Detection Of K-ras Point Mutation Research Articles

High-resolution and quantitative spatial analysis reveal intra-ductal phenotypic and functional diversification in pancreatic cancer.

A 'classical' and a 'basal-like' subtype of pancreatic cancer have been reported, with differential expression of GATA6 and different dosages of mutant KRAS. We established in situ detection of KRAS point mutations and mRNA panels for the consensus subtypes aiming to project these findings to paraffin-embedded clinical tumour samples for spatial quantitative analysis. We unveiled that, next to inter-patient and intra-patient inter-ductal heterogeneity, intraductal spatial phenotypes exist with anti-correlating expression levels of GATA6 and KRASG12D . The basal-like mRNA panel better captured the basal-like cell states than widely used protein markers. The panels corroborated the co-existence of the classical and basal-like cell states in a single tumour duct with functional diversification, i.e. proliferation and epithelial-to-mesenchymal transition respectively. Mutant KRASG12D detection ascertained an epithelial origin of vimentin-positive cells in the tumour. Uneven spatial distribution of cancer-associated fibroblasts could recreate similar intra-organoid diversification. This extensive heterogeneity with functional cooperation of plastic tumour cells poses extra challenges to therapeutic approaches. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Genotyping of KRAS Mutational Status by the In-Check Lab-on-Chip Platform.

The KRAS oncogene is involved in the pathogenesis of several types of cancer, particularly colorectal cancer (CRC). The most frequent mutations in this gene are associated with poor survival, increased tumor aggressiveness and resistance to therapy with anti-epidermal growth factor receptor (EGFR) antibodies. For this reason, KRAS mutation testing has become increasingly common in clinical practice for personalized cancer treatments of CRC patients. Detection methods for KRAS mutations are currently expensive, laborious, time-consuming and often lack of diagnostic sensitivity and specificity. In this study, we describe the development of a Lab-on-Chip assay for genotyping of KRAS mutational status. This assay, based on the In-Check platform, integrates microfluidic handling, a multiplex polymerase chain reaction (PCR) and a low-density microarray. This integrated sample-to-result system enables the detection of KRAS point mutations, including those occurring in codons 12 and 13 of exon 2, 59 and 61 of exon 3, 117 and 146 of exon 4. Thanks to its miniaturization, automation, rapid analysis, minimal risk of sample contamination, increased accuracy and reproducibility of results, this Lab-on-Chip platform may offer immediate opportunities to simplify KRAS genotyping into clinical routine.

A label-free colorimetric isothermal cascade amplification for the detection of disease-related nucleic acids based on double-hairpin molecular beacon

K-Ras mutations at codon 12 play an important role in an early step of carcinogenesis. Here, a label-free colorimetric isothermal cascade amplification for ultrasensitive and specific detection of K-Ras point mutation is developed based on a double-hairpin molecular beacon (DHMB). The biosensor consists of DHMB probe and a primer-incorporated polymerization template (PPT) designed partly complementary to DHMB. In the presence of polymerase, target DNA is designed to trigger strand displacement amplification (SDA) via promote the hybridization of PPT with DHMB and subsequently initiates cascade amplification process with the help of the nicking endonuclease. During the hybridization and enzymatic reaction, G-quadruplex/hemin DNAzymes are generated, catalyzing the oxidation of ABTS2− by H2O2 in the presence of hemin. Utilizing the proposed facile colorimetric scheme, the target DNA can be quantified down to 4 pM with the dynamic response range of 5 orders of magnitude, indicating the substantially improved detection capability. Even more strikingly, point mutation in K-ras gene can be readily observed by the naked eye without the need for the labeling or expensive equipment. Given the high-performance for K-Ras analysis, the enhanced signal transduction capability associated with double-hairpin structure of DHMB provides a novel rout to screen biomarkers, and the descripted colorimetric biosensor seems to hold great promise for diagnostic applications of genetic diseases.

Abstract B18: Molecular cancer testing of KRAS and miR-21 from EUS-guided biopsies of pancreatic tissue: Utility of aspirates vs. cytology

Abstract Introduction: Currently, there is a lack of reliable markers useable in diagnosis and/or management of the pancreatic ductal adenocarcinoma (PDAC). Although detection of KRAS point mutations for the differential diagnosis and determination of miR-21 expression for diagnostic and prognostic purposes have been widely investigated with promising results, neither KRAS nor miR-21 testing have become routine in clinical practice so far. One of the reasons is that analysis of KRAS mutations and miRNA expression is mainly performed on resected pancreatic tissue the use of which is relevant only for a minority of PDAC patients undergoing of surgical treatment. The present study describes development of a reliable methodology for analysis of KRAS mutations and miR-21 expression from samples acquired by endoscopic ultrasound-guided fine-needle biopsy (EUS-FNB), applicable to all PDAC patients. Consequently, the success rates and clinical validity of KRAS mutation and miR-21 expression analysis in two common types of EUS-FNB samples, i.e. native aspirates and cytology specimens are compared. Experimental: The study included 118 patients with a confirmed diagnosis of pancreatic ductal adenocarcinoma (PDAC). Each patient underwent EUS-FNB and the sample was divided into two parts, one part was stored in a stabilizing solution as native aspirate and the rest was processed into the cytology specimen. DNA/RNA was extracted and then analyzed for KRAS mutations and miR-21 expression. For both sample types, the yields of DNA/RNA and success rates for KRAS and miR-21 testing were compared along with evaluation of mutant cell fractions. Finally, confirmation of miR-21 prognostic role was tested by Kaplan-Meier analysis. Data Summary: The overall amount of isolated DNA/RNA from native aspirates was significantly lower compared to the cytology specimens (147 ng vs. 642 ng for DNA, 10 ng vs 164 ng for RNA). The success rates for subsequent KRAS and miR-21 analysis was 100% for both sample types. The KRAS-mutant detection rates in native aspirates were 12% lower than in cytology specimens (78% vs. 90%). The average fraction of KRAS-mutant cells was lower in native aspirates (31%) compared to the cytology specimens (56%). The prognostic role of miR-21 in native aspirates did not reach statistical significance (p = 0.06), but was confirmed in cytology specimens (p = 0.02). Conclusions: Although both types of EUS-FNB samples are suitable for DNA/RNA extraction and subsequent DNA mutation and miRNA expression analysis, reliable results with clinical validity were only obtained for cytological specimens. Native aspirates exhibit a low and undefined fraction of tumor cells leading to false interpretation of molecular testing results. EUS-FNB samples in the form of cytology specimens are a perspective source for the study of molecular markers in virtually all PDAC patients, including patients in inoperable stages. Supported by Internal Grant Agency of the Czech Ministry of Health (IGA) grant No. NT 13638 Citation Format: Lucie Benesova, Tereza Halkova, Bohus Bunganic, Barbora Belsanova, Eva Traboulsi, Miroslav Zavoral, Marek Minarik.{Authors}. Molecular cancer testing of KRAS and miR-21 from EUS-guided biopsies of pancreatic tissue: Utility of aspirates vs. cytology. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B18.

Chronocoulometric biosensor for K-ras point mutation detection based on E. coli DNA ligase and AuNPs amplification effects

We report a novel method for the amplified detection of K-ras point mutation by using a chronocoulometric DNA sensor based on Escherichia coli DNA ligase and AuNPs amplification effects. Our method is based on a ‘sandwich’ detection strategy, involving capture probe immobilized on a gold electrode (GE) and reporter probe loaded on Au nanoparticles (AuNPs probe). Each probe flanks one of two fragments of the target DNA sequence. Successive hybridization event brings AuNPs, along with hundreds of reporter probes to form the ‘sandwich’ structure. After high-fidelity E. coli DNA ligase reaction and denaturing at an elevated temperature, We then employ chronocoulometry to detect [Ru(NH3)6]3+ electrostatically bounding to the DNA probe strands. If there is an allele mismatch, no ligation takes place. In this way, the novel approach had successfully distinguish the K-ras mutant DNA and the K-ras wild DNA. The charge variable (ΔQ) is proportional to the logarithmic K-ras mutant DNA concentration from 1.0nM to 0.1pM, with the detection limit of 0.01pM. Owing to its ease of operation and low detection limit, it is expected that the proposed approach may hold great promise in both research-based and clinical genomic assays.

Detection of K-ras Point Mutation in Pancreatic Cancer Electrochemiluminescence-Polymerase Chain Reaction

An electrochemiluminescence-polymerase chain reaction (ECL-PCR) method for rapid detection of K-ras point mutation was developed. Briefly, the target gene was amplified by a Ru(bpy) 3 2+-labeled forward primer and a biotin-labeled reverse primer, and then followed by digestion with a restriction enzyme, MvaI, which only cut the wild-type amplicon containing its cutting site. Digested product was detected by the ECL assay after adsorption of the resulting DNA duplex to the solid phase. Thirteen pancreatic cancer samples were analyzed by the ECL-PCR assay. The positive rate of K-ras point mutation was 92.3%. The ECL-PCR method is useful in point mutation detection owing to its sensitivity, rapidness, simplicity, and safety. It can be used to detect a point mutation that creates or destroys a restriction site in any gene.

Detection of K-Ras Point Mutation Increases the Sensitivity of EUS-Guided FNA for the Diagnosis of Pancreatic Adenocarcinoma

Background: in larger series, EUS-guided FNA sensitivity for the diagnosis of pancreatic adenocarcinoma (PA) is around 85 and 90%. Mutational activation of the K-ras at codon 12 has been demonstrated in 70-100% of the cases of PA. Aim: to evaluate the efficacy of EUS-guided FNA biopsy combined with K-ras point mutational analysis for the diagnosis of PA. Patients and Methods: From May/02 to April/04, 78 consecutive patients were submitted to EUS-FNA for the presence of a solid pancreatic mass. Five patients were excluded because the final diagnosis were peripancreatic malignant lymph nodes or tumors.

K-RAS POINT MUTATION DETECTION BY PCR/RFLP ANALYSIS VERSUS HISTOPATHOLOGY IN HAMSTER EXPERIMENTAL PANCREATIC CANCER MODEL—WHICH SENSITIVITY IS HIGHER IN DETECTION OF METASTASES?

We have previously established a curative resection model in hamster experimental pancreatic cancer. Pathological examination should be enriched by sensitive methods to detect residual disease. Thus, a method of early diagnosis would be helpful for better follow-up. K-ras mutation has a relationship > 80% with pancreatic cancer. The purpose of this study was to clarify whether the sensitivity of histopathological (H) and molecular level (ML) differs in the diagnosis of metastatic sites. HaP-T1, a cell line derived from BHP induced pancreatic cancer was used in these experiments. A tissue derived from subcutaneously implanted cancer cells was implanted orthotopically. Partial pancreatectomy and splenectomy were done. Hamsters were divided in 3 groups: 1. Positive control (n=33), 2. Surgery performed at Day 7 (n=30), and 3. Surgery performed at Day 14 (n=27). Three animals of each group were sacrificed every 7 days until Day 77 and necropsy was performed. Surgically-resected and necropsied specimens were sent for histopathology and for detection of K-ras point mutation by PCR/RFLP analysis. Positive controls showed metastases in the H starting from Day 35 in 13 cases (39.4%) and in the ML starting from Day 21 in 22 cases (66.6%). Groups 2 and 3 showed free margins in the surgically-resected specimens. In Group 2, tumoral recurrence was detected from Day 42 in one case (3.3%) in the H and in 2 cases (6.6%) in the ML. In Group 3, metastases were detected from Day 35 in one animal (3.3%) in the H and ML in 2 cases (6.6%). PCR/RFLP analysis sensitivity rate was higher when compared with histopathological findings. These experiments showed the importance of possible use of this method for better staging and follow up of pancreatic cancer.

Detection of K-ras Point Mutation Increases the Efficacy of EUS-FNA for the Diagnosis of Ductal Pancreatic Adenocarcinoma

Detection of K-ras Point Mutation Increases the Efficacy of EUS-FNA for the Diagnosis of Ductal Pancreatic Adenocarcinoma

Detection of K-ras point mutation and telomerase activity during endoscopic retrograde cholangiopancreatography in diagnosis of pancreatic cancer

To study the value of monitoring K-ras point mutation at codon 12 and telomerase activity in exfoliated cells obtained from pancreatic duct brushings during endoscopic retrograde cholangiopancreatography (ERCP) in the diagnosis of pancreatic cancer. Exfoliated cells obtained from pancreatic duct brushings during ERCP were examined in 27 patients: 23 with pancreatic cancers, 4 with chronic pancreatitis. K-ras point mutation was detected with the polymerase chain reaction and restriction fragment-length polymorphism (PCR-RFLP). Telomerase activity was detected by PCR and telomeric repeat amplification protocol assay (PCR-TRAP-ELISA). The telomerase activities in 27 patients were measured in 21 exfoliated cell samples obtained from pancreatic duct brushings. D450 value of telomerase activities in pancreatic cancer and chronic pancreatitis were 0.446+/-0.27 and 0.041+/-0.0111, respectively. Seventy-seven point eight percent (14/18) of patients with pancreatic cancer and none of the patients with chronic pancreatitis showed telomerase activity in cells collected from pancreatic duct brushings when cutoff value of telomerase activity was set at 2.0. The K-ras gene mutation rate (72.2%) in pancreatic cancer was higher than that in chronic pancreatitis (33.3%) (P<0.05). In considering of both telomerase activities and K-ras point mutation, the total positive rate was 83.3%(15/18), and the specificity was 100%. Changes of telomerase activities and K-ras point mutation at codon 12 may be an early event of malignant progression in pancreatic cancer. Detection of telomerase activity and K-ras point mutation at codon 12 may be complementary to each other, and is useful in diagnosis of pancreatic cancer.

Detection of K-ras point mutations in sputum from patients with adenocarcinoma of the lung by point-EXACCT.

AbstractLung cancer is a disease with high incidence and mortality (1). The prognosis for patients with lung cancer is most favorable when tumors are detected early in a surgically resectable stage of non-small cell lung cancer (NSCLC). Methods that can increase the percentage of cases of lung cancer detected in early stage may theoretically lead to a decrease in mortality. However, in the past, screening programs for early detection of lung cancer that used chest X-ray of the thorax with or without sputum cytology have not been successful in reducing the high numbers of lung cancer deaths (2–5). Therefore, new approaches that use genetic alterations as potential biomarkers may be beneficial for early detection. KeywordsSputum SampleRelative Optical DensityOptical Density SignalSputum SpecimenReconstruction ExperimentThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Curative resection of orthotopically implanted pancreatic cancer in Syrian golden hamsters.

Homologous orthotopic implantation models of pancreatic ductal cancer in Syrian golden hamsters can mimic the clinical features of the disease in humans. However, there have been no studies attempting a surgical approach to this animal model. Our aim was to clarify the possibility of curative resection in these tumor-bearing animals. A nitrosamine-induced pancreatic ductal adenocarcinoma cell line, HaP-T1, was used for the experiments. Hamsters underwent two surgeries: first, the cells or the tissue previously grown subcutaneously were implanted into the pancreas, and second, pancreatectomy and splenectomy were performed within the following 4 wk after implantation. Animals were examined until death, when necropsies were performed. Surgically resected and necropsied specimens were studied histopathologically and at the molecular level by detection of K-ras point mutation at codon 12. At the second surgery, the success of implantation was 100%, and the curative resection rate was 75%. After observation for 400 d, 5 of 12 hamsters, which underwent second surgery within 15 d, were still alive. They were sacrificed and were found to be tumor-free. Orthotopic homologous implantation model of hamster pancreatic cancer combined with surgical resection may be useful for further research of adjuvant or neoadjuvant therapy for pancreatic carcinoma.

Detection of K-ras point mutations in sputum from patients with adenocarcinoma of the lung by point-EXACCT.

Kirsten ras (K-ras) point mutations are found in 30% to 56% of pulmonary adenocarcinomas by means of highly sensitive techniques. Recently, the Point-EXACCT (point mutation detection using exonuclease amplification coupled capture technique) method was described, which detected one cell with a mutation in 15,000 normal cells. The aim of this study was to examine whether K-ras point mutations could be found with this rapid method in the sputum of patients with adenocarcinoma of the lung. DNA from paraffin-embedded adenocarcinoma and corresponding sputum samples were analyzed for mutations of the K-ras gene. Twenty-eight biopsy specimens and 54 sputum samples of 22 patients were used for amplification and K-ras codon 12 point mutation detection. In 11 of 22 patients (50%), a mutation in K-ras codon 12 was shown in the tumor sample. In five of 11 patients (45%) with a K-ras mutation in the tumor, the same type of mutation was identified in at least one sputum sample. A mutation could not be detected in any of the sputum samples from patients with a K-ras-negative tumor. Time between K-ras point mutation detection in sputum and clinical diagnosis of lung cancer varied from 1 month to almost 4 years. In two of the five patients with K-ras-positive sputum specimens, malignant cells were found with cytologic examination. Point-EXACCT is suitable for the detection of K-ras point mutations in sputum samples of patients with adenocarcinoma of the lung. This approach may be an important adjunct to cytology in the early diagnosis of lung cancer.

Detection of K-ras point mutations in the supernatants of peritoneal and pleural effusions for diagnosis complementary to cytologic examination.

To determine whether DNA analysis can be performed using the supernatants of body fluids after centrifugation at 2,000 rpm for 10 minutes, peritoneal or pleural effusions or bile were examined for K-ras mutations in 34 cases of pancreatic, colorectal, gastric, esophageal, or hepatocellular carcinoma and 15 noncancer cases. The polymerase chain reaction products for K-ras gene codons 2 to 97 of exons 1 and 2 were generated with 41 (93%) of 44 body cavity fluid and 5 (100%) of 5 bile samples. By the single strand conformation polymorphism method, point mutations were detected in the ascites supernatants of 8 (89%) of 9 cases of pancreatic carcinoma. In the remaining case, no point mutation was demonstrated because few malignant cells were present in the ascites fluid. Furthermore, K-ras point mutations were observed in the ascites supernatants of 2 cases of colorectal carcinoma and 1 case of gastric carcinoma. The DNA analysis of the supernatant of ascites fluid showed a K-ras point mutation in 3 cases of false-negative cytologic diagnosis (2 cases of pancreatic carcinoma and 1 case of colorectal carcinoma). Direct sequencing confirmed identical point mutations in the supernatants, whole cell pellets, malignant cells from the cytologic smears of ascites fluid, and cancer tissues. This novel method allows simultaneous testing for genetic abnormalities in supernatants of body fluid, after removing cells for cytologic diagnosis.

Detection of K-raspoint mutation by enriched PCR–colorimetric plate assay

Point mutations in the K-rasgene are frequently observed in a variety of human malignancies, including colorectal and pancreatic cancers. In this paper, we describe a sensitive procedure for the detection of point mutations of codon 12 of the K-rasgene. The assay employs a single-tube enriched PCR procedure, coupled to colorimetric detection. In the enriched PCR procedure, the first round of amplification introduces a restriction enzyme site in the wild type, but not in mutant K-rasPCR product. The wild type products are then digested and the second round of PCR enriches for the mutant sequences by amplifying the resistant products. The second round of amplification allows the incorporation of biotin and a substrate binding tag at opposite ends of the mutant product, thus allowing detection of the product by a simple colorimetric assay. The assay has been validated using DNA from a variety of cell lines known to contain either mutant or wild type K-ras. Under these conditions, the assay has proved both reproducible and sensitive, with the ability to detect one mutant molecule in a background of 1000 wild type molecules. The assay allowed discrimination of mutant from wild type K-rasin samples from colonic adenocarcinomas and normal colonic mucosa. The use of a colorimetric detection system reduces observer bias and facilitates analysis of large numbers of samples. As such, the assay may have specific application in the sensitive detection of K-rasmutations in a variety of clinical samples.

Detection of K-ras point mutations in mesenteric venous blood from colorectal cancer patients by enriched polymerase chain reaction and single-strand conformation polymorphism analysis.

In order to confirm the presence of cancer cells in mesenteric venous blood and to examine their relationship with the occurrence of liver metastases, we attempted to detect K-ras codon 12 point mutations in perioperative mesenteric blood using enriched polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP) analysis in 25 patients with primary colorectal tumors carrying K-ras point mutations. Among these patients, three with synchronous liver metastases were included. The same K-ras point mutation (substitution of GAT for GGT) was detected in both the blood and the primary tumor in a Dukes' C patient. We confirmed this result by colony hybridization and estimated the tumor-to-normal cell ratio to be 1:400. This patient has no liver metastases two years after surgery and her carcinoembryonic antigen (CEA) level remains normal. We demonstrated that considerable numbers of cancer cells can be found in mesenteric venous blood during colorectal cancer surgery. However, their potential role in the formation of liver metastases remains unclear.

Detection of K-ras point mutations at codon 12 in pure pancreatic juice for the diagnosis of pancreatic cancer by PCR-RFLP analysis.

The present study was undertaken to detect K-ras point mutations at codon 12 in pure pancreatic juice (PPJ) for the diagnosis of pancreatic cancer (PC) using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. PPJ was collected through a cannula under a duodenal fiberscope from 26 patients with PC and 32 patients with chronic pancreatitis (CP). DNA was extracted from PPJ and was used as the template for PCR. Analysis of PPJ by PCR-RFLP with BstNI revealed that the incidence of K-ras point mutations at codon 12 was 81% (21/26) in patients with PC and 6% (2/32) in those with CP. With reference to the location of PC, the incidence of K-ras mutations was 79% (11/14) in the head, 86% (6/7) in the body, and 80% (4/5) in the tail of the pancreas. The incidence of K-ras mutants was 50% (1/2) in tumor size 1 (TS1; < or = 2.0 cm in size), 71% (5/7) in TS2 (2.1 to < or = 4.0 cm), 89% (8/9) in TS3 (4.1 to < or = 6.0 cm), and 88% (7/8) in TS4 (> 6.1 cm). These results suggested that analysis of K-ras point mutations at codon 12 in PPJ using the PCR-RFLP method is a promising new genetic test for the diagnosis of PC.

Detection of K-ras oncogene mutations in bronchoalveolar lavage fluid for lung cancer diagnosis.

Lung cancer is the leading cause of cancer deaths in the United States. A long-standing goal of cancer researchers has been to develop tests that would facilitate earlier diagnosis and treatment of lung cancer and thereby decrease mortality from this disease. Because cancer results from the accumulation of a variety of genetic events (e.g., mutations, rearrangements, and deletions) in genes controlling cell growth and differentiation, these changes might serve as diagnostically useful molecular markers. Activation of the K-ras oncogene by point mutations in codon 12, which occurs in many cases of lung adenocarcinoma, may serve as one such clinically useful molecular marker. For detection of K-ras point mutations in bronchoalveolar lavage fluid, in which small numbers of malignant cells are mixed with a population of predominantly genetically normal cells, the sensitivity of commonly used assays for ras mutations risks false-negative results. By applying a highly sensitive assay, we investigated whether detection of K-ras codon 12 mutations in samples of bronchoalveolar lavage fluid could be clinically useful in diagnosing lung cancer. We developed a highly sensitive assay for detecting K-ras codon 12 mutations based on an enriched polymerase chain reaction (PCR) technique. This technique was applied to 87 specimens of bronchoalveolar lavage fluid specimens that were obtained from 86 patients, and associated tumor biopsy specimens obtained from 35 of these patients who underwent diagnostic bronchoscopy for clinically suspected lung cancer. Statistical comparisons were performed by using the two-tailed Fisher's exact test [corrected]. Of 52 patients with confirmed lung cancer, samples of bronchoalveolar lavage fluid from 16 patients contained K-ras codon 12 mutations, including 14 (56%) of 25 patients with lung adenocarcinomas, one (33%) of three with bronchoalveolar carcinomas, one (20%) of five with large-cell carcinomas, and none of the 14 with squamous cell carcinomas. Mutations were detected in four additional cases in which cancer was suspected but had not been histologically confirmed. Tissue samples from 35 of the patients all yielded the identical K-ras codon 12 genotype found in the corresponding samples of bronchoalveolar lavage fluid. No mutation was found in any sample from 30 patients with diagnoses other than non-small-cell lung cancer. Thus, for those cases in which tissue was available and tested, the sensitivity and specificity of detecting K-ras mutations in bronchoalveolar lavage fluid for diagnosing K-ras mutation-positive lung cancer were both 100%. For nine patients, K-ras mutations were detected in bronchoalveolar lavage fluid obtained during otherwise nondiagnostic bronchoscopies. Our data demonstrate that sensitive detection of K-ras codon 12 mutations can serve as an important adjunct to cytology in the diagnosis of lung cancer. Detection of these mutations could lead to earlier cancer diagnosis and less need for invasive diagnostic procedures.

Diagnosis of pancreatic lesions using fine needle aspiration cytology: detection of K-ras point mutations using solid phase minisequencing.

To improve the diagnostic value of fine needle aspiration biopsy of pancreatic lesions using a simple mutation detection method based on the polymerase chain reaction (PCR). Fine needle aspirates from 21 suspected pancreatic lesions were analysed for K-ras codon 12 point mutations using solid phase minisequencing. A point mutation in codon 12 of the K-ras gene was detected in 14 of 17 cases of pancreatic carcinoma. No false positive results were recorded. The concordance of the result with routine cytology was 78%. All patients diagnosed as having malignant disease on cytology also had a K-ras point mutation. Additional information on the presence of malignancy was obtained using molecular genetic analysis in two cases. PCR based minisequencing is a promising method for the analysis of cytological material. K-ras point mutation analysis was modified to enable it to be carried out in a clinical laboratory. Advantages of the method include its simplicity and speed. Adequate sampling guidance is important but analysis can be performed even with small amounts of cellular material.

K-ras point mutations in routinely processed tissues: non-radioactive screening by single strand conformational polymorphism analysis.

To develop a non-radioactive method to screen routinely fixed, paraffin wax embedded specimens for the occurrence of point mutations; to evaluate the single strand conformational polymorphism (SSCP) analysis technique for the detection of K-ras point mutations as a result of electrophoretic mobility shifts. DNA was extracted from archival specimens of colon cancer and from established colon cancer cell lines with known point mutations. A K-ras gene fragment containing codons 12 and 13 of exon 1 was amplified with the polymerase chain reaction (PCR). Denatured DNA fragments were run on 10% polyacrylamide gels under non-denaturing conditions. After electrophoresis DNA was blotted and the single stranded DNA was detected using a digoxigenin labelled ras probe. The nature of the detected point mutations was identified and confirmed by sequencing and hybridisation with oligonucleotides using 32P labelling. Wild type and aberrant alleles were detected caused by mobility shifts after electrophoresis of the PCR products. Commonly occurring mutations in the K-ras gene--in the first two positions of codon 12--could easily be detected in DNA from archival paraffin wax embedded colon cancer tissue. In all the colon tumour samples studied wild type gene alleles were also found, presumably derived from normal cells in the specimen. The SSCP method permits rapid non-radioactive screening of adenomas or carcinomas for the occurrence of point mutations in the K-ras gene. But if a mutation is detected by an electrophoretic mobility shift, its identification requires confirmation by sequencing or oligonucleotide hybridisation.