Differential diagnosis between chronic pancreatitis and pancreatic cancer: value of the detection of KRAS2 mutations in circulating DNA

KRAS2 mutations in codon 12 have been detected in about 80% of pancreatic cancers. The aim of this study was to evaluate the value of KRAS2 mutations detection in circulating deoxyribo nucleic acid to differentiate pancreatic cancer from chronic pancreatitis. Circulating deoxyribonucleic acid was isolated from serum in 47 patients with histologically proven pancreatic adenocarcinomas (26 males, median age 65 years) and 31 controls with chronic pancreatitis (26 males, median age 48 years). Mutations at codon 12 of KRAS2 gene were searched for using polymerase chain reaction and allele specific amplification. Serum carbohydrate antigen 19.9 levels were also determined. KRAS2 mutations were found in 22 patients (47%) with pancreatic cancer and in four controls with chronic pancreatitis (13%) (P<0.002). None of the latter developed a pancreatic cancer within the 36 months of median follow-up. The sensitivity, specificity, positive and negative predictive values of serum serum KRAS2 mutations for the diagnosis of pancreatic cancer were 47, 87, 85 and 52%, respectively. KRAS2 mutations were not related to age, gender, smoking habit, tumour stage, or survival. Among the 26 patients with normal or non-contributive (due to cholestasis) serum carbohydrate antigen 19.9 levels, 14 (54%) had KRAS2 mutations. The combination of KRAS2 and carbohydrate antigen 19.9 gave a sensitivity, specificity, positive and negative predictive values for the diagnosis of pancreatic cancer of 98, 77, 87 and 96%, respectively. Detection of KRAS2 mutations in circulating deoxyribo nucleic acid has a low sensitivity but a specificity about 90% for the diagnosis of pancreatic cancer. It seems particularly useful when serum carbohydrate antigen 19.9 levels are normal or inconclusive. A combined normal serum carbohydrate antigen 19.9 and absence of circulating KRAS2 mutations makes the diagnosis of pancreatic cancer extremely unlikely.     

High Frequency of KRAS Codon 146 and FBXW7 Mutations in Thai Patients with Stage II-III Colon Cancer

Background: KRAS, NRAS, and BRAF gene mutations are the most clinically relevant and frequently reported incolorectal cancer (CRC). Although data on these genes are frequently reported in several counties, data specific to thesegenes among Thai population are scarce. The aim of this study was to investigate and identify molecular alterationsassociated with colon cancer in Thai population, and to determine the impact of these genetic aberrations on clinicaloutcome. Methods: DNA from 108 archived formalin-fixed, paraffin-embedded (FFPE) tissue samples that histologicallyconfirmed adenocarcinoma of stage II-III colon cancer between 2010 and 2012 at Siriraj Hospital (Bangkok, Thailand)were extracted. Gene mutational analysis was performed by next-generation sequencing (NGS) using an OncomineSolid Tumor DNA kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Results: A total of 22 somatic genemutations were detected. The mutation frequency observed in KRAS, NRAS, BRAF, PIK3CA, and FBXW7 mutationswas 47.2%, 1.9%, 1.9%, 12%, and 14.8%, respectively. KRAS mutation codon 12, 13, 59, 61, 117, and 146 mutationswere identified in 29.6%, 8.3%, 1.8%, 0.9%, 0.0%, and 8.3%, respectively. KRAS Exon 4 had better DFS comparedwith Exon 2 and 3. Conclusions: This study is the first to comprehensively report hotspot mutations using NGS in Thaicolon cancer patients. The most commonly identified gene mutation frequencies among Thai patients (KRAS, NRAS,BRAF, TP53, and PIK3CA) were similar to the gene mutation frequencies reported in Western population, except forsubgroup of KRAS codon 146 and FBXW7 mutations that had a slightly higher frequency.     

High Frequency of Codon 12 but not Codon 13 and 61 K-ras Gene Mutations in Invasive Ductal Carcinoma of Breast in a South Indian Population

Background: Ras genes are thought to play an important role in human cancer since they have been foundto be activated frequently in several types of tumors including breast cancer, where the overall incidence ofK-RAS oncogene activation is 0-10%. Evaluation of K-RAS gene not only for mutational frequency but also formutation types in this downstream signaling gene pathway is necessary to determine the mechanisms of action.The present study was conducted to test the hypothesis that K-RAS activation is involved in breast cancer riskof south Indian population. Materials and Methods: A total of 70 paired pathologically confirmed tumor andnon-tumor tissues from the same breast cancer patients were analysed for most common K-RAS mutationsof codon 12,13 and 61 by polymerase chain reaction followed by restriction digestion and direct nucleotidesequencing method. Results: We found that a high rate of homozygous and heterozygous mutations of codon 12,but not codon 13 and 61, may influence the invasive ductal carcinoma of breast risk in this study. Conclusions:Our study indicated that only codon 12 may be involved in initiating breast carcinogenesis in India.     

Detection of ALK and KRAS Mutations in Circulating Tumor DNA of Patients With Advanced ALK-Positive NSCLC With Disease Progression During Crizotinib Treatment

BackgroundIn patients with anaplastic lymphoma kinase (ALK)-positive non–small-cell lung cancer (NSCLC), disease progression occurs after a median of 9 to 10 months of crizotinib treatment. Several mechanisms of resistance have been identified and include ALK mutations and amplification or the activation of bypassing signaling pathways. Rebiopsy in NSCLC patients represents a critical issue and the analysis of circulating cell-free DNA (cfDNA) has a promising role for the identification of resistance mechanisms.Patients and MethodsTwenty patients with advanced ALK-positive NSCLC were enrolled after disease progression during crizotinib treatment; cfDNA was analyzed using digital droplet polymerase chain reaction (BioRad, Hercules, CA) for ALK (p.L1196M, p.G1269A, and p.F1174L) and Kirsten rat sarcoma (KRAS) (codons 12 and 13) mutations.ResultsALK secondary mutations (p.L1196M, p.G1269A, and p.F1174L) were identified in 5 patients; 1 patient had 2 ALK mutations (p.L1196M and p.G1269A). Overall, 10 patients presented KRAS mutations (7 p.G12D, 2 p.G12V, and 1 p.G12C mutations, respectively). In 3 patients KRAS mutations were associated with ALK mutations. cfDNA was monitored during the treatment with second-generation ALK inhibitors and the amount of ALK as well as KRAS mutations decreased along with tumor regression.ConclusionALK and KRAS mutations are associated with acquired resistance to crizotinib in ALK-positive NSCLC. In particular, ALK acquired mutations can be detected in plasma and could represent a promising tumor marker for response monitoring.     

DNA修复基因及其TP53共突变在肺腺癌免疫治疗疗效预测中的价值

目的 基于二代测序技术,探索DNA修复基因(DRGs)对肺腺癌免疫治疗疗效的预测价值.方法 选取癌症基因组图谱中肺腺癌两个独立数据集(分别为测试集和验证集).测试集中,依据肿瘤突变负荷评分15为阈值,分为低突变负荷组和高突变负荷组,分析不同肿瘤突变负荷与肺腺癌总生存的关系,并以KRAS/TP53共突变作为标准参照,分析...     

Detection of mutant KRAS and TP53 DNA in circulating exosomes from healthy individuals and patients with pancreatic cancer

Pancreatic cancer presents with a dismal mortality rate and is in urgent need of methods for early detection with potential for timely intervention. All living cells, including cancer cells, generate exosomes. We previously discovered double stranded genomic DNA in exosomes derived from the circulation of pancreatic cancer patients, which enabled the detection of prevalent mutations associated with the disease. Here, we report a proof-of-concept study that demonstrates the potential clinical utility of circulating exosomal DNA for identification of KRAS^G12D and TP53^R273H mutations in patients with pancreas-associated pathologies, including pancreatic ductal adenocarcinoma (PDAC), chronic pancreatitis (CP) and intraductal papillary mucinous neoplasm (IPMN), and in healthy human subjects. In 48 clinically annotated serum samples from PDAC patients, digital PCR analyses of exosomal DNA identified KRAS^G12D mutation in 39.6% of cases, and TP53^R273H mutation in 4.2% of cases. KRAS^G12D and TP53^R273H mutations were also detected in exosomal DNA from IPMN patients (2 out of 7 with KRAS^G12D, one of which also co-presented with TP53^R273H mutation). Circulating exosomal DNA in 5 out of 9 CP patients enabled the detection of KRAS^G12D mutation. In 114 healthy subject-derived circulating exosomal DNA, 2.6% presented with KRAS^G12D mutation and none with TP53^R273H mutation. This study highlights the value of circulating exosomal DNA for a rapid, low-cost identification of cancer driving mutations. The identification of mutations in IPMN patients and healthy subjects suggests that liquid biopsies may allow potential assessment of cancer risk but with a cautionary note that detection of clinical cancer cannot be assumed.     

DNA repair gene O6-methylguanine-DNA methyltransferase: promoter hypermethylation associated with decreased expression and G:C to A:T mutations of p53 in brain tumors

The DNA repair enzyme O(6)-methylguanine-DNA methyltransferase (MGMT) removes alkylating adducts from the O(6) position of guanine and protects cells from cytotoxic and mutagenic effects. Expression of MGMT is decreased in some cancers, which may be the result of methylation of CpG islands of both the promoter and coding regions of the gene. We studied the methylation status of the MGMT promoter in a very large collection of brain tumors (85) using methylation-specific polymerase chain reaction (PCR). Aberrant methylation occurred in 48% of 85 human brain tumor samples. Quantitative real-time PCR showed that expression of MGMT mRNA levels was significantly decreased (P < 0.001) in those brain tumors that had methylation of the promoter region of their MGMT gene. MGMT can prevent G to A mutations by removing alkyl groups from the O(6) position of guanine. We found a significantly increased frequency of G:C to A:T mutations of the p53 gene in brain tumors having a methylated MGMT promoter compared with those having an unmethylated MGMT promoter (P < 0.05), and all the non-CpG dinucleotide G:C to A:T mutations of p53 were in samples with a methylated MGMT promoter.