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DNA ploidy patterns in cervical intraepithelial neoplasia grade III, with and without synchronous invasive squamous cell carcinoma. Measurements in nuclei isolated from paraffin-embedded tissue.

This study presents the results of cytophotometric (CPM) and flow cytometric (FCM) DNA ploidy measurements in cervical intraepithelial neoplasias grade III (CIN III) with and without synchronous invasive squamous cell carcinoma. Hysterectomy and biopsy material from 21 patients 35 years of age or younger and from 18 patients age 50 years or older was studied. The DNA analysis was performed in nuclei isolated from specific areas of paraffin-embedded tissue. There were significant differences in the distribution of DNA patterns between the two age groups. About 80% of CIN III lesions in women 50 years of age or older, with or without a coexisting invasive cancer were aneuploid. In the group of younger women a diploid DNA pattern was found in about 60% of CIN III with concomitant invasive cancer. In the absence of an invasive cancer, CIN III lesions were mostly polyploid. The DNA pattern of invasive cancers was generally identical with the adjacent CIN, thus suggesting that the two lesions were related. Although the prognostic value of DNA ploidy measurements in cervical intraepithelial lesions in women in these two age groups has to be further evaluated, these results are at considerable variance with previously published data on DNA values in CIN and invasive carcinoma. In four CIN III lesions without invasive cancer, in women of the group of 35 years of age or younger, human papilloma virus common antigen could be demonstrated by immunochemical procedure. In three of these cases a polyploid DNA pattern was present; the fourth case showed a bimodal aneuploid pattern.

Adult↗

Analysis of DNA extracted from formalin-fixed, paraffin-embedded tissues by enzymatic amplification and hybridization with sequence-specific oligonucleotides.

The "polymerase chain reaction" (PCR) procedure for amplifying specific gene sequences has recently been combined with sequence-specific oligonucleotide (SSO) probe hybridization to develop a highly sensitive, rapid, and simple method for analyzing allelic variations in genomic DNA. In the present study we have used PCR/SSO to analyze partially purified DNA extracted from formalin-fixed, paraffin-embedded tissue specimens. We report that this DNA, including samples that were partially degraded, proved to be suitable for analysis by the PCR/SSO procedure.

Base Sequence↗

Detailed phenotypic analysis of B-cell lymphoma using a panel of antibodies reactive in routinely fixed wax-embedded tissue.

Sixty-three well characterized B-cell lymphomas, with diagnoses previously established by conventional cryostat immunocytochemistry or limited paraffin immunocytochemistry, were studied. The tumors encompassed most of the Kiel subtypes and included the newly recognized entity, sclerosing mediastinal B-cell lymphoma. by the avidin-biotin peroxidase complex technique, each tumor was stained with a panel of monoclonal and polyclonal antibodies reactive in routinely fixed wax-embedded tissues. The panel included four reagents recognizing probable T-cell and B-cell restricted leukocyte common moieties (UCHL1, MT1, MB1, 4KB5), three antibodies to B-cell-related antigens (KiB3, MB2, LN1), and one to a macrophage-related antigen (Mac411). Other antibodies employed included anti-mu chain, anti-kappa, anti-lambda, and seven antibodies to non-phenotype-associated antigens, including HLA-DR (TAL-1B5, LN3, LN2, MB3), CD15 (C3D-1), and CD30 (BER-H2). Monotypic surface or perinuclear space and cytoplasmic immunoglobulin were detected in 80% of cases. Distinctive immunocytochemical profiles were demonstrable in many tumor categories by means of the panel of antibodies, thus facilitating the differential diagnosis of tumors of similar morphology. These results, together with our work on T-cell lymphoma in paraffin sections, show that accurate phenotypic analysis of lymphoma is now possible in routinely processed tissues.

Antibodies, Monoclonal↗

Immunophenotypic classification of canine malignant lymphoma on formalin-mixed paraffin wax-embedded tissue by means of CD3 and CD79a cell markers.

Canine malignant lymphoma (CML) is a common lymphoid tumour. Identification of the immunophenotype is of prognostic importance: T-cell lymphomas have a worse prognosis than B-cell lymphomas. Until recently, identification of T- or B-cell lymphomas was undertaken by means of flow cytometry or fluorescent immunocytochemistry on frozen sections. Whilst valid in the research field, these methods are impractical for routine diagnostic histopathology in CML. Commercially available CD3 antibody has been successfully employed in T-cell identification in dogs in formalin-fixed paraffin wax-embedded tissue sections, but the lack of a B-cell marker has been a hindrance until the recent introduction of a commercially available pan-B cell marker, CD79a (DAKO M7051), suitable for diagnostic application upon formalin-fixed paraffin wax-embedded material. Antibody markers to CD3 and CD79a show cross-reactivity across species lines for B cells and T cells respectively. In this group of five selected canine cases, two were identified as B-cell and the other three as T-cell lymphoma, by means of CD3 and CD79a. To the best of our knowledge application of CD79a in cases of CML has not been reported.

Animals↗

Demonstration of URR-duplication variants of human papillomavirus type 6 in paraffin-embedded tissue sections of one condyloma acuminatum and one Buschke-Loewenstein tumour.

Human papillomavirus type 6 (HPV 6) induces condylomata acuminata and laryngeal papillomas. Occasionally, HPV 6 may also be found in low-grade verrucous carcinomas. In some tumours, genetic analysis revealed the presence of HPV 6 variants with rearrangements, mostly DNA duplications, within the upstream regulatory region (URR). In this study, we analysed 98 formalin-fixed, paraffin-embedded condylomata acuminata obtained from 54 patients for the presence of URR-duplication variants of HPV 6. HPV 6 DNA could be amplified by polymerase chain reaction (PCR) from 40 samples. One condyloma acuminatum contained a HPV 6 genome with rearranged URR. Analysis using restriction enzyme cleavage suggested a DNA duplication within the URR of approximately 200 bp, spanning the Hpa II site at nt 7863 and the Dde I site at nt 7843 but not involving the Rsa I site at nt 7633. In addition, we analysed the distribution of the already characterized URR-duplication variant HPV 6AC1B within different paraffin-embedded tissue sections of a Buschke-Loewenstein tumour. No correlation could be demonstrated between the presence of the rearranged genome and malignant histological changes. This result and the demonstration of an URR-duplication variant in a typical condyloma acuminatum suggest that duplications within the URR of HPV 6 are not directly related to malignant progression of HPV 6-induced tumours.

Base Sequence↗

Ras oncogene mutations in thyroid tumors: polymerase chain reaction-restriction-fragment-length polymorphism analysis from paraffin-embedded tissues.

Ras mutations have been found in thyroid lesions. Different studies have shown different frequencies of mutations among benign and malignant lesions. The presence of point mutations in codons 12 and 13 of the c-K-ras, c-H-ras, and N-ras genes was studied in 58 thyroid lesions (10 nodular goiters, 10 follicular adenomas, and 15 papillary, 10 follicular, and 13 anaplastic carcinomas). DNA was extracted from formalin-fixed paraffin-embedded tissue, and target sequences were amplified in vitro by the polymerase chain reaction. Mutations were detected by the presence of restriction-fragment-length polymorphisms either occurring naturally or introduced artificially by the use of mutant primers. No characterization of the mutations was performed. Results were correlated with clinicopathologic features and patient follow-up. One goiter showed a mutation at codon 13, c-K-ras. All follicular adenomas, including three hyalinizing trabecular adenomas, were negative. Four papillary carcinomas presented mutations (one at codon 13, c-K-ras; three at codon 12, N-ras). Two follicular carcinomas showed mutations at codon 12, N-ras. Five anaplastic carcinomas showed mutations (two at codon 12 and two at codon 13, c-K-ras; one at codon 12, N-ras). In summary, the results confirm that ras oncogenes play a role in thyroid tumorigenesis, probably at an early step. Ras mutations appear not to be related to prognosis.

Adenoma↗

Clonal analysis by study of X chromosome inactivation in formalin-fixed paraffin-embedded tissue.

Analysis of clonality by X chromosome inactivation has proven to be a powerful strategy in the study of neoplastic and preneoplastic disorders (P. J. Fialkow, Biochim. Biophys. Acta, 458: 283-321, 1976; B. Vogelstein et al., Cancer Res., 47: 4806-4813, 1987). Recently, the gene for the androgen receptor has been shown to be a highly polymorphic locus in which methylation of DNA correlates with inactivation of one or the other X homologue (R. C. Allen et al., Am. J. Hum. Genet., 51: 1229-1239, 1992). Unlike other loci used for analysis of X inactivation, the methylation sites within the androgen receptor gene lie close to the region of DNA containing the polymorphism. Consequently, it should be possible to use methylation-sensitive restriction enzymes and polymerase chain reaction to study differential methylation among alleles of this gene in formalin-fixed and paraffin-embedded archival tissue specimens. To investigate this question, we performed clonal analysis on a variety of randomly selected, formalin-fixed, paraffin-embedded tumor tissues from 15 women. Thirteen of the women were found to be heterozygous for polymorphisms at the androgen receptor locus. Among these women, 11 tumors were clearly clonal in assays of methylation of the androgen receptor gene. Interpretation of results for the remaining two cases was complicated because of a skewed pattern of X chromosome inactivation found in normal control tissues. We conclude that analysis of methylation in the androgen receptor gene should allow study of clonality in most formalin-fixed, paraffin-embedded tissue specimens from women, including small preneoplastic lesions or rare conditions for which sufficient material is not available for analysis by other techniques.

Base Sequence↗

Detection of hepatitis C by RT-PCR in formalin-fixed paraffin-embedded tissue from liver transplant patients.

The histopathologic alterations of hepatitis C virus (HCV) infection of the liver overlap with those of other diseases, making interpretation of liver biopsy specimens in some cases insufficient to render a diagnosis. Although HCV infection can be confirmed by detection of circulating anti-HCV antibodies, immunocompromised liver transplant recipients are often unable to mount an immunologic response to the virus, resulting in false-negative serologic testing. We describe the comparison of reverse transcription-polymerase chain reaction (RT-PCR) with histopathology, serology, and immunohistochemistry for the diagnosis of HCV. Sixty-three formalin-fixed, paraffin-embedded tissue samples (40 needle biopsy specimens and 23 native liver resection specimens) from 35 transplant patients were analyzed by use of a novel method of RNA extraction followed by nested PCR for HCV as well as albumin mRNA as an internal control. HCV was detected by RT-PCR in 50 of 51 (98%) paraffin sections of liver from transplant patients with circulating anti-HCV antibodies, 15 of which lacked characteristic histologic features of HCV infection. Overall, there were no false-negative results in 36 needle biopsy specimens from patients with hepatitis C infection, but three negative results were seen in end-stage cirrhotic native livers resected from HCV-infected patients. No false-positive test results were seen among 21 negative controls (10 liver samples from immunocompetent patients with abnormalities unrelated to hepatitis C and 11 liver biopsies from immunocompetent patients without histologic evidence of liver disease). In comparison, immunohistochemistry using antibody TORDJI-22 was positive for HCV in only 15 of 32 (47%) needle biopsies positive by RT-PCR. Our results indicate that RT-PCR is a more sensitive and specific method of detecting hepatitis C in routinely processed paraffin sections of formalin-fixed liver biopsy specimens than histopathologic examination or immunohistochemistry.

Formaldehyde↗

Optimization of biotinyl-tyramide-based in situ hybridization for sensitive background-free applications on formalin-fixed, paraffin-embedded tissue specimens.

BACKGROUND: Over the past five years in situ hybridization techniques employing tyramide amplification reagents have been developed and promise the potential detection of low/single-copy nucleic acid sequences. However the increased sensitivity that tyramide amplification brings about may also lead to problems of background staining that confound data interpretation. METHODS: In this study those factors enabling background-free biotinyl-tyramide based in situ hybridization assay of formalin-fixed paraffin-embedded tissues have been examined. SiHa, HeLa and CaSki cell lines known to contain HPV integrated into the cell genome, and archival cervical pre-invasive lesions and carcinomas have been successfully assessed using biotinylated HPV and centromeric probes. RESULTS: The single most important factor both for sensitivity and clean background was a tissue unmasking regimen that included treatment with 10 mM sodium citrate pH 6.0 at 95 degrees C followed by digestion with pepsin/0.2 M HCl. Concentrations both of probe and primary streptavidin-peroxidase conjugate and pH of hybridization mix and stringency washes were also critical for sensitivity. Certain probes were more associated with background staining than others. This problem was not related to probe purity or size. In these instances composition of hybridization mix solution was especially critical to avoid background. 3-amino-9-ethylcarbazole was preferred over 3,3'-diaminobenzidene as a chromogen because background was cleaner and the 1-2 copies of HPV16 integrated in SiHa cells were readily demonstrable. HPV detection on metaphase spreads prepared from SiHa cells was only successful when a fluorescent detection method was combined with tyramide reagent. 'Punctate' and 'diffuse' signal patterns were identified amongst tissues consistent with the former representing integration and 'diffuse' representing episomal HPV. Only punctate signals were detected amongst the cell lines and were common amongst high-grade pre-invasive lesions and carcinomas. However it remains to be determined why single/low-copy episomal HPV in basal/parabasal cells of low-grade lesions is not also detectable using tyramide-based techniques and whether every punctate signal represents integration. CONCLUSIONS: A tyramide-based in situ hybridization methodology has been established that enables sensitive, background-free assay of clinical specimens. As punctate signals characterize HPV in high-grade cervical lesions the method may have potential for clinical applications.

Journal Article↗

Argyria: microanalytic-morphologic correlation using paraffin-embedded tissue.

Scanning electron microscopes, which have energy dispersive spectroscopy (x-ray microanalysis) capability, can provide accurate identification of the composition of inorganic deposits in tissue. We report a case of florid argyria in which the same deparaffinized 4-micron sections were sequentially examined by light microscopy, scanning electron microscopy, backscattered electron imaging, and x-ray microanalysis to confirm the clinical impression of argyria. We recommend the use of x-ray microanalysis of paraffin-embedded tissue as a simple, rapid and inexpensive means of precise identification of inorganic deposits of heavy elements in tissue sections.

Administration, Intranasal↗

A case report of synovial sarcoma with translocation (X;18). Application of fluorescence in situ hybridization to paraffin-embedded tissue.

A 57-year-old female patient with synovial sarcoma in her right foot had a chromosome abnormality defined as translocation (X;18). The tumour was located in the subcutis, and histological investigation showed monophasic proliferation of oval to spindle-shaped cells with a fascicular arrangement lacking an epithelial component. Immunostaining disclosed no cytokeratin or epithelial membrane antigen in tumour cells. Karyotypic analysis revealed translocation (X;18) in addition to other nonspecific aberrations. Fluorescence in situ hybridization was carried out on paraffin-embedded tissue, using DNA probes for the centromeres of chromosomes X and 18 with whole chromosome painting probes for X and 18. The free nuclei showed two signals at a rate of 83-85% with the X and 18 centromeric probes, in contrast to three signals at a rate of 68-70% with the X and 18 painting probes.

Chromosomes, Human, Pair 18↗

Policy guidelines for the utilization of formalin-fixed, paraffin-embedded tissue sections: the UNC SPORE experience. University of North Carolina Specialized Program of Research Excellence.

Paraffin blocks represent a valuable resource that has allowed investigators to apply today's technology to address scientific questions in a shorter period of time and in more diverse populations than would have been possible with fresh or frozen tissue. However, in addition to being an exhaustible resource, there is concern regarding the appropriate use of these tissues, both with respect to medical or legal considerations and quality control and quality assurance practices. We describe policy guidelines to address these concerns, including: safeguards to address medical/legal and patient confidentiality issues, quality control and quality assurance for tissue sectioning, processing and storage, database management for sample tracking, and scientific review for utilization of specimens. These policies and procedures have been developed and implemented by the University of North Carolina (UNC) Specialized Program of Research Excellence (SPORE) in the Breast Cancer Immunohistochemistry (IHC) Core laboratory, in collaboration with our study pathologists, participants, and research investigators. It is our hope that the information and experience described here may stimulate discussion that can ultimately lead to a uniform policy for handling formalin-fixed paraffin-embedded tissues in research.

Breast Neoplasms↗

Amplification of c-erbB-2 in gastric cancer: detection in formalin-fixed, paraffin-embedded tissue by fluorescence in situ hybridization.

A total of 396 adenocarcinomas of the stomach were examined immunohistochemically using antibodies specific for the internal domain of human c-erbB-2 protein. Forty of these (consisting of 35 well-differentiated and 5 undifferentiated types) overexpressed c-erbB-2, as evidenced by cytoplasmic membrane staining; these tumors were further examined by fluorescence in situ hybridization using a cosmid probe for 17q11.2-12 (c-erbB-2 locus) on formalin-fixed, paraffin-embedded tissues. This technique enabled us not only to detect c-erbB-2 amplification on a cell-by-cell basis, but also to compare the gene amplification with the protein expression, observe topographical distribution, and establish quantitative pictures of the amplification in vivo condition. In all 40 tumors, we observed gene amplification and found that the cancer cell with the amplification corresponded to the cells overexpressing c-erbB-2. In eight mucosal carcinomas, signals were coalesced to one or two clusters, indicating that the amplified gene resided in a homogeneous staining region (HSR). Among 32 carcinomas invading beyond muscularis mucosae, approximately half were mostly composed of cells with the amplification; the others had populations of tumor cells with and without amplification, which were sometimes in separate zones or areas and sometimes mixed together. In 22 cancers, the amplified genes were exclusively in HSR; however, cancer cells with multiple scattered signals-suggesting that the amplified genes were translocated to other chromosomes-were found exclusively in 6 tumors and concurrently with HSR in 4 tumors. These findings suggest that: (a) the amplification produces c-erbB-2 in the HSR form generally early in the carcinogenesis of gastric adenocarcinomas; and (b) in the process of cancer development, the amplified gene is lost in some cancer cells by uneven segregation of HSR in mitosis, whereas in others, it is kept in HSR or translocated to other chromosomal positions, thereby preserving overexpression of c-erbB-2 protein.

Adenocarcinoma↗

[Expression of proliferating cell nuclear antigen in superficial bladder cancer--application to paraffin-embedded tissue sections].

It is important to know the proliferating ability and the malignant potential of each tumor. We studied 56 cases of pTa to pT1 superficial bladder tumors using immunohistochemical staining for proliferating cell nuclear antigen (PCNA), and compared the results with the clinical course of each patient. We obtained the following results. 1) We detected the PCNA positive nuclei in all cases, and the PCNA positive rates varied within a range of 1.1-77.5% with a mean of 34.0%. 2) The PCNA positive rate showed no correlation with age, sex, duration of paraffin-embedded, or pathological stage, but showed a significant correlation with the number of tumors, pathological grade of malignancy, or non-recurrence rate. PCNA positive rate of Grade 1 cases (n = 19, 15.6%: mean) was significantly lower than those of Grade 2 cases (n = 27, 39.9%) or Grade 3 cases (n = 10, 53.1%) (P < 0.01). The recurrence rate of the cases with PCNA positive rates of more than 34% (n = 24) was significantly higher than that of the cases with a PCNA positive rate of less than 34% (n = 32) (P < 0.05). In conclusion, the method of counting the rate of PCNA positive nuclei is considered to be very useful because of its applicability to paraffin-embedded tissue sections and the simple and rapid techniques. Our results in bladder cancer tissues suggest that this method may also be useful for investigating the proliferating ability and the malignant potential of tumors in general.

Adult↗

A universal method for the mutational analysis of K-ras and p53 gene in non-small-cell lung cancer using formalin-fixed paraffin-embedded tissue.

The p53 tumor suppressor gene has been found to be altered in almost all human solid tumors, whereas K-ras gene mutations have been observed in a limited number of human cancers (adenocarcinoma of colon, pancreas, and lung). Studies of mutational inactivation for both genes in the same patient's sample on non-small-cell lung cancer have been limited. In an effort to perform such an analysis, we developed and compared methods (for the mutational detection of p53 and K-ras gene) that represent a modified and universal protocol, in terms of DNA extraction, polymerase chain reaction (PCR) amplification, and nonradioisotopic PCR-single-strand conformation polymorphism (PCR-SSCP) analysis, which is readily applicable to either formalin-fixed, paraffin-embedded tissues or frozen tumor specimens. We applied this method to the evaluation of p53 (exons 5-8) and K-ras (codon 12 and 13) gene mutations in 55 cases of non-small-cell lung cancer. The mutational status in the p53 gene was evaluated by radioisotopic PCR-SSCP and compared with PCR-SSCP utilizing our standardized nonradioisotopic detection system using a single 6-microns tissue section. The mutational patterns observed by PCR-SSCP were subsequently confirmed by PCR-DNA sequencing. The mutational status in the K-ras gene was similarly evaluated by PCR-SSCP, and the specific mutation was confirmed by Southern slot-blot hybridization using 32P-labeled sequence-specific oligonucleotide probes for codons 12 and 13. Mutational changes in K-ras (codon 12) were found in 10 of 55 (18%) of non-small-cell lung cancers. Whereas adenocarcinoma showed K-ras mutation in 33% of the cases at codon 12, only one mutation was found at codon 13. As expected, squamous cell carcinoma samples (25 cases) did not show K-ras mutations. Mutations at exons 5-8 of the p53 gene were documented in 19 of 55 (34.5%) cases. Ten of the 19 mutations were single nucleotide point mutations, leading to amino acid substitution. Six showed insertional mutation, and three showed deletion mutations. Only three samples showed mutations of both K-ras and p53 genes. We conclude that although K-ras and p53 gene mutations are frequent in non-small-cell lung cancer, mutations of both genes in the same patient's samples are not common. We also conclude that this universal nonradioisotopic method is superior to other similar methods and is readily applicable to the rapid screening of large numbers of formalin-fixed, paraffin-embedded or frozen samples for the mutational analysis of multiple genes.

Base Sequence↗

Single-cell immunohistochemical mutation load assay (SCIMLA) using human paraffin-embedded tissues.

It would be advantageous to measure mutation load in situ in order to determine the relationship between a high mutation load and increased risk for cancer or other diseases and to evaluate sources of possible mutagen exposure. Previously, in situ mutation detection assays have been plagued with multiple rounds of amplification and high rates of false-positives and false-negatives. The single cell immunohistochemical mutation load assay (SCIMLA) was developed to measure somatic mutation frequency, pattern, and spectrum in normal tissues with a single round of amplification. The P53 gene was utilized as a mutation reporter because of the unusual property that missense mutations often cause P53 protein to accumulate in the cell, allowing the mutant proteins to be detected by immunohistochemical staining. Alternative reporter genes with stabilized mutant proteins may be envisioned. Single cells that stain positively for P53 protein overabundance (red cells) were microdissected from ethanol-fixed and paraffin-embedded tissues. A novel stimulated-PCR (S-PCR) protocol permitted successful amplification of a 1.8-kb segment of the P53 gene (i.e., exons 5-9) in 87% of single mammary cells. Subsequent sequence analysis demonstrated that 35% of the amplified red-stained epithelial cells from normal breast tissue have missense mutations at evolutionarily conserved amino acids. Jackpot mutations, presumably due to clonal expansion, were common. False-positive missense mutations at conserved residues were observed in 3% of the clear cells (i.e., without red stain), presumably due to DNA polymerase error in early PCR cycles. The allele dropout rate was measured at 40% of the amplified cells. SCIMLA is applicable to a variety of tissues, utilizes a single amplification of an endogenous gene, displays mutant cells in situ, and may be adapted to other species.

Base Sequence↗

Analysis of N-ras gene mutations in medulloblastomas by polymerase chain reaction and oligonucleotide probes in formalin-fixed, paraffin-embedded tissues.

Precise data on the incidence of transforming ras oncogenes in pediatric tumors and the correlations with the histopathological properties of the tumors are very limited. Additionally the presence of ras activation in medulloblastomas has not been investigated so far. Using a combination of techniques including in vitro gene amplification by polymerase chain reaction (PCR) and detection of single base mutations by sequence-specific oligonucleotides we studied N-ras activation (mutations at codon 12, 13, and 61) in 32 medulloblastomas. DNA was isolated from 20 microns sections of formalin-fixed paraffin-embedded tissue. Mutations were found in 3 out of 32 examined medulloblastomas. In all cases only mutations of codon 61 were found: two of three mutations were C to A mutations at position 1 of the codon 61 (leading to a substitution of a glutamine residue for a lysine) and one was A to T mutation at position 3 in the same codon (glutamine-histidine). Our results indicate 10% incidence N-ras mutation in medulloblastoma, higher than in other CNS tumors studied so far. The main advantages of the procedure described are its greatly improved sensitivity, the increased speed with which tumor samples can be analyzed, and the possibility of using paraffin-embedded sections to analyze various rare tumors in retrospect.

Adolescent↗

H-ras-1 point mutation in malignant peripheral nerve sheath tumors: polymerase chain reaction restriction fragment length polymorphism analysis and direct sequencing from paraffin-embedded tissues.

It has been shown that the NF1 (neurofibromatosis type 1) gene encodes a tumor suppressor which inactivates ras proteins. Among malignant mesenchymal tumors, H-ras-1 mutations have been found in malignant fibrous histiocytoma, leiomyosarcoma and embryonal rhabdomyosarcoma. However, studies on H-ras-1 mutation of many cases of malignant peripheral nerve sheath tumors (MPNST) have not been documented. Therefore, we investigated H-ras-1 mutations of MPNST. In 45 cases of MPNSTs of our files, DNA was extracted from the formalin-fixed paraffin-embedded tissue, and the mutations of the H-ras-1 gene were detected by using PCR-RFLP (polymerase chain reaction- restriction fragment length polymorphisms) method and direct sequencing. We found two cases with H-ras-1 point mutation in MPNST for the first time. Both cases showed the same mutation in codon 13.1 [GGT(Gly) to AGT(Ser) transition]. Interestingly, both cases were associated with NF1. It is possibile that the mutation of the H-ras-1 gene occurred after the mutation of the NF1 gene in the MPNST.

Adolescent↗