H-ras and K-ras gene mutations in primary human soft tissue sarcoma: concomitant mutations of the ras genes.

ras gene mutations have been described with varying frequency in several types of human malignancies. To determine the incidence and type of ras mutations in human soft tissue tumors, we studied 45 sarcomas, including 27 malignant fibrous histiocytomas (MFHs), 10 liposarcomas, 2 rhabdomyosarcomas, and 6 leiomyosarcomas. Al of the tumors were investigated by direct sequence analysis with the automated DNA sequencing of polymerase chain reaction-amplified ras sequences. Twenty (44%) of the sarcomas examined harbored K-ras mutations, 18 (90%) of which were MFHs. All of the K-ras mutations were G-to-A transition mutations in the second position of codon 13 (glycine --> aspartic acid). Of the samples with K-ras activation, 7 (16% of the total of 45 tumors), including 6 MFHs and 1 leiomyosarcoma, also contained H-ras mutation. All of the tumors that showed H-ras alteration had G-to-T transversion mutations in the second base of codon 12 (glycine --> valine). These data possibly implicate that ras gene activation may be a relatively uncommon event in soft tissue tumors, with the exception of MFH. It is suggested that the oncogenic process underlying the development of tumors between these groups may be different and that ras gene mutations may play a role in the etiology and/or progression of MFH. It is noteworthy that when ras gene activation occurs in sarcoma, it predominantly affects the K-ras gene, particularly codon 13. Moreover, H-ras mutations in our samples were detected only in association with tumors that also displayed K-ras-mutated genes. This study demonstrates for the first time concomitant mutations in two different members of the ras gene family in sarcoma     

Denaturing gradient gel electrophoresis (DGGE) assay for K-ras and N-ras genes: detection of K-ras point mutations in human lung tumour DNA

Point mutations in the ras oncogenes are very common in lungcancers as well as in many of the other solid tumours. To effectivelyexamine the occurrence of these mutations in a large numberof tumour samples, we have applied denaturing gradient gel electrophoresis(DGGE) for the analysis of point mutations of the K-ras andN-ras genes, using GC-clamped, PCR-amplified DNA fragments.Among the 68 tumour DNA samples, we detected 14 mutations inthe K-ras gene. This was 78% of the mutations identified byoligonucleotide hybridization. Altogether, eight of the ninedifferent kinds of base substitutions found in the tumour sampleswere detected by the DGGE assay, representing substitutionsat codons 12, 13, and 61 of the K-ras gene. Six of the detectedmutations were guanine to thymine transversions at codon 12;this was the most common type of alteration. On the basis ofour experience, the present non-radioactive DGGE analysis seemsto be readily applicable for detection of the mutations in theK-ras and N-ras genes. Types of ras gene mutations frequentin adenocarcinomas of the lung are also discussed.     

A novel point mutation at codon 146 of the K-ras gene in a human colorectal cancer identified by the polymerase chain reaction

In this report, point mutations of the K-ras gene at codon 146 were analyzed in 25 cases of colon cancer, 4 cases of lung cancer, and 41 cases of lymphoid malignancy. A codon 146 mutation substituting threonine (ACA) for alanine (GCA) was detected in the tumor tissue of a patient with colon cancer and was not detected in the normal tissue of the same patient. Any additional mutations of theras gene family were not detected in this patient. These results suggest that the codon 146 mutation of the K-ras gene could be involved in the development of naturally occurring human malignancies.     

Correlation between the presence of high-risk human papillomaviruses and Id gene expression in Syrian women with cervical cancer

Infection by high-risk human papillomaviruses (HPVs) is considered to be the central cause of invasive cervical cancer. Previously reported studies have shown that Id genes regulate cell invasion and metastasis in several human carcinomas including cervical cancer. In order to investigate the correlation between high-risk HPVs and Id genes in human cervical cancer, the presence of high-risk HPVs and their association with Id gene expression was examined using PCR methods and tissue microarray analyses in a cohort of 44 cervical cancer patients from Syria. This study showed that high-risk HPVs were present in 42 samples (95%) that represent invasive cervical cancers and that the most frequent high-risk HPV types in Syrian women were 33, 16, 18, 45, 52, 58, 35, 51 and 31. Furthermore, the expression of E6 oncoprotein of high-risk HPVs was found to correlate with overexpression of Id-1, but not of Id-2, Id-3 or Id-4 in the majority of invasive cervical cancer tissue samples. These data suggest that high-risk HPVs can enhance the progression of human cervical cancer through Id-1 regulation.     

Oncogenic point mutations in the human retinoblastoma gene: their application to genetic counseling

Mutations of the retinoblastoma gene, most of which cannot be detected by conventional Southern blotting, are known to cause both the nonhereditary and hereditary forms of retinoblastoma and have been implicated in the development of other cancers. Nonhereditary retinoblastoma is caused by a somatic mutation. Hereditary retinoblastoma is caused by a germ-cell mutation, most often a new one, and thus there is usually no family history of the disease. Unlike patients with the nonhereditary disease, those with the hereditary form are at risk for additional retinoblastomas, and their progeny are at risk for the tumors. We used a sensitive technique of primer-directed enzymatic amplification, followed by DNA sequence analysis, to identify mutations as small as a single nucleotide change in tumors from seven patients with simplex retinoblastoma (with no family history of the disease). In four patients the mutation involved only the tumor cells, and in three it involved normal somatic cells as well as tumor cells but was not found in either parent; thus, these mutations appeared to be new, germ-cell mutations. In addition, we found point mutations in cells from a bladder carcinoma, a small-cell carcinoma of the lung, and another retinoblastoma. We conclude that the technique that we have described can distinguish hereditary from nonhereditary retinoblastoma and that it is useful in risk estimation and genetic counseling.     

K-ras gene mutations: an unfavorable prognostic marker in stage I lung adenocarcinoma

Activation of K-ras gene by point mutations, a common finding in lung adenocarcinomas, has been suggested to decrease patient survival. We investigated 109 lung adenocarcinomas, mostly small, peripheral, stage I tumours (81/109) for presence of K-ras gene mutations at codons 12 and 13. Mutations were detected by denaturing gradient gel electrophoresis analysis of specific sequences amplified by polymerase chain reaction from DNA extracted from archival pathological material. Thirty-three of 109 (30.3%) tumours showed mutations at codon 12 (28/33, 84.8%) or 13 (5/33, 15.2%) of the gene. Mutations and type of nucleotide substitutions were differently distributed among cytological subtypes, being more prevalent among less differentiated (G2 and G3) tumours and among bronchial than bronchiolo-alveolar type adenocarcinomas. Survival analysis showed an adverse effect of K-ras mutation on survival, restricted to stage I tumours. Median survival for 81 stage I patients was 30 months for non-mutated tumours versus 20 months for mutated tumours (p=0.016). Multivariate analysis showed that age of patient (p=0.001) and K-ras mutation status (p=0.04) were the only independent factors influencing survival significantly. These data strengthen the hypothesis that K-ras gene mutations may be useful in identifying a subgroup of patients with poor outcome.     

Comparison of different methods of molecular-genetic analysis of somatic mutations in K-ras gene in patients with colorectal cancer

Two approaches to somatic point mutations in 12 and 13 codones of K-ras gene were analyzed: PCR/SSCP/ACRS/sequencing and allele-specific PCR in the real-life regimen (Russian set "KRAS-7M"). The comparison was carried out on 62 examples of genomic DNA extracted from frozen colon carcinomas, which underwent manual dissection. The results obtained in two attempts were consistent in 95,2% (N=59). Specificity and sensitivity of K-ras mutations detection using "KRAS-7M" set were 100 and 96,4% respectively, and 94,1 and 100% respectievly using PCR/SSCP/ACRS/automatic sequencing. False positive results were absent when detecting with "KRAS-7M" and accounted for 2 cases (5,9%) when using PCR/SSCP/ ACRS/automatic sequencing. The only false negative response (3,6%) was obtained analyzing mutations using "KRAS-7M".     

Detection of point mutations of BCL10 gene in hepatocellular carcinoma tissues: report of 46 cases

BCL10 was found to have truncated mutations at a high frequency in MALT (mucosa-associated lymphoid tissue) B cell lymphomas. We examined the mutations of BCL10 gene in human primary liver cancer using non-isotopic PCR-SSCP. Three exons were examined in both cancer and non-HCC adjacent liver tissues. For each exon, six PCR products with abnormal bands were sequenced to verify those mutations. 56.5% samples were revealed a C to G mutation at position 5744 (g5744C>G) of the first exon of BCL10 gene; 54.3% samples were revealed a T deletion mutation at position 11311 (g11311delT) of the second exon of BCL10 gene; 45.7% samples were revealed a C to T mutation at position 14116 (g14116C>T) of the third exon of BCL10 gene. Similar mutation types were found in tumor-adjacent tissues at a lower frequency. The single base changes result in a truncated BCL10 protein expression. Serum alpha-fetoprotein (AFP) level, the tumor size had no significant relationship with BCL10 mutation.     

High-degree tumor budding and podia-formation in sporadic colorectal carcinomas with K-ras gene mutations

In vitro ras activation enhances the epithelial-mesenchymal transition of colorectal carcinoma cells. But ras effects are known to be highly dependent on cell types and the tissue context. Therefore, this study was made to test the hypothesis that in clinical colorectal carcinoma specimens, aggressive invasion phenotypes, specifically tumor budding and podia formation, would correlate with K-ras gene mutations. In a series of 95 clinically sporadic primary colorectal carcinomas collected ad hoc, tumor budding and podia formation were counted using pan-cytokeratin immunohistochemistry, and K-ras gene mutations in codons 12 and 13 were determined. Consistent with the hypothesis, tumor budding and podia formation were observed to be significantly higher in the 32 (34.7%) of the tumors with K-ras gene mutations (29 mutations in codon 12, 3 in codon 13), and this correlation was observed independent of the patterns of invasion (expansive versus infiltrative). Microsatellite status, numbers of losses of heterozygosity, adenomatous polyposis coli and p53 gene mutations, and degree of promoter methylations (CIMP status) were not associated with K-ras gene mutations. Besides their effects on the tumor cell cycles, oncogenic K-ras gene mutations in colorectal carcinomas could be important for aggressive tumor invasion. This may be important in metastasizing disease and could provide a rationale for developing drugs that interrupt ras-signaling cascades.     

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

AIMS--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. METHODS--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. RESULTS--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. CONCLUSIONS--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.     

肾血管平滑肌脂肪瘤31例临床病理分析

目的：探讨肾血管平滑肌脂肪瘤（angiomyolipoma，AML）的临床病理特征。方法：对31例肾AML的临床病理特征进行分析，并做免疫组化检测，其中4例做电镜观察。结果：31例肾AML按其组织形态可分为典型型（67.7%）、非典型型(6.5%)、平滑肌瘤样型（9.7%）、脂肪瘤样型（9.7%）、炎症型（6.5%）5型。免疫表型：肿瘤细胞特征性表达HMB45、Melan-A、gp-100、HHF35、SMA、CD68。其中1例电镜下肿细胞内可见黑色素小体。结论：肾AML的组织形态多样，特征性免疫级化标记为诊断提供依据。掌握形态特征及其鉴别诊断要点有助于临床的正确治疗。     

多种恶性肿瘤p21基因启动子及编码区点突变的检测

目的 探讨ｐ２１基因点突变是否与恶性肿瘤的发生发展过程有关,特别是与那些没有ｐ５３基因突变的恶性肿瘤发病的关系。方法 结合ＳＳＣＰ分析及ＤＮＡ序列测定技术,检测３４６例９种不同组织学类型的恶性肿瘤ｐ２１基因的启动子和主要的编码区。结果 ３４６例恶性肿瘤的ｐ２１基因启动子未检测到突变,但在Ｐ５３蛋白结合位点不游及下游处存在多态性变异。仅１例膀胱癌有一杂合性４４ｂｐ缺失突变。此外,在编码区还发现了１个     

Nucleotide sequence of human papillomavirus type 31: a cervical neoplasia-associated virus

The nucleotide sequence of human papillomavirus (HPV) 31 DNA (7912 bp) was determined and used to deduce the genomic organization of this cervical cancer-associated virus. Based on comparisons of the HPV 31 DNA sequence to other sequenced HPVs, HPV 31 is a typical papillomavirus most related to HPV 16 (70% identical nucleotides). The E6 and E7 open reading frames (ORF) of HPV 31 contain several potential DNA binding motifs (Cys-X-X-Cys), the locations of which are conserved in all HPVs. The E6 ORF also has the potential to code for an E6* protein. The E7 ORF of HPV31 encodes a polypeptide motif which appears to distinguish HPVs associated with cervical cancer, such as types 16, 18, 31, and 33, from HPVs found primarily in benign lesions, such as types 6 and 11.     

Extreme exertion rhabdomyolysis: a histopathologic study of 31 cases

Muscle biopsy specimens from 31 cases of rhabdomyolysis associated with prolonged repetitive exercise in a Marine recruit population were studied. A wide range of nonspecific histologic changes was seen. Symptoms and signs of muscle necrosis developed in the first days of recruit training, generally involving muscle groups not usually stressed and occurring particularly in men having a sedentary background. No relation to race, geographic origin, climate, or prior inoculations and vaccinations was apparent. Although dark urine, hemoglobinuria, or hematuria could be documented in all cases, myoglobinuria was not consistently demonstrable. The activities of serum enzymes (GOT, GPT, CPK, LDH) were greatly elevated. None of the patients had had previous signs or symptoms suggesting stress-induced muscle necrosis. The pathophysiology of this syndrome is not understood; however, the two contributing factors of major importance are a prior sedentary background and exhausting exercise.     

Genotyping of human papillomavirus in cervical intraepithelial neoplasia in a high-risk population

Infection with the human papillomavirus (HPV) is responsible for 99.7% of cervical cancers, the second most prevalent neoplasia in women worldwide and the fifth leading cause of death by cancer in this population. In Chile, the incidence rate is 14.4 cases per 100,000 women per year and it is considered a significant public health problem. The natural history of cervical cancer begins gradually from low-grade and high-grade squamous intraepithelial lesions to an invasive disease. In this study the frequency of HPV types was determined by HPV genotyping with reverse line blot hybridization in 200 cytobrushes of women with preneoplastic lesions in a high-risk population. HPV DNA was found in 89% of the lesions (83.3% of low-grade squamous intraepithelial lesions and 93.6% of high-grade squamous intraepithelial lesions). Multiple HPV infections were found in 14.4% and 15.5% of low- and high-grade lesions, respectively. HPV 16 was the most frequent genotype in single infections, followed by HPV 18. These results show that most of the preneoplastic lesions of the cervix (60%) were associated with HPV 16 and/or HPV 18, supporting the implementation of an HPV vaccination program in this high-risk population.     

The potential role of self-sampling for high-risk human papillomavirus detection in cervical cancer screening

High-risk human papillomavirus (hr-HPV) detection will become an important tool in the screening for cervical cancer. Self-sampling is an inexpensive and well-accepted method for HPV detection that will increase participation of nonresponders in current screening programs. Even more, because self-collected samples are as good as physician-collected samples for HPV detection, self-sampling might be a suitable method for future primary cervical cancer screening.