Ras Point Mutations Research Articles

Seventh Japanese-German workshop on molecular and cellular aspects of carcinogenesis

Seventh Japanese-German workshop on molecular and cellular aspects of carcinogenesis

EUS and K- ras analysis of pure pancreatic juice collected via a duodenoscope after secretin stimulation for diagnosis of pancreatic mass lesion: a prospective study

Background: The early diagnosis of pancreatic cancer remains problematic. This prospective study assessed the utility of a combination of endoscopic ultrasound (EUS) and genetic analysis of pure pancreatic juice in the diagnosis of pancreatic mass lesions. Methods: One hundred seventy-six patients with suspected pancreatic disease were enrolled and underwent ultrasonography (US), computed tomography (CT), endoscopic retrograde choleangiopancreatography (ERCP), and EUS. Pure pancreatic juice was collected endoscopically after secretin stimulation. K- ras point mutations at codon 12 in the juice were assayed by polymerase chain reaction–restriction fragment length polymorphism. Results: Thirty-six (20%) patients were found to have solid pancreatic masses including 19 with cancer (7 patients, ≤ 2 cm) and 17 patients with an inflammatory mass (13 patients, ≤ 2 cm). US and CT were both less sensitive, particularly in patients with small masses. In 13 patients, small masses (4 cancer and 9 inflammatory masses) were not delineated until EUS. The combination of EUS and K- ras analysis had a high sensitivity (100%; p < 0.05 vs. US) and its accuracy reached 94% ( p < 0.01 vs. US). Conclusions: EUS and K- ras determination in pure pancreatic juice collected via a duodenoscope may be useful for the diagnosis of pancreatic mass lesions. (Gastrointest Endosc 1999;50:797-803.)

Differences in K-ras Codon 12 Mutation Frequency between “High-Risk” and “Low-Risk” HPV-Infected Samples

Objectives. The current study was designed to determine the association between K-ras point mutations and the presence of high- and low-risk HPV types in noncancerous samples.Methods. One hundred fifty-five noncancerous cytological samples from genital specimens positive for HPV-6, -16, or -18 were analyzed for codon 12 ras point mutations. DNA samples were subjected to nested PCR for the detection of HPV genome. Mutations at the first and second bases of K-ras codon 12 were detected using specific enriched PCR.Results. Eleven percent of the HPV-positive samples analyzed showed mutation in K-ras codon 12 (17 of 155). Mutations were detected in 8 of 53 HPV-16- (15%) and 8 of 38 HPV-18-positive samples (21%). Nevertheless, when HPV-6-positive samples were screened for K-ras mutations only 1 of 64 samples was found mutated (1.56%). Statistical analysis using the χ2 test showed a highly significant difference (χ2 = 9.865, P < 0.01) when the rate of mutation for samples positive for high-risk HPV types (16 and 18) was compared with the frequency found in low-risk-infected samples (HPV-6).Conclusion. These results could be considered an indicator of the association between K-ras codon 12 mutations and the presence of high-risk HPV types.

Protein expression and genetic alterations of p53 and ras in intrahepatic cholangiocarcinoma

The significance of molecular and genetic alterations of p53 and ras in the development and progression as well as the histological differentiation of intrahepatic cholangiocarcinoma (ICC) was evaluated. We examined immunohistochemically ras p21 protein and p53-related products (p53 protein, WAF-1 and mdm-2) in 43 cases of ICC. In addition, point mutations of ras and p53 were examined genetically in selected ICC cases by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and direct sequence analysis. Point mutation of K-ras gene codon 12 was detected in three of 14 cases and one of 15 cases by PCR-RFLP and direct sequence analysis, respectively. Immunoreactivity of ras p21 protein was not detected. Point mutation of p53 was detected in three of 15 cases. p53 protein was immunohistochemically detectable in 33 of 43 cases. Immunoreactivity of p53 was more frequent in well-differentiated and less frequent in poorly differentiated cases. Immunoreactivity of WAF-1 and mdm-2 was seen in 16 and eight of 43 cases, respectively. Both proteins were frequently detected in the cases positive for p53 protein. These results suggest that dysregulation of ras is involved in at least 20% of ICC and expression of p53 protein is more significantly involved in ICC, particularly in the well and moderately differentiated cases. While some cases of p53 expression may be explainable by point mutation of p53, there may be some epigenetic phenomena that stabilize p53 protein in ICC. That is, wild type p53 may be stabilized and then detectable by forming complexes with other molecules of p53 downstream effector genes, such as WAF-1 and mdm-2.

Ras gene mutations in ethmoid sinus adenocarcinoma: prognostic implications.

The presence of mutations of the 3 ras proto-oncogenes in 31 cases of ethmoid sinus adenocarcinoma, an uncommon tumor type epidemiologically related to professional exposure to wood dust, was studied. The authors studied 31 patients with ethmoid sinus adenocarcinoma. The polymerase chain reaction was used to amplify ras specific sequences of DNA isolated from paraffin embedded tumor samples. ras point mutations were subsequently detected with mutation specific oligonucleotide probes. H-ras was found to be mutated in 5 cases (16%). It is noteworthy that all of these mutations were identical and consisted of a G-for-T transversion at the second base of codon 12. H-ras mutations were related to a worse prognosis, with shorter tumor free survival (P = 0.04) and overall survival (P = 0.008). T classification was a significant clinical factor related to survival (P = 0.01 for disease free survival and P = 0.006 for overall survival). The prognostic value of H-ras mutation was consistent after adjustment for T classification. H-ras mutations showed no association with patients' previous exposure to wood dust. K-ras was found to be transformed in a single case; this was the only patient in the series to develop lymph node metastases. In this case, both the nasal tumor and the lymph nodes showed the GAT-for-GGT mutation at codon 12 of K-ras. No activation of the N-ras gene was detected. The presence of H-ras point mutations defines a subgroup of patients with ethmoid sinus adenocarcinomas for whom the prognosis is very poor. The finding that all of these mutations are identical emphasizes the peculiarity of this type of tumor.

Ras Gene mutations in ethmoid sinus adenocarcinoma

BACKGROUND The presence of mutations of the 3 ras proto-oncogenes in 31 cases of ethmoid sinus adenocarcinoma, an uncommon tumor type epidemiologically related to professional exposure to wood dust, was studied. METHODS The authors studied 31 patients with ethmoid sinus adenocarcinoma. The polymerase chain reaction was used to amplify ras specific sequences of DNA isolated from paraffin embedded tumor samples. ras point mutations were subsequently detected with mutation specific oligonucleotide probes. RESULTS H-ras was found to be mutated in 5 cases (16%). It is noteworthy that all of these mutations were identical and consisted of a G-for-T transversion at the second base of codon 12. H-ras mutations were related to a worse prognosis, with shorter tumor free survival (P = 0.04) and overall survival (P = 0.008). T classification was a significant clinical factor related to survival (P = 0.01 for disease free survival and P = 0.006 for overall survival). The prognostic value of H-ras mutation was consistent after adjustment for T classification. H-ras mutations showed no association with patients' previous exposure to wood dust. K-ras was found to be transformed in a single case; this was the only patient in the series to develop lymph node metastases. In this case, both the nasal tumor and the lymph nodes showed the GAT-for-GGT mutation at codon 12 of K-ras. No activation of the N-ras gene was detected. CONCLUSIONS The presence of H-ras point mutations defines a subgroup of patients with ethmoid sinus adenocarcinomas for whom the prognosis is very poor. The finding that all of these mutations are identical emphasizes the peculiarity of this type of tumor. Cancer 1999;86:255–64. © 1999 American Cancer Society.

H-ras and K-ras mutations in soft tissue sarcoma

The authors' recent investigation of Korean patients with sarcoma has suggested that ras gene activation may play a role in oncogenesis. The authors attempted to extend the mutation analysis to sarcomas in American patients to determine whether there were racial or geographic factors relevant to the initiation or progression of sarcoma. H-ras and K-ras genes were examined in sarcomas obtained from patients in the midwestern U. S. using the polymerase chain reaction technique and direct automated sequencing analysis. Tumors studied included 29 malignant fibrous histiocytomas, 7 liposarcomas, 5 rhabdomyosarcomas, and 9 leiomyosarcomas. Of the 50 sarcomas evaluated, only 1 (2%) definable mutation was found; a GGC to AGC transition at codon 12 of H-ras was found in a rhabdomyosarcoma. None of the patients had a K-ras mutation. The rates of incidence of ras point mutations in these samples were much lower (H-ras: 2%; 95% confidence interval [95% CI], 0-11.5% and K-ras: 0%) than described for both genes in Korean studies (H-ras: 16%; 95% CI, 5.2-26.8% and K-ras: 44%; 95% CI, 29.5-58.5%). Although the reason for this discrepancy is not clear, there were no major differences found in histology or clinical stages. Based on this study of 50 sarcoma samples from American patients and the authors' previous study of 45 Korean tumor samples, the authors conclude that differing genetic and/or environmental mechanisms can affect sarcoma development or progression. Mutation of the H-ras and K-ras genes appears to be uncommon in sarcomas occurring in American patients, suggesting that the activation by point mutations of the H-ras and K-ras genes does not play a significant role in the pathogenesis or progression of sarcoma in these patients.

H‐ras and K‐ras mutations in soft tissue sarcoma

BACKGROUND The authors' recent investigation of Korean patients with sarcoma has suggested that ras gene activation may play a role in oncogenesis. The authors attempted to extend the mutation analysis to sarcomas in American patients to determine whether there were racial or geographic factors relevant to the initiation or progression of sarcoma. METHODS H-ras and K-ras genes were examined in sarcomas obtained from patients in the midwestern U. S. using the polymerase chain reaction technique and direct automated sequencing analysis. Tumors studied included 29 malignant fibrous histiocytomas, 7 liposarcomas, 5 rhabdomyosarcomas, and 9 leiomyosarcomas. RESULTS Of the 50 sarcomas evaluated, only 1 (2%) definable mutation was found; a GGC to AGC transition at codon 12 of H-ras was found in a rhabdomyosarcoma. None of the patients had a K-ras mutation. The rates of incidence of ras point mutations in these samples were much lower (H-ras: 2%; 95% confidence interval [95% CI], 0-11.5% and K-ras: 0%) than described for both genes in Korean studies (H-ras: 16%; 95% CI, 5.2-26.8% and K-ras: 44%; 95% CI, 29.5-58.5%). CONCLUSIONS Although the reason for this discrepancy is not clear, there were no major differences found in histology or clinical stages. Based on this study of 50 sarcoma samples from American patients and the authors' previous study of 45 Korean tumor samples, the authors conclude that differing genetic and/or environmental mechanisms can affect sarcoma development or progression. Mutation of the H-ras and K-ras genes appears to be uncommon in sarcomas occurring in American patients, suggesting that the activation by point mutations of the H-ras and K-ras genes does not play a significant role in the pathogenesis or progression of sarcoma in these patients. Cancer 1999;86:58–63. © 1999 American Cancer Society.

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.

Sensitive Methods for the Detection of ras Mutations in Lung Cancer: Some Answers, More Questions

The ras family consists of three evolutionarily conserved genes, H- ras , N- ras , and K- ras . The ras genes code for 21-kDa proteins that mediate signal transduction between cell surface receptors and intracellular regulatory molecules. The proteins attach to the inner surface of the cytoplasmic membrane via a posttranslationally added farnesyl group. This lipid modification is essential for function, and considerable recent interest has focused on agents that inhibit the enzyme responsible, farnesyl transferase, as a mechanism to block the growth of tumors having ras mutations (1). Ras is homologous to a larger family of “G proteins” that exist in two states, GDP- and GTP-bound. Only the GTP-bound form is effective at mediating growth response, and there is a dynamic interconversion between the two forms. Point mutations of ras are the most frequent dominant oncogene mutation found in human tumors and usually target only three codons: 12, 13, and 61. Most activating mutations are defective in GTPase activity and thus are locked into the GTP-bound form, resulting in continuous growth stimulation. Detection of ras mutations in tumors enables us to understand cancer biology and pathogenesis and may be of clinical importance by providing information useful for early diagnosis and prognosis and the development of novel therapeutic approaches (1)(2). As discussed below, ras mutations may be detected by a large variety of methodologies, both conventional and, as reported in this issue of Clinical Chemistry , supersensitive (3). Although about 20–25% of human tumors have ras mutations, especially involving the K- ras gene, the distribution demonstrates considerable tumor type specificity (4)(5). Thus, although K- ras mutations are found in nearly 90% of pancreatic carcinomas and about 50% of colorectal carcinomas, they are rare in breast cancers. Lung cancers are an example of the heterogeneity …

Mutations of the NF1 Gene in Children With Juvenile Myelomonocytic Leukemia Without Clinical Evidence of Neurofibromatosis, Type 1

Juvenile myelomonocytic leukemia (JMML) is a pediatric myelodysplastic syndrome that is associated with neurofibromatosis, type 1 (NF1). The NF1 tumor suppressor gene encodes neurofibromin, which regulates the growth of immature myeloid cells by accelerating guanosine triphosphate hydrolysis on Ras proteins. The purpose of this study was to determine if the NF1 gene was involved in the pathogenesis of JMML in children without a clinical diagnosis of NF1. An in vitro transcription and translation system was used to screen JMML marrows from 20 children for NF1 mutations that resulted in a truncated protein. Single-stranded conformational polymorphism analysis was used to detect RAS point mutations in these samples. We confirmed mutations of NF1 in three leukemias, one of which also showed loss of the normal NF1 allele. An NF1 mutation was detected in normal tissue from the only patient tested and this suggests that JMML may be the presenting feature of NF1 in some children. Activating RAS mutations were found in four patients; as expected, none of these samples harbored NF1 mutations. Because 10% to 14% of children with JMML have a clinical diagnosis of NF1, these data are consistent with the existence of NF1 mutations in approximately 30% of JMML cases.

Detection of codon 12 point mutations of the K-ras gene from mouse lung adenocarcinoma by 'enriched' PCR

Purpose: Recent studies have shown that chemical carcinogens induce a high frequency of point mutations in the K- ras oncogene from mouse lung tumours at codons 12, 13 and 61. These experiments were performed to identify K- ras mutations in tissues from control and radiation-exposed mice. Materials and methods: By modifying the technique of the 'enriched' polymerase chain reaction (PCR), it was possible to detect point mutations at codon 12 of the K- ras oncogene from 25-year-old paraffin-embedded normal lungs and lung adenocarcinomas from mice exposed to radiation. Together, a total of 120 lung tissues were screened for point mutations at codon 12 of the K- ras oncogene in this study. Results: A significant increase in K- ras codon 12 point mutations was observed in the normal lungs from mice exposed to 24 onceweekly neutron irradiations (100%), compared with normal lungs from mice with sham-irradiation (50%) (p 0.05). Lung adenocarcinomas from mice receiving 24 once-weekly neutron irradiations also had a significantly higher frequency of K- ras codon 12 point mutations (100%) than the lung adenocarcinomas of mice receiving 24 or 60 once-weekly gamma -ray irradiations (50%), but the higher frequency was not significantly different from that in spontaneous lung adenocarcinomas from mice (75%; p 0.05). The validity of the technique was confirmed by sequencing two of the mutants. In doing so, a K- ras 13(Asp) point mutation was observed. Conclusions: The data suggest that high-linear energy transfer (LET) neutron radiation was more effective than low-LET gamma -rays in inducing K- ras point mutations at codon 12 in the lungs of B6CF1 mice.

Synergistic effect of MNU and DMBA in mammary carcinogenesis and H- ras activation in female Sprague–Dawley rats

The combined application of N-methyl- N-nitrosourea (MNU) and 7,12-dimethylbenz[ α]anthracene (DMBA) was compared with the administration of each carcinogen alone as to the effectiveness of the induction of mammary carcinomas and the influence of H- ras oncogene activation in female Sprague–Dawley rats. At 50 days of age, group 1 received 30 mg/kg MNU intraperitoneally (i.p.), group 2 received 30 mg/kg DMBA i.p., group 3 received 60 mg/kg MNU i.p., group 4 received 60 mg/kg DMBA i.p., group 5 received 30 mg/kg MNU followed by 30 mg/kg DMBA i.p., group 6 received 30 mg/kg MNU i.p. and then 30 mg/kg DMBA intravenously (i.v.) and group 7 remained untreated. Animals were killed when the largest mammary tumor reached 1–2 cm in diameter or were necropsied when they were 30 weeks of age. MNU i.p. produced no deaths (groups 1 and 3), however, the i.p. administration of DMBA induced death due to peritonitis (groups 2, 4 and 5), whereas the i.v. administration of DMBA suppressed the death (group 6). All of the tumors produced by MNU were adenocarcinomas of mammary origin. In contrast, DMBA produced tumors of other than mammary origin. The combined treatment with DMBA and MNU increased the mammary carcinogenic effect; it significantly increased the mean number of mammary cancers per rat. With either carcinogen alone and in combination, the mammary carcinomas produced identical adenocarcinoma histology. Of the mammary carcinomas induced by the combined application of MNU and DMBA (group 6), all 11 tumors from five rats showed the GGA to GAA transitional mutation in H- ras codon 12 (38%) and all 18 tumors from the other 10 rats remained as wild-type. An H- ras point mutation at codon 61 was not detected.

Detection of colorectal cancer K- ras mutations using a simplified oligonucleotide ligation assay

It has been suggested that some mutations in codons 12 and 13 of the K- ras gene are associated with the progression of colorectal adenomas to carcinomas. The aim of this study was to develop a rapid, colorimetric assay for K- ras point mutations commonly associated with colorectal cancer. K- ras exon 1 was amplified from colorectal tumor DNA and K- ras activating mutations detected using an oligonucleotide ligation assay (OLA) in combination with immunological and colorimetric detection. Using the OLA with oligonucleotides specific to individual K- ras mutations, 6 (of 17 total colorectal adenomas/carcinomas) were found to have K- ras mutations. The assay could detect as little as 10% mutant allele. A simplified OLA designed to test for either the presence (+) or absence (−) of any of the K- ras activating mutations was developed. The assay was further streamlined by use of a dipstick methodology for colorimetric development. If required, assay sensitivity can be increased by the use of the recently described EDNA–ELCA detection system. The simplified (+/−) mutation OLA in combination with a dipstick or EDNA–ELCA detection system provides a rapid, sensitive assay for K- ras point mutations suitable for use as part of the clinical assessment of colorectal cancer.

The RET proto-oncogene in medullary and papillary thyroid carcinoma. Molecular features, pathophysiology and clinical implications.

The evolution of cancer is a multistep phenomenon, and multiple cellular genetic lesions are involved in the emergence of the malignant neoplasm. Several early events have been implicated in the neoplastic transformation of thyrocytes, and recent reports have described the involvement of specific genetic alterations in different types of thyroid neoplasms: ras point mutations are frequently observed in tumours with follicular histology, gsp-the mutated form of the alpha subunit of the Gs-protein-is encountered in up to 73% of papillary or follicular thyroid carcinomas, and a high prevalence of p53 point mutations has been found in anaplastic thyroid carcinomas but not in differentiated follicular tumours. More recent studies revealed that the RET proto-oncogene is involved in the oncogenesis of medullary thyroid carcinoma (MTC) and papillary thyroid carcinoma (PTC) by activation of its tyrosine kinase either by point mutation or rearrangement. In this review the most important recently published data on alterations of the RET proto-oncogene in heritable and sporadic MTCs and in PTCs will be summarized. Emphasis will be directed to the pathophysiological mechanisms of tumour initiation, the indications and limitations of DNA testing, and the clinical implications of identified RET defects in thyroid lesions.

Detection of K- ras point mutation by in situ PCR in cell suspensions: Comparison of the indirect and direct methods

In situ PCR is a new technique for the localization of low copy number sequences. We report here a method for the in situ visualization of a point mutation in K- ras codon 12 by indirect in situ PCR. Twenty-five primers were examined to select mutant-specific primers. Harvested cell lines were fixed and suspended in PCR mixture. Forty cycles of PCR in cell suspension was performed in a thermal cycler using a hot start method. Cells were cytocentrifuged onto slides, and post-fixation was performed. The specimens on the slides were then hybridized with a digoxigenin-labeled probe, followed by color reaction. Both Calu-1 (mutated: TGT) and NCI-H460 (wild type: GGT) cells had strong hybridization signals in the nuclei with general primers. But with mutant-specific primers, only Calu-1 cells had hybridization signals. No signal was observed without primers or Taq DNA polymerase. Southern blotting of the same preparation confirmed desired amplification. We also applied direct in situ PCR, but this method failed to detect the point mutation. We conclude that our indirect in situ PCR method shows the feasibility of in situ identification of single cells carrying point mutations.

Expression of the placenta-specific, 100 kDa ras GTPase activating protein in several human cancer cell lines and normal human tissues.

The ras GTPase activating protein (ras GAP), a regulator of Ras activity, has two isoforms; ras GAP 120 and ras GAP 100. The latter, whose molecular size is about 100 kDa, is generated alternative splicing from the ras GAP 120 gene and is considered placenta-specific, while the former is expressed ubiquitously. As point mutations of ras are frequently observed in human tumors, we investigated the expression of ras GAP in several human cancer cell lines and samples of human colon cancer using immunoprecipitation and immunoblot analysis with an anti-GAP monoclonal antibody, B4F8, as well as reverse transcription-polymerase chain reaction (RT-PCR). ras GAP 100 protein was detected in 4 of 9 colonic, 1 of 6 gastric and 1 of 4 lung cancer cell lines as well as ras GAP 120, but not in colon cancer specimens. In contrast, ras GAP 100 mRNA was present in all tested cell lines and colon cancer specimens. Then, we investigated ras GAP 100 expression in normal tissues, ras GAP 100 protein was not detected in human normal tissues except placenta. Contrary, ras GAP 100 message was expressed in normal tissues derived from liver, stomach, colon and lymphocyte although the level of which was smaller than that in placenta. These findings demonstrate that ras GAP 100, reportedly placenta-specific, is distributed in other normal tissues at least at mRNA level and its expression is augmented in some cancer cell lines.

Cytogenetics and molecular genetics of carcinomas arising from thyroid epithelial follicular cells.

Cytogenetic and molecular analyses of thyroid tumors have indicated that these neoplasms represent a good model for analyzing human epithelial cell multistep carcinogenesis. They comprise, in fact, a broad spectrum of lesions with different phenotypes and variable biological and clinical behavior. Molecular analysis has detected specific genetic alterations in the different types of thyroid tumors. In particular, the well-differentiated carcinomas of the papillary type are characterized by activation of the receptor tyrosine kinases (RTKs), RET and NTRK1 proto-oncogenes. Cytogenetic analysis of these tumors has contributed to defining the chromosomal mechanisms leading to RTK oncogenic activation. In the majority of cases, intrachromosomal inversions of chromosome 10 and chromosome 1 led to the formation of RET-derived and NTRK1-derived oncogenes, respectively. Interestingly, molecular analysis of these oncogenes revealed their nature of chimeric fusion proteins all sharing the tyrosine kinase (TK) domains of the respective proto-oncogenes. Moreover, the sequencing of the oncogenic rearrangements led to the identification of a breakpoint cluster region in both RTK proto-oncogenes. Exposure to ionizing radiation is associated with papillary carcinomas and RET activation has been suggested to be related to this event. Conversely, RAS point mutations are frequently observed in tumors with follicular histology and have been associated with metastatic dissemination. Iodide-deficient areas seem to provide a higher frequency of RAS positive follicular carcinomas. Finally, a high prevalence of TPS3 point mutations has been detected only in undifferentiated or anaplastic carcinomas and found to correlate inversely with 8CL2 expression. All of these findings are contributing to the definition of genetic and environmental factors relevant for the pathogenesis of thyroid tumors. Moreover, the characterization of specific genetic lesions could provide significant molecular tools for a better differential diagnosis and for the development of novel therapeutic avenues for thyroid cancer.

Cytogenetics and molecular genetics of carcinomas arising from thyroid epithelial follicular cells

Cytogenetic and molecular analyses of thyroid tumors have indicated that these neoplasms represent a good model for analyzing human epithelial cell multistep carcinogenesis. They comprise, in fact, a broad spectrum of lesions with different phenotypes and variable biological and clinical behavior. Molecular analysis has detected specific genetic alterations in the different types of thyroid tumors. In particular, the well-differentiated carcinomas of the papillary type are characterized by activation of the receptor tyrosine kinases (RTKs), RET and NTRKI proto-oncogenes. Cytogenetic analysis of these tumors has contributed to defining the chromosomal mechanisms leading to RTK oncogenic activation. In the majority of cases, intrachromosomal inversions of chromosome 10 and chromosome I led to the formation of RET-derived and NTRKI-derived oncogenes, respectively. Interestingly, molecular analysis of these oncogenes revealed their nature of chimeric fusion proteins all sharing the tyrosine kinase (TK) domains of the respective proto-oncogenes. Moreover, the sequencing of the oncogenic rearrangements led to the identification of a breakpoint cluster region in both RTK proto-oncogenes. Exposure to ionizing radiation is associated with papillary carcinomas and RET activation has been suggested to be related to this event. Conversely, RAS point mutations are frequently observed in tumor with follicular histology and have been associated with metastatic dissemination. Iodide-deficient areas seem to provide a higher-frequency of RAS positive follicular carcinomas. Finally, a high prevalence of TP53 point mutations has been detected only in undifferentiated or anaplastic carcinomas and found to correlate inversely with BCL2 expression. All of these findings are contributing to the definition of genetic and environmental factors relevant for the pathogenesis of thyroid tumors. Moreover, the characterization of specific genetic lesions could provide significant molecular tools for a better differential diagnosis and for the development of novel therapeutic avenues for thyroid cancer. Genes Chromosom Cancer 16:1–14 (1996). © 1996 Wiley-Liss, Inc.

Mutagenic effects of tumorigenic neutron radiation

A fraction of thymic lymphomas induced by high LET neutron radiation contains activating mutations (single-base substitutions) in the ras genes. To determine whether such mutations are the result of the interaction of high LET radiation with cellular DNA, we have utilized an in vitro model system to screen and isolate neutron-radiation-induced mutants. With that aim, we irradiated the PL61 hamster cell line with 0.4 MeV neutrons. This cell line contains linked copies of the gpt and neo(r) genes, which permits selection for large or small alterations, depending on the selection imposed. Mutants selected for large alterations represented 98.2% of the total. When selection for small mutations was imposed, 9 clones grew. The molecular and biochemical analysis of these clones revealed that 5 of them had identifiable mutations in the gpt gene, consisting of small insertions and deletions, but no single-base substitutions were detected. This represents the first sequence characterization of neutron-induced mutants. The results obtained are consistent with the notion that the ras point mutations identified in the neutron-induced tumors are most likely detected due to the strong selective advantage that they confer to the host cell, but they probably arose during tumour evolution, since they represent a negligible proportion of the total number of alterations induced by neutron radiation.