Frequency Of KRAS Gene Mutations Research Articles

Fusobacterium nucleatum-Mediated Alteration in Expression of VEGF and CCL3 Genes and KRAS Mutation in Colorectal Cancer Patients

Background: Colorectal cancer (CRC) is the third most common cancer worldwide, and its development is influenced by genetic and environmental factors, including the gut microbiota. Recent studies have reported an association between Fusobacterium nucleatum abundance and CRC. Objectives: This study aimed to investigate the abundance of F. nucleatum in CRC and polyp patients and its association with the expression of chemokine ligand -3(CCL3), vascular endothelial growth factor (VEGF), and nuclear factor-kappa B (NF-KB11) genes and the presence of deoxyribonucleic acid (DNA) mutations and polymorphisms in the Kirsten rat sarcoma viral oncogene homolog (KRAS) gene. Methods: A total of 80 biopsy samples were collected from CRC, polyp, and colitis patients. Moreover, F. nucleatum abundance was measured by quantitative polymerase chain reaction (qPCR). The expression of CCL3, VEGF, and NF-KB11 genes was measured by reverse transcription polymerase chain reaction (RT-PCR). Additionally, KRAS gene mutations and polymorphisms were detected by the Mutation Surveyor software (V5.1.2). Results: The results showed that F. nucleatum abundance was significantly higher in CRC and polyp patients than in colitis patients (P < 0.05). The expression of CCL3 and VEGF genes was also significantly higher in F. nucleatum-positive samples (P < 0.05). However, NF-KB11 gene expression was non-significant. Fusobacterium nucleatum-positive biopsy samples had a higher frequency of KRAS gene mutations and polymorphisms than F. nucleatum-negative CRC patients (odds ratio = 3). Most of the mutations observed in the positive samples were (6144A>AT,31E>E) at exon 2 of the KRAS gene. Conclusions: The study findings suggest that F. nucleatum might play a role in CRC and polyp development and contribute to KRAS gene mutations. Therefore, targeting F. nucleatum in the gut microbiota could be a potential therapeutic strategy for preventing CRC and polyp development.

STUDY OF THE RELATIONSHIP BETWEEN KRAS GENE MUTATIONS AND GENDER, AGE AND RACE IN COLORECTAL CANCER PATIENTS RESIDING IN THE REPUBLIC OF KAZAKHSTAN

In 2016 3158 patients with colorectal cancer (CRC) were registered in the Republic of Kazakhstan, out of them, 1,484 patients died. Colorectal and colon cancers are the 8th and the 5 th leading causes of mortality, respectively. One of the most significant events in the molecular pathogenesis of CRC is an activating mutation in the KRAS oncogene. Recently, studies of the KRAS gene mutation and analysis of its relation with the clinical course of CRC have been carried out in different countries. The effect of gender and age on the KRAS gene status in CRC remains a subject for discussion. The purpose of this study was to study the relationship between the KRAS gene status and gender, age and race in colorectal cancer patients residing in the Republic of Kazakhstan. Material and methods. Data on 332 patients with CRC for the period from 2010 to 2014 were studied. KRAS test was performed using BioLink kits to detect mutations in 12 and 13 codons of 2 exon using allele-specific PCR method. R esults. In our study, the frequency of KRAS gene mutations in CRC patients residing in the Republic of Kazakhstan was 44.9 %. The frequency of mutations in the studied codons among women and men was the same. When analyzing the mutation frequency of the KRAS gene, it was revealed that among both sexes the G12D mutation was more often observed. The least common mutations were G12C, G12S, which occurred in both sexes (up to 5 % of cases). The analysis of the dependence of the mutation on the race of the patients revealed some predominance of the wild type in the Asian group – 94 (51.4 %), while Europeans were more often detected with the KRAS mutation – in 81 (54.4 %) patients. Mutation of the KRAS gene was more frequently observed in the group of older patients.

G12V and G12C mutations in the gene KRAS are associated with a poorer prognosis in primary colorectal cancer.

The increased incidence of colorectal cancer (CRC) has necessitated the development of novel prognostic and predictive factors from which new diagnostic tests could evolve. Evidence suggests the KRAS gene represents such a factor; its mutations are considered to be early indicators of CRC progression. This study assessed the prognostic impact of specific known KRAS codon 12/13 mutations on survival in patients with CRC. Formalin-fixed paraffin-embedded tissue blocks or sections from primary were obtained from patients registered between 2014 and 2016 for genomic DNA extraction. KRAS gene was analyzed by direct sequencing or Luminex assay. The primary endpoint was the frequency of KRAS gene mutations and the secondary endpoints were differences in KRAS mutation rates by various stratification factors. Univariate and multivariate analyses were performed to investigate relationships between KRAS mutation rates and patient background factors. Sequencing of 200 CRC primary tumor samples demonstrated 74 (37.5%) with KRAS mutations in codons 12 (77%; 57/74) and 13 (23%; 17/74), all of which were TNM stages I-III. Tumors with KRAS mutations were more frequently located in the right side of the colon. Multivariate analysis indicated that G12V or G12C mutations were associated with poor prognosis [hazard ratio (HR) = 3.77, 95% confidence interval (CI), 1.54-8.39 and HR = 6.57; 95% CI, 1.90-17.7, respectively] in terms of recurrence-free survival. KRAS codon 12G-to-V or G-to-C mutations are independent prognostic factors in patients with stage I-III CRC.

No Correlation between KRAS Status and Advanced Pancreatic Adenocarcinoma Survival

Erlotinib plus gemcitabine is one of the standard chemotherapies for unresectable pancreatic cancer. Pancreatic cancer has the highest frequency of KRAS gene mutations among human cancers, and some studies suggest that KRAS status might be a predictive biomarker for anti-epidermal growth factor receptor treatment. However, the reliability of this biomarker has not been confirmed. Here, we evaluated the impact of KRAS mutations in pancreatic cancer patients treated with first line gemcitabine-based chemotherapy. 23 patients treated with gemcitabine-based chemotherapy whose KRAS status could be examined from primary or metastatic lesions were enrolled. KRAS mutations were analyzed by sequencing codons 12 and 13. We retrospectively evaluated the correlation between KRAS status, and prognosis and treatment efficacy. Patient characteristics were as follows: median age 68 years, male/female=6/17, PS 0/1=9/14, TNM stage III/IV=1/22, and gemcitabine alone/erlotinib plus gemcitabine=13/10. Among the 23 patients, KRAS codon 12 was mutated in 15, one of whom also had mutation on codon 13. Median progression-free survival (PFS) and overall survival (OS) of all patients were 4.3 months (95% confidence interval (CI): 3.1 to 5.4) and 8.1 months (95% CI: 5.9 to 10.0; events in 96%), respectively. KRAS status showed no association with PFS (p=0.310), OS (p=0.934), or the efficacy of treatment with (p=0.833) or without erlotinib (p=0.478). Thus, in this study, there was no correlation between KRAS status and the efficacy of first line chemotherapy with gemcitabine with or without erlotinib. Identification of a rationale for personalized medicine in pancreatic cancer will require further exploratory prospective studies.

Roles of EGFR and KRAS and their downstream signaling pathways in pancreatic cancer and pancreatic cancer stem cells.

Pancreatic cancer is currently the fourth most common cancer, is increasing in incidence and soon will be the second leading cause of cancer death in the USA. This is a deadly malignancy with an incidence that approximates the mortality with 44,000 new cases and 36,000 deaths each year. Surgery, although only modestly successful, is the only curative option. However, due the locally aggressive nature and early metastasis, surgery can be performed on less than 20% of patients. Cytotoxic chemotherapy is palliative, has significant toxicity and improves survival very little. Thus new treatment paradigms are needed desperately. Due to the extremely high frequency of KRAS gene mutations (>90%) detected in pancreatic cancer patients, the roles of the epidermal growth factor receptor (EGFR), Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTORC1/GSK-3 pathways have been investigated in pancreatic cancer for many years. Constitutively active Ras can activate both of these pathways and there is cross talk between Ras and EGFR which is believed to be important in driving metastasis. Mutant KRAS may also drive the expression of GSK-3 through Raf/MEK/ERK-mediated effects on GSK-3 transcription. GSK-3 can then regulate the expression of NF-kappaB which is important in modulating pancreatic cancer chemoresistance. While the receptors and many downstream signaling molecules have been identified and characterized, there is still much to learn about these pathways and how their deregulation can lead to cancer. Multiple inhibitors to EGFR, PI3K, mTOR, GSK-3, Raf, MEK and hedgehog (HH) have been developed and are being evaluated in various cancers. Current research often focuses on the role of these pathways in cancer stem cells (CSC), with the goal to identify sites where therapeutic resistance may develop. Relatively novel fields of investigation such as microRNAs and drugs used for other diseases e.g., diabetes, (metformin) and malaria (chloroquine) have provided new information about therapeutic resistance and CSCs. This review will focus on recent advances in the field and how they affect pancreatic cancer research and treatment.

KRAS gene somatic mutations in Chilean patients with colorectal cancer

The molecular testing of KRAS mutation status in metastatic colorectal cancer patients is mandatory to identify patients eligible for anti-epidermal growth factor receptor monoclonal antibody therapy. To report the frequency of KRAS gene mutations in Chilean patients with colorectal cancer (CRC). A cohort of 262 Chilean patients with CRC aged 26 to 90 years (53% males), was studied. KRAS mutation status was analyzed by real-time polymerase chain reaction and correlated with clinicopathological data. Ninety-eight patients (37%) were positive for KRAS mutations. G12D was the most common mutation with a frequency of 36.7%, followed by G12V (25.5%), G13D (17.3%), G12A (7.1%), G12C (6.1%), G12S (5.1%) and G12R (2%). The frequency of the mutation in left, right colon and rectal tumors was 37.8, 32.6 and 44.9%, respectively. Among tumors with mutations, 86.7% were well or moderately differentiated tumors and the rest were poorly differentiated. No significant associations between KRAS gene mutations and other clinicopathological features of the tumor were observed. The frequencies of KRAS mutations reported in this study are similar to frequencies reported for European and North-American populations, lower than in a Spanish study and higher than in a Peruvian study.

Clinicopathologic features of patients with KRAS wild-type pancreatic adenocarcinoma.

192 Background: Pancreatic adenocarcinoma (PC) has the highest frequency of KRAS gene mutations among human cancers. A better understanding of disease biology may help improve targeted therapies for this disease. There is limited data about clinical, pathological features and treatment outcomes in patients (Pts) with wild type KRAS tumors (KRASwt). Methods: Retrospective review of 71 pts with histologically confirmed PC between 2009 and 2012 was undertaken. The KRAS codon 12 and 13 and BRAF codon 600 mutations were assessed in diagnostic tumor tissues (TrimGen Mutector II Shifted Termination Assay). Differences were evaluated with t-test and Fisher exact test. Results: KRAS mutations were identified in 50 tumors (70%), while 21 tumors (30%) had KRASwt genotype. Almost all (96%) mutations occurred at codon 12, there were no mutations in codon 13, and none of the KRASwt tumors harbored BRAF mutations. Pts with KRASwt tumors were mostly white (76%), females (57%), and did not have diabetes mellitus (80%). At diagnosis, pts with KRASwt tumors had a tendency to present at younger age (median age of 60 vs. 66 y; P = 0.1) and with lower CA19-9 values (median CA19-9: 392 vs. 3371 U/mL; P=0.042). Head of the pancreas was the primary site of disease in the majority of pts with KRAS wt tumors (58%), while body or tail was the primary site of disease in the majority of pts with KRAS mutant tumors (53%). There was no difference in the histologic differentiation. Although the majority of pts with KRAS wt (55%) and mutant tumors (57%) presented with distant metastases or locally advanced disease (40% and 39%; respectively), liver involvement was seen in only 55% of pts with KRAS wt tumors vs 92% in the pts with mutant tumors (p=0.016). In univariable analysis, age was an independent predictor of overall survival (HR 1.14, p=0.037) in pts with KRAS wt tumors, while level of bilirubin at presentation was a predictor of overall survival (HR 1.86 (0.77, 4.53); P <0.0001) in pts with KRAS wttumors who had distant metastasis. Conclusions: The frequency of pts with PC who had KRASwt tumors was higher than previously described. Pts with KRASwt tumors may have a disease that is biologically different than those with mutated KRAS. Further prospective characterization is warranted.

KRAS Mutational Status in Japanese Patients with Colorectal Cancer: Results from a Nationwide, Multicenter, Cross-sectional Study

KRAS gene mutations are a useful predictive factor for the efficacy of anti-epidermal growth factor receptor therapeutics. Since there were no large-scale studies among Asian populations, we designed an observational nationwide study in Japan. Formalin-fixed paraffin-embedded tissue blocks or sections from primary or metastatic lesions were obtained from patients registered between 2009 and 2010 for genomic DNA extraction. KRAS gene was analyzed by direct sequencing or Luminex assay. The primary endpoint was the frequency of KRAS gene mutations and the secondary endpoints were differences in KRAS mutation rates by various stratification factors. Univariate and multivariate analyses were performed to investigate relationships between KRAS mutation rates and patient background factors. We analyzed 5790 eligible samples out of 5887 registered. The overall KRAS mutation rate was 37.6%, with 29.9% in codon 12 and 7.7% in codon 13, and wild type was 62.4%. A significant relationship with the KRAS mutation rate was found for gender, age, the year that the sample was prepared and the site of the primary lesion. The KRAS mutation rate of Japanese colorectal cancer patients was 37.6%. Gender, age, the site of the primary lesion and the year that the sample was prepared were independent risk factors for KRAS mutations.

Detection of KRAS Mutations and Their Associations with Clinicopathological Features and Survival in Chinese Colorectal Cancer Patients

Mutation of the KRAS (v-Kiras2 Kirsten rat sarcoma viral oncogene homologue) gene plays an important role in colorectal tumorigenesis. This study examined associations between KRAS gene mutations and clinicopathological and survival data in Chinese patients with colorectal cancer (CRC). CRC patients were recruited for the detection of KRAS gene mutations using polymerase chain reaction and DNA sequencing. Data on clinicopathological features and survival times were collected. The study included 78 CRC patients. The overall mutation frequency of the KRAS gene at codons 12 and 13 was 33.3% (26/78). KRAS gene mutations were significantly associated with poor tumour differentiation and liver metastasis. Patients with the wild-type KRAS gene had significantly higher median survival times than patients with KRAS gene mutations (35.05 months versus 25.72 months). Those with KRAS gene mutations at codons 12 or 13 did not have significantly different median survival times (25.69 months versus 20.67 months, respectively). These findings suggest that a high frequency of KRAS gene mutations exists in Chinese patients with CRC, and that such mutations are associated with poor survival, tumour differentiation and liver metastasis in CRC patients.

Association of KRAS G12D mutation with outcome in patients with pancreatic cancer after surgery.

221 Background: The frequency of KRAS gene mutations in pancreatic adenocarcinoma is high and most mutations occur in codon 12 and 13. However, the association of KRAS mutation status with clinical outcome of pancreatic cancer patients after surgery is not clearly understood. The aim of present study was to clarify whether presence of mutation in KRAS is of prognostic significance, and to determine whether the different type of KRAS mutation have different impact on clinical outcome in pancreatic cancer. Methods: A total of 62 pancreatic adenocarcinoma tissues from patients underwent curative surgery were analyzed for KRAS mutation. DNA was extracted from the tissue microarray. The domain containing the KRAS gene codons 12 and 13 was amplified by PCR, and KRAS mutation was detected by loop hybrid mobility shift assay (LH-MSA). Results: KRAS mutation was detected in 41 of 62 tumors (66.1%). Mutations of KRAS codon 12 and codon 13 were detected in 37 of 62 tumors (59.7%) and in 4 of 62 tumors (6.5%), respectively. G12D mutation were detected in 35.5% (22/62) and G12V mutation in 19.4% (12/62) of all. The patients with KRAS mutations had a significantly shorter overall survival (OS) (log rank, p=0.0375) and disease free survival (DFS) (p=0.0175). When the type of mutation were evaluated, G12D mutation was significantly associated with both shorter OS (p=0.0410) and DFS (p=0.0214), whereas G12V mutation was associated with neither OS (p=0.6727) nor DFS (p=0.9854). Conclusions: The presence of KRAS mutation was significantly associated with poor outcome of pancreatic cancer patients after surgery. A specific mutation G12D demonstrated significant association with shorter OS and DFS in pancreatic cancer. The different type of KRAS mutation might have different impact on clinical outcome in pancreatic cancer.

Cetuximab in small bowel adenocarcinoma: a new friend?

Sir, Small bowel adenocarcinoma (SBA) is a rare and aggressive tumour. SBA in the United States increased from 5.7 cases per million in 1973 to 7.3 cases per million in 2004 (Surveillance Epidemiology and End Results (SEER), 1973–2004 database; Jemal et al (2009). Surgery is the mainstay of treatment, even if chemotherapy in advanced disease has been associated with an increased survival. The most effective agents include 5-FU, irinotecan, platinum agents and gemcitabine (Fishman et al, 2006; Speranza et al, 2010). The molecular characterisation of this cancer could help to improve prognosis. Specifically, the frequency of KRAS gene mutations is similar than in colorectal cancer (Ari et al, 1997). The role of targeted therapy, specifically of epidermal growth factor receptor (EGFR) inhibitors, has never been investigated in patients with SBA. A recent report showed that a high percentage of tumours express both EGFR and VEGF-A suggesting that these patients could benefit from therapeutic strategies targeting EGFR and VEGF receptors (Overman et al, 2010). In our institution four patients with advanced SBA (three men and one woman) were enrolled to receive cetuximab in combination with chemotherapy. The primary site of disease was duodenum in two patients and jejunum in the other two patients. All patients had peritoneal carcinosis, whereas one presented also bone, abdominal lymph nodes and liver metastases. Cetuximab (250 mg mq−1, with a loading dose of 400 mg mq−1) was associated with CPT-11-based chemotherapy in first- (two patients) or second-line (two patients) therapy for metastatic disease. The patients previously treated progressed with folfiri during first-line chemotherapy. The median number of weekly cetuximab cycles was 14 (range: 8–28). One patient obtained a complete peritoneal response, two patients a partial peritoneal response and one patient stable disease in all the disease localisations. Two patients have a time to progression with cetuximab of 3 months, whereas the other two are still on cetuximab after 3 and 10 months. Two patients are still alive (overall survival of 7 and 17 months), the other two had an overall survival of 35 and 19 months. Treatment was well tolerated, with one patient presenting grade 3 neutropenia and grade 3 diarrhoea. Nobody needed to have a dose modification. In three patients KRAS status was tested, resulting in KRAS wild-type mutational status. The same patients presented a G2 skin rash, that was associated with the two partial responses and the complete response. Table 1 shows the main characteristics and the results. Table 1 Patients' characteristics and results obtained with cetuximab These case reports show that anti-EGFR therapy may have a role in SBA, especially in those patients harbouring a wild-type KRAS status. A prospective trial is needed to explore and to support these preliminary observations.

K-RAS gene mutations in patients with non-small cell lung cancer

背景与目的最近研究显示存在K-RAS基因突变的非小细胞肺癌患者难以从辅助化疗中获益，并且对表皮生长因子受体（epidermal growth factor receptor, EGFR）酪氨酸激酶抑制剂（tyrosine kinase Inhibitors, TKIs）耐药。这些发现提示K-RAS基因突变情况可作为EGFR TKIs疗效的预测指标。本研究中分析了中山大学肿瘤防治中心非小细胞肺癌患者肺癌组织中K-RAS基因突变情况。方法收集52例非小细胞肺癌患者的新鲜组织标本，采用PCR技术扩增K-RAS基因，然后进行DNA测序并进行相应分析。结果在52例患者中，2例患者肿瘤组织中的K-RAS基因的12号密码子存在突变（2/52, 3.8%）。统计学分析未发现K-RAS基因突变与性别、病理类型、吸烟情况以及肿瘤分化程度和分期间存在相互关系。结论非小细胞肺癌患者K-RAS基因突变率较低，与亚裔患者相近，而低于白种人患者。

PTU-079 K-ras gene mutations in pancreatic cancer and chronic pancreatitis patients in North Indian population

<h3>Introduction</h3> Pancreatic cancer is one of the most common malignant tumours in the digestive tract in both sexes. Pancreatic adenocarcinoma shows the highest frequency of K-ras gene mutations among the common human cancers but the reasons are unknown. There is lack of knowledge of prevention, early diagnosis, and effective therapy. The molecular mechanism of pancreatic carcinogenesis remains to be fully explored from India. The study was designed to clarify the sensitivity and validity of K-ras point mutational analysis at codon 12 and codon 13 in North Indian patients with pancreatic diseases, and the possible correlation between the presence of mutation and histopathological findings. <h3>Methods</h3> Sixty-five North Indian patients with pancreatic ductal adenocarcinoma, and fifty chronic pancreatitis were enrolled in this study. Out of 65 pancreatic cancer, 24 patients were female (36.92%) and 41 patients were male (63%) having an average age of 47.42±11.32 years (range 22–70 years), 50 chronic pancreatitis only seven were female (14%) and 43 were male (86%) having an average age of 36.02±10.55 years (range 20–63 years). Codon 12 K-ras mutations were examined using the two step polymerase chain reaction (PCR) with RFLP, followed single-strand conformation polymorphism (SSCP) and automated DNA sequencing. <h3>Results</h3> The sensitivity of K-ras mutational analysis in surgically resected tissue samples of the pancreatic carcinoma was 72.30% (47/65) and mutations were found in 63% which was significantly higher than the chronic pancreatitis 33% (5/15) (P&lt;0.01). The mutation pattern of K-ras codon 12 in pancreatic carcinoma 63% while it was 12% in chronic pancreatitis. The nucleotide sequences of K-ras showed the following transitions at codon12 GAG to GTG, GGC to GGG. The control group showed wild type GGA and GGC at codon12. It was also found that K-ras mutation rate was progressively increased from normal duct at the tumour-free resection margin to pancreatic carcinoma. <h3>Conclusion</h3> Overall, our findings support the observations that K-ras mutations play a key role in dysregulated growth of pancreatic cancer and may explain at least in part the aggressive behaviour of this tumour type.

Mutación del gen K-ras en el cáncer de la vesícula biliar

Different K-ras mutation frequencies in gallbladder cancer have been reported. To study the frequency of K-ras gene mutations in advanced gallbladder carcinoma not associated to anomalous junction of pancreatic-biliary duct (AJPBD). 33 formalin fixed paraffin embedded samples of gallbladder carcinoma (30 women, age range 32-86 years) were selected. Pancreatic cancer tissue with K-ras mutations was used as control. DNA was extracted from the histological section by mean of microdissection and K-ras mutations in codon 12 were detected by polymerase chain reaction and restriction fragment length polymorphism (RFLP), using previously reported technique. Most cases were poorly differentiated adenocarcinomas. K-ras mutation was detected in 10 cases (30%) samples. No differences in K-ras mutation frequency between subserous and serous tumors were detected and no relation between histological features and the mutation was observed. K-ras mutation in codon 12 is present in 30% in our advanced gallbladder carcinomas. The study of K-ras mutation in preneoplastic lesions and early carcinonmas will be important to determine the role of this gene in the gallbladder carcinogenesis in Chile.

DOC-2/hDab2 Expression Is Up-Regulated in Primary Pancreatic Cancer but Reduced in Metastasis

DOC-2/hDab2 (DOC-2) has tumor suppressive functions in ovarian cancer and choriocarcinoma. In these tumors, it negatively influences mitogenic signal transduction of growth factors and blocks ras activity. Pancreatic cancer exhibits a high frequency of K-ras gene mutations; however, it is not known whether DOC-2 expression is altered in these tumors. Therefore, we investigated DOC-2 expression in 22 pancreatic adenocarcinomas and in 6 pancreatic cancer cell lines. Findings in human tumors were compared with normal controls and correlated with clinicopathological data. Additionally, the influence of K-ras on DOC-2 transcription was investigated. Northern blot and Western blot analyses both demonstrated an increase of DOC-2 mRNA and protein levels in primary pancreatic cancers in comparison with normal controls. In situ hybridization showed DOC-2 mRNA expression in the majority of cancer cells of primary tumors, as well as in chronic pancreatitis-like lesions surrounding the cancer mass. Immunohistochemistry mirrored the in situ hybridization findings. In contrast, levels of expression of DOC-2 in lymph node metastases were markedly decreased in comparison with levels in primary tumors. In addition, in 5 metastatic pancreatic cancer cell lines, DOC-2 mRNA and protein levels were low, whereas quantitative RT-PCR demonstrated relatively higher levels in a nonmetastatic pancreatic cancer cell line. In conclusion, DOC-2 is overexpressed in primary pancreatic adenocarcinoma but down-regulated in metastatic disease, suggesting a tumor suppressor function of DOC-2 in the late steps of pancreatic carcinogenesis.

Analysis of K-ras gene mutations in human pancreatic cancer cell lines and in bile samples from patients with pancreatic and biliary cancers

The ras family of oncogenes are the most frequently activated group of dominant transforming genes in both human and experimental cancers. The ras family of genes encode highly similar proteins with molecular weights of 21 kDa which are thought to play a key role in signal transduction. Activation in vivo by point mutations results in the ras p21 protein being maintained in the activated form and stimulating cellular proliferation autonomously. Point mutations at codon 12 of K-ras have been observed in >75% of cases of adenocarcinomas of the exocrine pancreas. The type and frequency of K-ras gene mutations in pancreatic cancer cell lines and in bile samples from patients with cytologically-proven biliary tract malignancies and from patients with non-malignant disorders of the biliary tract were determined. Codons 12, 13 and 61 of the K-ras gene were analysed by using restriction fragment length polymorphisms created through mismatched primers during polymerase chain reaction (PCR) of genomic DNA. A mutation of codon 12 of K-ras was detected in 10 of 13 (77%) human pancreatic cancer cell lines. The amino-acid substitutions were glycine to aspartate (5 samples), arginine (2), valine (2) and cysteine (1). No mutations were found at codons 13 or 61. A mutation at codon 12 of K-ras was detected in 9 of 18 (50%) of bile samples analysed. Eleven bile samples had positive cytology for malignancy of pancreaticobiliary origin, and 4 (36%) of these had a codon 12 mutation. Mutations were detected in 5 of the 7 (71%) cytologically-negative bile samples, although malignancy was subsequently diagnosed in 2 of these patients on further histology, and was suspected in 3 other cases on clinical and radiological criteria. This method provides a rapid determination of K-ras gene mutations in bile samples for patients with pancreatic and biliary tract diseases, which may be useful when considering future therapy directed at inhibition of activated ras-induced signal transduction pathways.