and corresponding tissues in an initial set of 12 patients with newly diagnosed primary colorectal carcinomas and 20 patients with newly diagnosed colorectal adenomas, using methylation-specific polymerase chain reaction. Results were replicated in a set of 82 patients (20 healthy subjects, 16 patients with inflammatory bowel disease (IBD), 20 patients with adenomas, and 26 patients with carcinomas), using MS-MCA analyses.ResultsIn the initial set, ≥ 1 positive methylation marker was detected in the stools of 9 of 12 patients (75%) with carcinomas and 12 of 20 patients (60%) with adenomas, with no false-positive results. Stool analyses missed 7 methylated lesions (25%). In the replication set, stool DNA testing detected 16 of 26 carcinomas (62%) and 8 of 20 adenomas (40%). The MS-MCAs missed 14 methylated tumors (37%). No aberrant methylation was evident in healthy subjects, but the RARB2 marker was positive in 2 of 15 stool samples (13%) of patients with IBD.ConclusionAnalysis via MS-MCA of a panel of methylation markers in stool DNA may offer a good alternative in the early, noninvasive detection of colorectal tumors.     

KRAS mutation detection and prognostic potential in sporadic colorectal cancer using high-resolution melting analysis

Background:

The development of targeted therapies has created a pressing clinical need for molecular characterisation of cancers. In this retrospective study, high-resolution melting analysis (HRMA) was validated and implemented for screening of 164 colorectal cancer (CRC) patients to detect KRAS hot-spot mutations and to evaluate its prognostic value. Direct sequencing was used to confirm and characterise HRMA results.

Methods:

After establishing its sensitivity, HRMA was validated on seven cell lines and inter- and intra-variation were analysed. The prognostic value of KRAS mutations in CRC was evaluated using survival analysis.

Results:

HRMA revealed abnormal melting patterns in 34.1% CRC samples. Kaplan–Meier survival curves revealed a significantly shorter overall (OS) and disease-free survival (DFS) for CRC patients harbouring a KRAS mutation. In the Cox regression analysis, only when colon and rectal cancer were analysed separately, KRAS mutation was a negative predictor for OS in patients with rectal cancer and DFS in those with stage II colon cancer.

Conclusions:

HRMA was found to be a valid screening method for KRAS mutation detection. The KRAS mutation came forward as a negative predictive factor for OS in patients with rectal cancer and for DFS in stage II colon cancer patients.     

The KRAS StripAssay for detection of KRAS mutation in Egyptian patients with colorectal cancer (CRC): A pilot study

BackgroundEpidermal growth factor receptor (EGFR) and its downstream factors KRAS and BRAF are mutated in several types of cancer, affecting the clinical response to EGFR inhibitors. Mutations in the EGFR kinase domain predict sensitivity to the tyrosine kinase inhibitors gefitinib and erlotinib in lung adenocarcinoma, while activating point mutations in KRAS and BRAF confer resistance to the anti-EGFR monoclonal antibody cetuximab in colorectal cancer. The development of new generation methods for systematic mutation screening of these genes will allow more appropriate therapeutic choices.PurposeDetection of KRAS mutation in Egyptian colorectal cancer (CRC) patients by the KRAS StripAssay.MethodsExamination of 20 colorectal cancer (CRC) patients is done to detect KRAS mutations by KRAS StripAssay. For the StripAssay, a mutant-enriched PCR was followed by hybridization to KRAS-specific probes bound to a nitrocellulose strip.ResultsAmong 20 patients, KRAS mutations were identified in 80% of patients by the KRAS StripAssay.ConclusionsOur preliminary results suggest that KRAS StripAssay is an alternative to protocols currently in use for KRAS mutation detection.     

Detection of K-ras mutations in stools of patients with colorectal cancer by mutant-enriched PCR

Mutant-enriched PCR was applied to the detection of mutations at codons 12 and 13 of K-ras genes in the stools of patients with colorectal cancer. Mutations were analyzed in stool samples obtained prior to surgery. Resected tumor specimens were screened for K-ras mutations by PCR-mediated RFLP analysis. Using normal stool samples, assay conditions were adjusted to optimal sensitivity and specificity. The following specimens were included in the study: 16 stool samples corresponding to carcinomas in which K-ras mutations had been identified; 7 randomly selected stool samples corresponding to carcinomas which were negative for K-ras mutations; 1 stool sample from a patient with non-Hodgkin's lymphoma. In 13 of the 16 stool samples (81%) corresponding to tumors in which K-ras mutations had been identified previously, K-ras mutations were detected. In 2 of the 7 stool samples corresponding to tumors in which K-ras mutations had not been detected by previous PCR-mediated RFLP analysis, K-ras mutations were also present. Reanalyses of the tumors corresponding to these 2 positive stool samples by mutant-enriched PCR revealed a K-ras mutation in one of the tumors. The stool and tumor of the patient with non-Hodgkin's lymphoma were negative for K-ras mutations. DNA sequence analysis revealed that, for each of the K-ras mutations identified in stool samples, identical base substitutions were present in the corresponding tumor tissue. The results indicate that tumor cells harboring K-ras mutations can be detected in the stools of patients with colorectal cancer by mutant-enriched PCR with high sensitivity and specificity. Because of the simplicity of the technique, it may be suitable for screening of stool samples for mutations of the K-ras gene. © 1996 Wiley-Liss, Inc.     

Detecting colorectal cancer in stool with the use of multiple genetic targets

BACKGROUND: Colorectal cancer cells are shed into the stool, providing a potential means for the early detection of the disease using noninvasive approaches. Our goal was to develop reliable, specific molecular genetic tests for the detection of colorectal cancer in stool samples. METHODS: Stool DNA was isolated from paired stools and primary tumor samples from 51 colorectal cancer patients. Three genetic targets-TP53, BAT26, and K-RAS-were used to detect tumor-associated mutations in the stool prior to or without regard to the molecular analyses of the paired tumors. TP53 gene mutations were detected with a mismatch-ligation assay that detects nine common p53 gene mutations. Deletions within the BAT26 locus were detected by a modified solid-phase minisequencing method. Mutations in codons 12 and 13 of K-RAS were detected with a digital polymerase chain reaction-based method. RESULTS: TP53 gene mutations were detected in the tumor DNA of 30 patients, all of whom had the identical TP53 mutation in their stools. Tumors from three patients contained a noninherited deletion at the BAT26 locus, and the same alterations were identified in these patients' stool specimens. Nineteen of 50 tumors tested had a K-RAS mutation; identical mutations were detected in the paired stool DNA samples from eight patients. In no case was a mutation found in stool that was not also present in the primary tumor. Thus, the three genetic markers together detected 36 (71%) of 51 patients (95% confidence interval [CI] = 56% to 83%) with colorectal cancer and 36 (92%) of 39 patients (95% CI = 79% to 98%) whose tumors had an alteration. CONCLUSION: We were able to detect the majority of colorectal cancers by analyzing stool DNA for just three genetic markers. Additional work is needed to determine the specificity of these genetic tests for detecting colorectal neoplasia in asymptomatic patients and to more precisely estimate the prevalence of the mutations and sensitivity of the assay.     

Heterogeneity of KRAS,NRAS, BRAF and PIK3CA mutations in metastatic colorectal cancer and potential effects on therapy in the CAPRI GOIM trial

BackgroundEvidence suggests that metastatic colorectal carcinoma (mCRC) has a high level of intratumor heterogeneity. We carried out a quantitative assessment of tumor heterogeneity for KRAS, NRAS, BRAF and PIK3CA mutations, in order to assess potential clinical implications.Patients and methodsTumor samples (n = 182) from the CAPRI-GOIM trial of first-line cetuximab + FOLFIRI in KRAS exon-2 wild-type mCRC patients were assessed by next-generation sequencing that allows quantitative assessment of mutant genes. Mutant allelic frequency was normalized for the neoplastic cell content and, assuming that somatic mutations usually affect one allele, the Heterogeneity Score (HS) was calculated by multiplying by 2 the frequency of mutant alleles in neoplastic cells. Therefore, HS virtually corresponds to the fraction of neoplastic cells carrying a specific mutation.ResultsThe KRAS HS ranged between 12 and 260 with mean value of 87.1 and median value of 84.4, suggesting that in most CRC, the majority of neoplastic cells carry mutant KRAS. Similar findings were observed for NRAS (HS range 35.5–146.7; mean 102.8; median 117.1). In contrast, in BRAF (HS range 17.1–120; mean 54.8; median 54.3) and PIK3CA (HS range 14.3–120; mean 59.5; median 47.3) mutant cases, only a fraction of neoplastic cells seem to carry the mutant allele. The response rate was 70% in KRAS mutant patients with an HS <33 (low KRAS; n = 10) and 45.7% in KRAS HS >33 patients (high KRAS; n = 35); median progression-free survival were 7.97 and 8.37 months, respectively. Low-KRAS tumors had a higher frequency of additional mutations in PIK3CA when compared with high-KRAS (6/10 versus 8/35).ConclusionsKRAS and NRAS mutations are usually present in the majority of neoplastic cells, whereas BRAF and PIK3CA mutations often affect a limited fraction of transformed cells. Resistance to cetuximab in low-KRAS patients might be driven by the complex mutational profile rather than KRAS mutation load.     

银染PCR-SSCP检测粪便中结肠直肠癌抑癌基因突变的研究

目的：建立无创伤性结肠直肠癌筛查方法。方法：应用8％银染非变性聚丙烯酰胺凝胶（丙烯酰胺：甲叉丙烯酰胺＝29：1）检测粪便中抑癌基因点突变。结果：45例结肠直肠癌患者中,高分化癌15例,中分化癌18例,低分化癌12例;Dukes分期中A期15例,B期16例,C期9例,D期5例;淋巴结转移14例,无淋巴结转移31例。正常肠粘膜组织为对照组。粪便中APC（adenomatous polyposis coli)及／或MCC（mutated in colorectal cancer)基因突变28例（62．2％）,占有APC及／或MCC基因突变的结直肠癌组织的93．3％（28／30例）;粪便APC及MCC基因突变分别占APC及MCC基因突变的结直肠癌组织的91．3％及84．6％。粪便中基因突变与肿瘤组织学类型、Dukes分期、淋巴结转移、粪便潜血是否阳性及有无便血史、肿块大小、病灶部位及肿瘤形态统计学上无显著差异（P＞0．05）。结论：该方法可望成为无创伤性结肠直肠癌普查、筛选、疗效观察及预后判断的方法。     

KRAS mutation detection by high-resolution melting analysis significantly predicts clinical benefit of cetuximab in metastatic colorectal cancer

Background:Anti-epidermal growth factor receptor (EGFR) monoclonal antibodies are restricted to KRAS wild-type (WT) metastatic colorectal cancers (mCRCs), usually identified by direct sequencing, that may yield false negative results because of genetic heterogeneity within the tumour. We evaluated the efficiency of high-resolution melting analysis (HRMA) in identifying KRAS-mutant (MUT) tumours.Methods:We considered 50 mCRC patients scored as KRAS-WT by direct sequencing and treated with cetuximab-containing chemotherapy, and tested the correlations between HRMA findings and response rate (RR), progression-free (PFS) and overall survival (OS).Results:Aberrant melting curves were detected in four (8%) cases; gene cloning confirmed these mutations. Response rate (RR) of HRMA KRAS-WT patients was 28.3%. There was no response in HRMA KRAS-MUT patients. Disease control rate (responsive plus stable disease) was 58.7% in HRMA KRAS-WT patients and 25% in HRMA KRAS-MUT patients. There was no correlation between HRMA KRAS status and RR (P=0.287) or disease control (P=0.219). Median PFS (4.8 vs 2.3 months; hazard ratio (HR)=0.29, P=0.02) and OS (11.0 vs 2.7 months; HR=0.11, P=0.03) were significantly longer for the HRMA KRAS-WT than for HRMA KRAS-MUT patients.Conclusions:High-resolution melting analysis identified 8% more KRAS-MUT patients not responding to cetuximab-containing regimens, suggesting that HRMA may be more effective than direct sequencing in selecting patients for anti-EGFR antibodies.     

Clinical activity of FOLFIRI plus cetuximab according to extended gene mutation status by next-generation sequencing: findings from the CAPRI-GOIM trial

BackgroundTreatment with antiepidermal growth factor receptor (anti-EGFR) monoclonal antibodies has been restricted to metastatic colorectal cancer (mCRC) patients with RAS wild-type tumors. Next-generation sequencing (NGS) allows the assessment in a single analysis of a large number of gene alterations and might provide important predictive and prognostic information.Patients and methodsIn the CAPRI-GOIM trial, 340 KRAS exon 2 wild-type mCRC patients received first-line FOLFIRI plus cetuximab. Tumor samples (182/340, 53.5%) were assessed by NGS to search for mutations in 22 genes involved in colon cancer.ResultsObjective responses in the NGS cohort were observed in 104/182 patients [overall response rate (ORR) 57.1%; 95% confidence interval (95% CI) 52% to 66.4%] with a median progression-free survival (mPFS) of 9.8 (95% CI 8.7–11.5) months. NGS analysis was successfully completed in all 182 samples. One or more gene mutations (up to five) were detected in 124/182 (68.1%) tumors within 14/22 genes for a total of 206 mutations. KRAS exon 2 mutations were identified in 29/182 (15.9%) samples, defined as wild type by local laboratory assessment. Frequently mutated genes were: TP53 (39.6%), KRAS exons 3/4 (8.8%), NRAS exons 2/3 (7.1%), PIK3CA exons 9/20 (13.2%), BRAF (8.2%). FOLFIRI plus cetuximab treatment determined ORR of 62.0% (95% CI 55.5% to 74.6%) with mPFS of 11.1 (95% CI 9.2–12.8) months in patients with KRAS and NRAS wild-type tumors. Conversely, ORR was 46.6% (95% CI 39.9–57.5%) with mPFS of 8.9 (95% CI 7.4–9.6) months in patients with KRAS or NRAS mutations. Similarly, the subgroup of patients carrying KRAS, NRAS, BRAF, or PIK3CA mutations showed a worse outcome, although this might be due to a prognostic effect.ConclusionsThis study demonstrates that NGS analysis in mCRC is feasible, reveals high level of intra and intertumor heterogeneity, and identifies patients that might benefit of FOLFIRI plus cetuximab treatment.     

Kirsten rat sarcoma (KRAS) oncogene mutation predicts magnitude of response and outcomes in hepatic arterial infusion pump therapy of unresectable colorectal liver metastases

BackgroundThe Kirsten rat sarcoma (KRAS) mutation predicts negative outcomes following resection of colorectal liver metastases (CRLM) and adjuvant hepatic arterial infusion (HAI) pump chemotherapy. Less is known on the effects of KRAS mutation on tumor response in patients with unresectable CRLM undergoing HAI chemotherapy with floxuridine.MethodsThis is a retrospective cohort study investigating the effects of KRAS mutation on tumor response in patients with unresectable CRLM treated with HAI chemotherapy. Primary endpoint was objective response rate (ORR), secondary endpoints included overall tumor response and conversion to resectability.ResultsTwenty-five patients with unresectable liver metastases from colorectal cancer were treated with HAI chemotherapy between 2017–2019. Median number of liver lesions was 12 (range, 1–59) and almost all (n=24) had prior chemotherapy before starting HAI therapy. Median number of cycles administered via HAI pump was 6 (range, 3–12). Overall decrease in liver tumor burden was 63.5% (median; range, −257–100%) with an ORR of 20/25 (80%) and 10 (40%) patients converting to resectable status. Eleven (44%) patients had KRAS positive tumors. When compared to wild-type, KRAS positive tumors had less overall percent decrease (58% vs. 70%; P=0.04) and ORR (7/11 vs. 13/13; P=0.03). Fewer patients with KRAS positive tumors converted to resectable status during HAI therapy (2/11 vs. 8/13; P=0.05). At a median follow-up of 14.6 months (range, 4.0–36.6 months), overall survival is 45% among KRAS-positive and 77% for wild type patients.ConclusionsKRAS mutational status in patients with unresectable liver metastases from colorectal cancer predicts worse response to HAI chemotherapy compared to wild type.     

Efficacy according to biomarker status of cetuximab plus FOLFOX-4 as first-line treatment for metastatic colorectal cancer: the OPUS study

BackgroundThe randomized phase II OPUS (Oxaliplatin and Cetuximab in First-Line Treatment of Metastatic Colorectal Cancer) study showed that tumor KRAS mutation status was predictive for outcome in patients receiving cetuximab plus FOLFOX-4 (oxaliplatin/5-fluorouracil/folinic acid) as first-line therapy for metastatic colorectal cancer (mCRC).Patients and methodsThe biomarker analysis was extended through the use of additional DNA samples extracted from stained tissue sections. KRAS and BRAF tumor mutation status was determined for new (and for BRAF, existing) samples using a PCR technique. Clinical outcome was reassessed according to mutation status. Overall survival data are presented.ResultsOf 315 KRAS evaluable patient samples (93%), 179 tumors (57%) were KRAS wild type. Eleven of 309 (4%) KRAS/BRAF evaluable tumors (all KRAS wild type) carried BRAF mutations. The addition of cetuximab to FOLFOX-4 significantly improved progression-free survival (hazard ratio 0.567, P = 0.0064) and response (odds ratio 2.551, P = 0.0027) in patients with KRAS wild-type tumors. A favorable effect on survival was also observed.ConclusionsThese results confirm the efficacy of cetuximab plus FOLFOX-4 in the first-line treatment of patients with KRAS wild-type mCRC and confirm KRAS mutation status as an effective predictive biomarker. The small number of tumors with BRAF mutations precluded the drawing of definitive conclusions concerning the predictive or prognostic utility of this biomarker.     

Fecal Genetic Mutations and Human DNA in Colorectal Cancer and Polyps Patients

Background: Colorectal cancer (CRC) is one of the most frequent cancers. Genetic mutations in CRC already described can be detected in feces. Microarray methods in feces can represent a new diagnostic tool for CRC and significant improvement at public health. Aim: to analyze stool DNA by human DNA quantify and microarray methods as alternatives to CRC screening. Method: Three methods were analyzed in stool samples: Human DNA Quantify, RanplexCRC and KRAS/BRAF/PIK3CA (KBP) Arrays. Results: KBP array mutations were presented in 60.7% of CRC patients and RanplexCRC Array mutations in 61.1% of CRC patients. Sensitivity and specificity for human DNA quantification was 66% and 82% respectively. Fecal KBP Array had 35% sensitivity and 96% specificity and RanplexCRC Array method had 78% sensitivity and 100% specificity. Conclusion: Microarray methods showed promise as potential biomarkers for CRC screening; however, these methods had to be optimized to improve accuracy and applicability by clinical routine.     

PTEN gene expression and mutations in the PIK3CA gene as predictors of clinical benefit to anti-epidermal growth factor receptor antibody therapy in patients with KRAS wild-type metastatic colorectal cancer

PurposeTo identify novel genetic markers predictive of clinical benefit from epidermal growth factor receptor–directed antibody therapy in patients with metastatic colorectal cancer.Patients and MethodsSeventy-six consecutive patients who received cetuximab or panitumumab, either alone or in combination with chemotherapy and with available tumor tissue were included. Tumor tissue was tested by pyrosequencing for mutations at known hot spots in the KRAS, BRAF, PIK3CA, PIK3R1, AKT1, and PTEN genes. PTEN promoter methylation status was analyzed by methylation-specific polymerase chain reaction, and expression was determined by immunohistochemistry (IHC). Forty-four patients had 4 weeks of therapy and were considered for clinical correlates.ResultsConsistent with previous studies, KRAS gene mutations were associated with a shorter progression-free survival (PFS) and overall survival (OS). Among the patients with wild-type KRAS, preservation of PTEN expression and PIK3CA wild-type status was associated with improved OS (median OS, 80.4 vs. 32.5 weeks; hazard ratio, 0.33; P = .0008) and a trend toward improved PFS (median PFS, 24.8 vs. 15.2 weeks; hazard ratio, 0.51; P = .06), compared with PTEN-negative or PIK3CA-mutant tumors. PTEN methylation was more common in the metastatic samples than in the primary samples (P = .02). The simultaneous presence of methylation and mutation in the PTEN gene was associated with IHC negativity (P = .026).ConclusionIn addition to KRAS mutation, loss of PTEN expression (by IHC) and PIK3CA mutation is likely to be predictive of a lack of benefit to anti-EGFR therapy in metastatic colorectal cancer. PTEN promoter methylation and mutation status was predictive of PTEN expression and may be used as an alternative means of predicting response to EGFR-targeted therapy.     

The BRAF V600E mutation is an independent prognostic factor for survival in stage II and stage III colon cancer patients

BackgroundMolecular markers in colon cancer are needed for a more accurate classification and personalized treatment. We determined the effects on clinical outcome of the BRAF mutation, microsatellite instability (MSI) and KRAS mutations in stage II and stage III colon carcinoma.Patients and methodsStage II colon carcinoma patients (n = 106) treated with surgery only and 258 stage III patients all adjuvantly treated with 5-fluorouracil chemotherapy were included. KRAS mutations in codons 12 and 13, V600E BRAF mutation and MSI status were determined.ResultsOlder patients (P < 0.001), right-sided (P = 0.018), better differentiated (P = 0.003) and MSI tumors (P < 0.001) were significantly more frequent in stage II than stage III. In both groups, there was a positive association between mutated BRAF and MSI (P = 0.001) and BRAF mutation and right-sided tumors (P = 0.001). Mutations in BRAF and KRAS were mutually exclusive. In a multivariate survival analysis with pooled stage II and stage III data, BRAF mutation was an independent prognostic factor for overall survival (OS) and cancer-specific survival [hazards ratio (HR) = 0.45, 95% confidence interval (CI) 0.25–0.8 for OS and HR = 0.47, 95% CI 0.22–0.99]. KRAS mutation conferred a poorer disease-free survival (HR = 0.6, 95% CI 0.38–0.97).ConclusionsThe V600E BRAF mutation confers a worse prognosis to stage II and stage III colon cancer patients independently of disease stage and therapy.     

Phase II Trial of Trametinib and Panitumumab in RAS/RAF Wild Type Metastatic Colorectal Cancer

IntroductionMEK inhibition may overcome resistance to EGFR inhibition in patients with RAS wildtype (wt) metastatic colorectal cancer (mCRC). We evaluated antitumor activity of trametinib (MEK1/2 inhibitor) with panitumumab (EGFR monoclonal antibody) in a phase II trial.MethodsPatients with KRAS, NRAS, and BRAF wt mCRC with prior 5-FU, irinotecan, oxaliplatin, +/- bevacizumab and no prior anti-EGFR therapy were treated with trametinib 1.5 mg oral daily and panitumumab 4.8 mg/kg IV every 2 weeks. Primary endpoint was clinical benefit rate (CB; CR, PR, or SD ≥24 weeks) by RECIST v1.1. A 2-stage minimax design was used. Serial plasma circulating free DNA (cfDNA) was collected and profiled using Oncomine Lung cfDNA assay.ResultsFourteen patients were enrolled from November 2015 to April 2019. CB rate was 38% (5/13) and median progression free survival (PFS) was 4.4 months (95% CI, 2.9-7.1). Confirmed overall response rate was 38% (5/13). Treatment-related AE (trAE) included acneiform rash (85%), diarrhea (62%), maculopapular rash (54%), mucositis (46%), and others. Dose modifications and interruptions of trametinib occurred in 69% and panitumumab in 54% of patients. The trial did not progress to stage II accrual due to tolerability and short duration of response. RAS or BRAF mutations cfDNA were detected in 3/13 patients (23%) before radiographic disease progression.ConclusionThe addition of trametinib to panitumumab led to a high rate of tumor shrinkage in RAS/RAF wt metastatic colorectal cancer, with poor tolerability due to a high incidence of skin toxicity. Median PFS was similar to panitumumab alone in historical control data.     

Effect of Coexisting KRAS and TP53 Mutations in Patients Treated With Chemotherapy for Non–small-cell Lung Cancer

BackgroundKRAS and TP53 are common mutations in non–small-cell lung cancer (NSCLC). The Lung Adjuvant Cisplatin Evaluation Biological Program group found adjuvant chemotherapy to be deleterious in patients with coexisting KRAS/TP53 mutations.Patients and MethodsTo validate these results, patients with NSCLC tested for KRAS and TP53 mutations and receiving chemotherapy for any stage NSCLC were selected. Mutation status was analyzed using next generation sequencing (Illumina) or multiplex recurrent mutation detection (MassARRAY, Agena Biosciences) assays, and was correlated with clinical and demographic data. Disease-free (DFS) or progression-free survival (PFS) was the main endpoint, and overall survival (OS) was the secondary endpoint.ResultsAmong 218 patients, 28 had coexisting KRAS/TP53 mutations, 77 TP53, 37 KRAS, 76 had neither KRAS nor TP53 mutation (WT/WT). There was no DFS/PFS difference for the KRAS/TP53 group versus all others among 99 patients who received adjuvant chemotherapy (hazard ratio [HR], 1.22; 95% confidence interval [CI], 0.61-2.44; P = .57), 27 stage III patients who received chemo-radiation (HR, 0.87; 95% CI, 0.32-2.38; P = .8), and 63 patients who received palliative chemotherapy (HR, 0.68; 95% CI, 0.31-1.48; P = .33). OS was longer in the WT/WT group compared with any other group (KRAS: HR, 1.87; 95% CI, 1.02-3.43; P = .043; TP53: HR, 2.17; 95% CI, 1.3-3.61; P = .0028; KRAS/TP53: HR, 2.06; 95% CI, 1.09-3.88; P = .026). No OS difference was seen for KRAS/TP53 compared with the other groups (HR, 1.26; 95% CI, 0.75-2.13; P = .38).ConclusionsThere was no significant difference in DFS/PFS between the 4 groups. However, OS was longer for patients with TP53 and KRAS wild-type NSCLC who received chemotherapy for any stage compared with patients with KRAS, TP53 mutation, or double mutant tumors.     

Detection of KRAS mutations in circulating tumour DNA from plasma and urine of patients with colorectal cancer

BackgroundCirculating tumour DNA (ctDNA) is very useful for purposes of cancer genetics; however, it has some limitations. Recently, ctDNA in body fluids, such as urine, sputum, and pleural effusion, has been investigated. The aim of this study was to evaluate the quantity of ctDNA derived from urine (trans-renal ctDNA) and the accuracy of KRAS mutation detection in relation to disease stage in colorectal cancer.MethodsUrine, plasma, and tissue samples were collected from consecutively resected colorectal cancer patients. DNA was extracted from each sample and the quantity was determined. From each DNA sample, KRAS mutations were detected using droplet digital PCR.Results200 patients participated and KRAS mutations were detected in 84 patients (42.0%) from tumour tissue. The concentration of trans-renal ctDNA (trtDNA) was significantly lower than that of plasma; however, there was no significant difference between the sensitivity using ctDNA and that using trtDNA (29.8% VS 33.3%, p = 0.62). Concordance between these two tests was only 17.5%. Combination analysis (ctDNA + trtDNA) improved the sensitivity to 53.6%, and sensitivity was significantly higher than that of corresponding single assays (p = 0.003). In early cancer stages, trtDNA had greater sensitivity for detecting KRAS mutations than ctDNA (37.7% vs. 21.3%, p = 0.047). Conversely, it was less useful for advanced cancer stages (21.7% vs. 52.2%, p = 0.07). Notably, KRAS mutations were detected using ctDNA or trtDNA in 12 of 116 (10.3%) patients who had no KRAS mutations in their tissue samples.Conclusions: trtDNA and ctDNA have equal potential and combination analysis significantly improved the sensitivity.     

Development of a novel PTT assay for mutation detection in <Emphasis Type="Italic">PALB2</Emphasis> large exons and <Emphasis Type="Italic">PALB2</Emphasis> screening in medullary breast cancer

Beyond BRCA1 and BRCA2 genes, PALB2 (Partner and localizer of BRCA2) emerges as the third breast cancer susceptibility gene due to its role in the same DNA repair pathway: homologous recombination. In most populations studied so far, PALB2 mutations are detected in 1–2 % of BRCA negative female patients. PALB2 gene contains 13 exons; exons 4 and 5 consist 65 % of the coding area. We developed a protein truncation test (PTT) for quick screening of truncating pathogenic mutations of these two large exons. Specific primers were de novo, in silico designed and the PTT-PCR products were translated in the presence of biotinylated lysine and detected colorimetrically. The assay was initially tested in 30 patients with hereditary breast cancer, negative for BRCA mutations and then, in 17 patients with the rare medullary breast cancer subtype. Small PALB2 exons were screened with high-resolution melting curve analysis (HRMA) and the large DNA rearrangements with multiplex ligation-dependent probe amplification (MLPA). Any alterations detected were verified by Sanger DNA Sequencing. The developed PTT methodology is highly specific for clinical significant mutations; positive control samples that produce truncated PALB2 peptides were correctly identified and the method was accurate when compared to DNA sequencing. We did not detect any deleterious PALB2 mutation in both groups of patients. HRMA and MLPA were also negative for all tested samples. However, our novel, fast and cost-effective PTT method for pathogenic mutation detection of the two large PALB2 exons can be applied in screening of a large number of breast cancer patients.     

Prevalence and clinical association of gene mutations through multiplex mutation testing in patients with NSCLC: results from the ETOP Lungscape Project

BackgroundReported prevalence of driver gene mutations in non-small-cell lung cancer (NSCLC) is highly variable and clinical correlations are emerging. Using NSCLC biomaterial and clinical data from the European Thoracic Oncology Platform Lungscape iBiobank, we explore the epidemiology of mutations and association to clinicopathologic features and patient outcome (relapse-free survival, time-to-relapse, overall survival).MethodsClinically annotated, resected stage I–III NSCLC FFPE tissue was assessed for gene mutation using a microfluidics-based multiplex PCR platform. Mutant-allele detection sensitivity is >1% for most of the ~150 (13 genes) mutations covered in the multiplex test.ResultsMultiplex testing has been carried out in 2063 (76.2%) of the 2709 Lungscape cases (median follow-up 4.8 years). FFPE samples mostly date from 2005 to 2008, yet recently extracted DNA quality and quantity was generally good. Average DNA yield/case was 2.63 µg; 38 cases (1.4%) failed QC and were excluded from study; 95.1% of included cases allowed the complete panel of mutations to be tested. Most common were KRAS, MET, EGFR and PIK3CA mutations with overall prevalence of 23.0%, 6.8%, 5.4% and 4.9%, respectively. KRAS and EGFR mutations were significantly more frequent in adenocarcinomas: PIK3CA in squamous cell carcinomas. MET mutation prevalence did not differ between histology groups. EGFR mutations were found predominantly in never smokers; KRAS in current/former smokers. For all the above mutations, there was no difference in outcome between mutated and non-mutated cases.ConclusionArchival FFPE NSCLC material is adequate for multiplex mutation analysis. In this large, predominantly European, clinically annotated stage I–III NSCLC cohort, none of the mutations characterized showed prognostic significance.