Point Mutations Of K-ras Gene Research Articles

Abstract 671: Prognostic importance of EGFR expression and Kras gene mutations in gallbladder carcinoma

Abstract Gallbladder carcinomas involve multiple cascades of oncogenes like EGFR and Kras. Activation of Kras downstream pathways predict the sensitivity to anti EGFR treatment. In 2012, for the first time it was established that GBC and cholangiocarcinomas have their unique somatic genomic landscape and thus be studied independently. This study evaluates whether EGFR is a prognostic factor in GBC and identifies the frequency of codon 12 and codon 13 Kras mutations along with its association with subject survival. Seventy two GBC curative resections from North India were immunostained with anti EGFR monoclonal antibody and assessed for codon 12 and 13 KRAS mutation by real time PCR. Strong EGFR expression was observed in 25.4percent cases. About 28/72; G12C, 17/72; G12R, 10/72; G12D and 18/72; G13D cases harboured point mutations in Kras gene, which was significantly higher as compared to control tissues. Multivariate analysis revealed that EGFR expression (p=0.01; HR=3.14; 95percent CI=1.28-7.71) and codon-13 mutation (p=0.001; HR=38.34; 95percent CI=4.90-300.01) had a significant impact on survival and were independent prognostic factors in GBC. Our study suggests that EGFR expression and codon 13 mutations are independent prognostic factors in the selected GBC cohort and showed a high frequency of Kras codon 12 mutations as well. Citation Format: Anjali Singh, Abul Kalam Najmi, Pramod Mishra, Majid Talikoti. Prognostic importance of EGFR expression and Kras gene mutations in gallbladder carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 671. doi:10.1158/1538-7445.AM2017-671

Additional K-ras mutation analysis and Plectin-1 staining improve the diagnostic accuracy of pancreatic solid mass in EUS-guided fine needle aspiration.

BackgroundOne of the major genetic alterations in pancreatic ductal adenocarcinoma (PDAC) is the point mutation of K-ras gene. Plectin-1 was also recently identified as PDAC specific biomarker. The aim of this study was to investigate the improvement of diagnostic accuracy of endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) by using additional K-ras mutation analysis and Plectin-1 staining in patients with pancreatic mass.MethodsA total of 85 study patients with pancreatic mass underwent EUS-FNA and the final diagnoses were as follows; PDACs: 70 patients, pancreas neuroendocrine tumor: 4, metastasis to pancreas: 5, autoimmune pancreatitis: 3, chronic pancreatitis: 1, tuberculous lymphadenitis: 1, pseudocyst: 1.ResultsSensitivity, specificity and accuracy of pathologic diagnosis in EUS-FNA specimen were 81%, 80% and 79% accordingly. When we combine K-ras gene mutation analysis with histological assessment, we could get the following results for sensitivity, specificity and accuracy; cytology and K-ras mutation analysis: 93%, 87%, and 92%, cytology, K-ras mutation analysis, and Plectin-1 staining: 96%, 93%, and 95%.ConclusionsTriple combinations of the techniques; cytology, K-ras gene mutation analysis, Plectin-1 staining could increase accuracy in diagnosis of PDACs. Further investigation of using minimal specimens from EUS-FNA may give us insight to understand the biological behavior of PDAC.

先天性胆道拡張症における K-ras がん遺伝子変異の解析

先天性胆道拡張症における K-ras がん遺伝子変異の解析

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.

Alterations of KRAS Exon 2 Codon 12/13 Mutation Status in Prostatic Adenocarcinoma; Bioinformatics Aspects

Prostate cancer is one of the most common cancers which develops by mutations or/and other genetic alterations in specific genes. According to the recent studies there are predominant mutations occur in KRAS gene in different types of cancers. Missense mutations in codon 12 and codon 13 of KRAS proto-oncogene are main point mutations that happen in about 20% of diverse malignancies in human. The aim of this study is to find out the prevalence of two known main point mutations of KRAS gene (p.G12V/c.35G>T and p.G13D/c.38G>A) in addition to bioinformatics survey of its in Iranian patients with prostatic adenocarcinoma. A total of 35 prostatic adenocarcinoma fresh tissue samples that enriched in neoplastic cells, were obtained from the Cancer Institute of Iran. The presences of mutations at codons 12 and 13 of KRAS were investigated by direct Sanger sequencing. To investigate of the role of the mentioned mutations on structure and protein function, bioinformatics assessments were performed by using SWISS-MODEL server and the PSIPRED Protein Sequence Analysis software. KRAS mutations were detected in 2 of 35 patients (5.7%). Two cases carried the homozygote mutations on exon 2 in codon 12 (G12V) and codon 13 (G13D), respectively. Several prediction programs such as SIFT, Mutation Taster and polyphen-2 classified them as pathogenic. However, the bioinformatics survey shows that amino acid changes on codons 12/13 do not cause any probable disorder on protein structure, but effect on protein function. The result indicated that the protein function is modified. Based on the group of patients with prostate adenocarcinoma our study indicates that p.G12V/c.35G>T and p.G13D/c.38G>A mutations in codons 12 and 13 KRAS are most frequently occur in prostate carcinomas. The bioinformatics results from this study demonstrate that KRAS may have effects on pathogenesis of prostate cancer.

An electrochemical sensor based on DNA polymerase and HRP-SiO2 nanoparticles for the ultrasensitive detection of K-ras gene point mutation

We developed a DNA sensor for the precise detection of point mutation of K-ras gene. The sensor was based on DNA replication, which employed the DNA polymerase I and the principle of base pairing, and can detect target DNA in mixture sample.

Highly sensitive quantitative detection of circulating tumor DNA in urine and plasma from advanced colorectal cancer patients in aid of early diagnosis of clinically relevant KRAS mutations.

654 Background: Acquisition of point mutations in KRAS gene is causally associated with the onset of development of a resistance to anti-EGFR therapy in colorectal cancer. Newly acquired KRAS mutations can be detected in blood plasma months before radiographic detection. The objective of this study was to demonstrate feasibility of an ultrasensitive non-invasive method for detection of KRAS mutations in urine and plasma of patients with advanced colorectal cancer. Methods: Archived, matched urine and plasma samples (stored between 3-5 years prior to ctDNA extraction) from 20 treatment naïve, advanced stage cancer patients with known tumor tissue KRAS mutations determined by an accredited clinical laboratory, were used in a retrospective setting for a blinded concordance study. KRAS status in urine and plasma was compared to that in tumor tissue in order to assess clinical sensitivity of the ctDNA assay. An ultrashort-amplicon (31bp) assay for KRAS mutation enrichment and detection in highly fragmented urinary and plasma ctDNA was developed. The assay detected 1 copy of KRASG12A/C/D/R/S/V or G13D mutant allele in a background of wild-type DNA with a verified analytical sensitivity of 0.007% (7 copies per ~100,000 genome equivalents). Results: In a pilot study of 20 advanced stage colorectal cancer patients, 15 of 16 evaluable archived urine samples (94%) had KRAS mutation that was concordant with tissue biopsy. Of 20 archived plasma samples evaluated, 19 (95%) displayed the KRAS mutation concordant with tumor tissue. Of 16 paired urine and plasma samples, 15 (94%) had concordant KRAS mutation calls. Conclusions: This study demonstrates high clinical sensitivity (≥94%) of concordant KRAS mutation detection between urine, plasma and tissue specimens from advanced colorectal cancer patients. Early detection and monitoring of acquired KRAS mutations in circulating tumor DNA, and in particular urinary ctDNA, opens the possibility of a new paradigm for a truly non-invasive method of individualized care for colorectal cancer patients.

EVALUATION OF DIAGNOSTIC SIGNIFICANCE OF MOLECULAR-GENETIC MARKERS IN РАNCREATIC ADENOCARCINOMA

The purpose of the study : to evaluate the diagnostic value of molecular genetic markers in pancreatic cancer. Subjects. Data on patients with pancreatic cancer and patients with chronic pseudo tumor-like pancreatitis. The point mutations in K-ras gene were analyzed using polymerase chain reaction (PCR); the mutations in BRCA1, 2 genes were determined, the status of methylation of promoter segment of р16INK4A gene was analyzed. 67 % patients with pancreatic cancer and 63,6 % patients with chronic pancreatitis detected the mutant K-ras gene. 6,7 % patients with pancreatic cancer revealed the BRCA1 gene mutations in the heterozygous state, 13 % patients revealed the gene BRCA2 mutations. Diagnosis of the K-ras gene mutations in patients with pseudo tumor-like pancreatitis is conjugated with high risk of transformation of the pancreas to adenocarcinoma. 41 % patients with pancreatic cancer detected aberrant hypermethylation of р16INK4A gene, which is indicative of its aggressive genotype and poor prognosis.

Association of epidermal growth factor receptor and K-Ras mutations with smoking history in non-small cell lung cancer patients

Lung cancer, a major health problem affecting the epithelial lining of the lower respiratory tract, is considered to be one of the deadliest types of cancer in males and females and it is well-known that smoking is the chief cause of lung cancer. In addition to smoking and environmental factors, genetic susceptibility may also contribute to the development of lung cancer. Previous studies have shown that certain non-small cell lung cancer (NSCLC) patients harbor gain-of-function mutations in the epidermal growth factor receptor gene (EGFR). Phosphorylated EGFR triggers the activation of intracellular signal transduction pathways, including the RAS-MAPK, PI3K-Akt and STAT pathways. However, K-Ras gene point mutations in codons 12, 13 or 61 cause the inactivation of GTPase activity which results in overstimulation of cellular growth and gives rise to neoplastic development. Our aim was to investigate the presence and association of EGFR and K-Ras mutations in 50 primary NSCLC patients with a smoking history by using real-time PCR and sequencing. EGFR mutations were detected in four patients (8%). Two of these mutations were L858R mutations and the remaining two were deletion mutations spanning between codons 746 and 750. The L858R mutation was significantly associated with smoking status (P=0.003). K-Ras codon 12 and 61 mutations were also observed in four patients. However, no association was observed between K-Ras mutations and the tumor staging, gender, histology and smoking status of the patients.

Diagnosis Value of Detection K-Ras Gene Mutations in Patients with Gastric Cancer

Mutations of K-ras gene have been proven to play important roles in human tumor progression. Point mutations of K-ras gene located in codons 61 are gene hot spot mutation regions in patients with gastric cancer. A simple and effective capillary electrophoresis (CE) method for point mutation detection in codon 61 of K-ras gene was established. The polymerase chain reaction (PCR) fragments included codon 61 of K-ras gene were analyzed by single-strand conformation polymorphism (SSCP)-CE method. The CE analysis was performed by using a 1×Tris–borate–EDTA (TBE) buffer containing 3.0% (w/v) poly (ethylene oxide) (PEO) (MW 300 000) under reverse polarity with 15 oC and voltage of 15 kV. Forty-two gastric cancer patients were analyzed using this developed method. K-ras gene mutation was found in 17 cases (40.5 %) and the results showed that K-ras gene mutation in gastric cancer tissue is a usual event, and the SSCP-CE was feasible for mutation detection of K-ras gene in populations. Detection of gene mutation of gastric cancer tissue by PCR - SSCP has a certain value to diagnosis of gastric cancer and to prevent gastric cancer associated with clinical and pathological features, living habits of different patients by detection mutation of K-ras gene codon 61.

Study on point mutations of K-ras gene in non-small cell lung cancer in Guangxi

背景与目的近期研究显示存在K-ras基因突变的非小细胞肺癌（non-small cell lung cancer, NSCLC）患者对表皮生长因子受体-酪氨酸激酶抑制剂（epidermal growth factor receptor-tyrosine kinase inhibitors, EGFR-TKIs）耐药。本研究检测广西地区NSCLC中K-ras基因密码子12、13及61点突变，旨在探讨K-ras基因突变与广西地区NSCLC的关系。方法采用聚合酶链反应-单链构象多态性（polymerase chain reaction-single-strand conformation polymorphism, PCR-SSCP）分析法与PCR-DNA序列分析法联合检测105例新鲜NSCLC手术切除标本和30例癌旁正常肺组织中K-ras基因密码子12、13及61点突变。结果105例NSCLC癌组织及30例癌旁正常肺组织中无1例发生K-ras基因点突变，本研究选取的NSCLC病例组的K-ras基因突变率为0（0/105）。结论广西地区NSCLC中K-ras基因野生型的比例高，提示广西地区NSCLC患者更能从EGFR-TKIs治疗中获益。

Rapid Screening Assay for KRAS Mutations by the Modified Smart Amplification Process

Previously, the smart amplification process version 2 (SMAP-2) was developed to detect mutations from tissue and in crude cell lysates and has been used for rapid diagnosis of specific somatic mutations with single-nucleotide precision. The purpose of this study was to develop a rapid and practical method to detect cancer and metastasis in specimens using the SMAP-2 assay. We developed modified SMAP-2 assays that enabled detection of any change in a single codon using a single assay. Rapid SMAP-2 screening assays are suitable for routine clinical identification of critical amino acid substitutions such as codon 12 mutations in KRAS. Primers bracketing the first two nucleotides of KRAS codon 12 were designed so that all possible alleles would be amplified by the SMAP-2 assay. In combination with the peptide nucleic acid (PNA) with exact homology to the wild-type allele, our assay amplified all mutant alleles except for the wild-type sequence. With this new assay design (termed PNA-clamp SMAP-2), we could detect KRAS mutations within 60 minutes, including sample preparation. We compared results from PNA-clamp SMAP-2 assay, polymerase chain reaction-restriction fragment length polymorphism, and direct sequencing of clinical samples from pancreatic cancer patients and demonstrated perfect concordance. The PNA-clamp SMAP-2 method is a rapid, simple, and highly sensitive detection assay for cancer mutations.

Rapid detection of K-ras gene point mutation in pancreatic adenocarcinoma by Pyrosequencing

Objective To establish a method for detecting K-rag gene point mutation in pancreatic adenocarcinoma based on the pyrosequencing and to compare its performance with that of Sanger sequencing.Methods Genomic DNA was extracted from formalin-fixed,paraffin-embedded pancreatic tissues including 49 pancreatic adenocarcinoma,10 normal pancreas,11 chronic pancreatitis,18 benign pancreatic tumor,7 insulin carcinoma,9 ampullary carcinoma,7 bile duct carcinoma and 7 duodenum papillary adenocarcinoma tissues.K-rag gene point mutations at codon 12 were analyzed by pyrosequencing and Sanger sequencing,respectively.Results No mutant K-ras gene Wag detected in normal pancreas,chronic pancreatitis,benign pancreatic tumor,insulin carcinoma,ampullary carcinoma,bile duct carcinoma and duodenum papillary adenocarcinoma tissues by either pyrosequencing or Sanger sequencing.K-rag gene point mutation was detection rate in pancreatic adenocarcinoma tissues was 71.4%(35/49)by pyrosequencing and 61.2%(30/49)by Sanger sequencing,respectively.Conclusions Pyrosequencing is more sensitive than Sanger sequencing and is also accurate,rapid and of high throushput in detecting mutant K-ras gene.It may serve as a practical method for fast batch analysis of clinical samples. Key words: Pancreatic neoplasms; Genes,rag; Point mutation; Phosphoric acids; Sequence analysis,DNA; Pyrosequencing

Enhanced therapeutic effects for human pancreatic cancer by application K-ras and IGF-IR antisense oligodeoxynucleotides

To investigate the combined effects of K-ras antisense oligodeoxynucleotide (K-ras ASODN) specific to GTT point mutation at codon 12 and type I insulin-like growth factor receptor (IGF-IR) antisense oligodeoxynucleotide (IGF-IR ASODN) on proliferation and apoptosis of human pancreatic cancer Patu8988 cells in vitro and in vivo. K-ras gene point mutation and its style at codon 12 of human pancreatic cancer cell line Patu8988 were detected by using polymerase chain reaction with special sequence primers (PCR-SSP) and sequence analysis. According to the mutation style, K-ras mutation ASODN specific to K-ras point mutation at codon 12 was designed and composed. After K-ras ASODN and IGF-IR ASODN treated on Patu8988 cells respectively or cooperatively, the proliferation and morphological change of Patu8988 cells were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, colony forming assay and transmission electron microscopy; the expression of K-ras and IGF-IR mRNA and protein in the treated cells was measured by reverse-transcript polymerase chain reaction (RT-PCR) and flow cytometry respectively; apoptosis was determined by flow cytometry. The combined antitumor activity of K-ras ASODN and IGF-IR ASODN was evaluated in BALB/c nude mice bearing human pancreatic cancer inoculated with Patu8988 cells. The results of PCR-SSP and sequence analysis showed that the human pancreatic cancer cell line Patu8988 had point mutation at codon 12, and the mutation style was GGT-->GTT. 2-32 microg/mL K-ras ASODN and 2-32 microg/mL IGF-IR ASODN could inhibit Patu8988 cells' growth, induce apoptosis and decrease the expression of K-ras and IGF-IR mRNA and protein alone. However, there was much more effective inhibition of growth and induction of apoptosis by their combination than by each one alone. In tumor bearing mice, the combination of K-ras ASODN and IGF-IR ASODN showed a significant inhibitory effect on the growth of transplanted pancreatic cancer, resulting in a statistically significant difference compared with each alone. It has been found that K-ras ASODN combined with IGF-IR ASODN could cooperatively inhibit the growth of Patu8988 cells, and induce their apoptosis via reinforcing specific down regulation of K-ras and IGF-IR mRNA and protein expression.

Relation between the Expression of K-ras in Hep-2 Cells and Development of Laryngeal Carcinoma

Objective: To investigate the expression of K-ras in human laryngeal squamous cell carcinoma cell lines (Hep-2) and its significance for establishing a solid foundation for further study of the relationship between human laryngeal squamous cell carcinoma and K-ras gene point mutations. Methods: The expression of K-ras in human laryngeal squamous cell carcinoma cell lines (Hep-2) and human pancreatic carcinoma cell lines (MIAPaCa-2) was detected by using RT-PCR. Results: The expression of K-ras mRNA in Hep-2 and MIAPaCa-2 was strong and positive. Conclusion: The expression of K-ras mRNA in human laryngeal squamous cell carcinoma cell lines (Hep-2) is positive. Development of laryngeal carcinoma might be related to the activation of K-ras gene point mutation.

Detection of K-ras Gene Point Mutation's Style in Human Pancreatic Cancer Cell Line PANC-1 by PCR-SSP

Objective: To detect the style of K-ras gene point mutation in human pancreatic cancer cell line PANC-1 and decide the bp sequence of Ras target position interfered by RNA. Methods: Three kinds of special sequence primers (SSP) for polymerase chain reaction (PCR) with regard to the mutation styles (GAT, CGT and GGT) at codon 12 of K-ras were used to study the human pancreatic cancer cell line PANC-1. The amplification products were studied with polyacrylamine gel electrophoresis to detect the style of point mutation. Results: The style of K-ras gene point mutation at codon 12 was GAT in human pancreatic cancer cell line. Conclusion: PCR-SSP is rapid, convenient and high specific. The results provide a basis for further gene therapy by RNA interference for pancreatic cancer.

Combined small‐cell carcinoma of the stomach: p53 and K‐ras gene mutational analysis supports a monoclonal origin of three histological components

Primary small-cell carcinoma (SmCC) is extremely rare in stomach. We reported an autopsy case of combined gastric SmCC with p53 and K-ras mutational analysis. Histologically, the tumour was composed of well-differentiated adenocarcinoma surrounding the central dominant SmCC component with scattered nests of squamous cell carcinoma. Immunohistochemically, all the neoplastic components revealed strong expression for p53 protein. Based on these findings, we hypothesized that these histologically different components originated from a same progenitor cell that possessed p53 mutation. Using Laser-capture microdissection technique and mutational analysis, we identified the same point mutations of p53 gene (A-->G transversion in codon 239) and K-ras gene (G-->A transversion in codon 13) in all the neoplastic components, but not in the adjacent normal gastric epithelium. Our results strongly support a hypothesis that the combined SmCC of stomach in this case was of monoclonal origin.

Survival regulation in pancreatic cancer cells by c-Jun

Over 90% of human pancreatic cancers harbor an activating point mutation in the K-ras gene at codon 12. However, it is not clear whether all downstream K-ras are activated and which downstream contributes to the cell survival and proliferation of pancreatic cancer cells. MEK kinase 1 (MEKK1)-c-Jun N-terminal kinase (JNK)-c-Jun pathway has an important role in cell proliferation, survival and apoptosis in various cells. We previously demonstrated that the dominant negative form of MEKK1 (DN-MEKK) inhibits the survival of human pancreatic cancer cell lines. In this study we investigated whether JNK-c-Jun, the downstream pathway of DN-MEKK, affects the survival of human pancreatic cancer cell lines. Colony formation assays indicated that c-Jun failed to inhibit the survival of pancreatic cancer cells, whereas c-Jun remarkably inhibited the cell survival of non-pancreatic cancer cells. Reporter gene assays using Gal4-c-Jun and gel retardation assays indicated that c-Jun functions were activated in growing pancreatic cancer cells. These results revealed that c-Jun activation does not prevent the cell survival of pancreatic cancer cells in contrast to non-pancreatic cancer cells. It appears that MEKK1-JNK-c-Jun pathway fails to act as a negative regulator for the cell survival of pancreatic cancer cells. Greater understanding of these mechanisms may be helpful in the treatment of pancreatic cancer.

“Low-risk” and “High-risk” HPV-infection and K-ras Gene Point Mutations in Human Cervical Cancer

To analyze the coexistence of human papilloma virus (HPV) infection and K-ras gene activation in cervical neoplasia, we investigated 31 (seven pre-invasive and 24 invasive) cervical carcinomas for "low-risk" (types 6 and 11) and "high-risk" (types 16 and 18) HPVs and K-ras point mutations using PCR-based technology. "Low-risk" HPVs were not detected in the group investigated; however, 20 of 31 (64%) cases were HPV 16 positive, while HPV 18 was found in only three (9.7%) samples (HPV 6/11 v. HPV 16/18, p < 0.0001; HPV 16 v. HPV 18, p < 0.0001; Fisher's exact test). There was a K-ras codon 12 point mutation in two of 31 (6.4%) neoplasms, with none of the cases showing a K-ras codon 13 point mutation. Two moderately differentiated squamous carcinomas showed K-ras exon 2 gene alterations. Interestingly, none of the pre-invasive cervical carcinomas displayed K-ras gene point mutations. The mean patient age did not differ significantly in the number of HPV-positive and -negative cases. A coexistence of "high-risk" human papillomavirus DNA with K-ras gene alterations was observed in three of 31 (9.7%) neoplasms (one IIA and two IB moderately differentiated cervical carcinomas). Our results suggest that "high-risk" HPVs coexist with K-ras gene alterations in a subset of moderately differentiated carcinomas of the cervix uteri.

“Low-risk” and “High-risk” HPV-infection and K-ras Gene Point Mutations in Human Cervical CancerA Study of 31 Cases

To analyze the coexistence of human papilloma virus (HPV) infection and K-ras gene activation in cervical neoplasia, we investigated 31 (seven pre-invasive and 24 invasive) cervical carcinomas for "low-risk" (types 6 and 11) and "high-risk" (types 16 and 18) HPVs and K-ras point mutations using PCR-based technology. "Low-risk" HPVs were not detected in the group investigated; however, 20 of 31 (64%) cases were HPV 16 positive, while HPV 18 was found in only three (9.7%) samples (HPV 6/11 v. HPV 16/18, p < 0.0001; HPV 16 v. HPV 18, p < 0.0001; Fisher's exact test). There was a K-ras codon 12 point mutation in two of 31 (6.4%) neoplasms, with none of the cases showing a K-ras codon 13 point mutation. Two moderately differentiated squamous carcinomas showed K-ras exon 2 gene alterations. Interestingly, none of the pre-invasive cervical carcinomas displayed K-ras gene point mutations. The mean patient age did not differ significantly in the number of HPV-positive and -negative cases. A coexistence of "high-risk" human papillomavirus DNA with K-ras gene alterations was observed in three of 31 (9.7%) neoplasms (one IIA and two IB moderately differentiated cervical carcinomas). Our results suggest that "high-risk" HPVs coexist with K-ras gene alterations in a subset of moderately differentiated carcinomas of the cervix uteri.