A 3'-UTR KRAS-variant is associated with cisplatin resistance in patients with recurrent and/or metastatic head and neck squamous cell carcinoma.

BACKGROUND: A germline mutation in the 3'-untranslated region of KRAS (rs61764370, KRAS-variant: TG/GG) has previously been associated with altered patient outcome and drug resistance/sensitivity in various cancers. We examined the prognostic and predictive significance of this variant in recurrent/metastatic (R/M) head and neck squamous cell carcinoma (HNSCC). PATIENTS AND METHODS: We conducted a retrospective study of 103 HNSCCs collected from three completed clinical trials. KRAS-variant genotyping was conducted for these samples and 8 HNSCC cell lines. p16 expression was determined in a subset of 26 oropharynx tumors by immunohistochemistry. Microarray analysis was also utilized to elucidate differentially expressed genes between KRAS-variant and non-variant tumors. Drug sensitivity in cell lines was evaluated to confirm clinical findings. RESULTS: KRAS-variant status was determined in 95/103 (92%) of the HNSCC tumor samples and the allelic frequency of TG/GG was 32% (30/95). Three of the HNSCC cell lines (3/8) studied had the KRAS-variant. No association between KRAS-variant status and p16 expression was observed in the oropharynx subset (Fisher's exact test, P = 1.0). With respect to patient outcome, patients with the KRAS-variant had poor progression-free survival when treated with cisplatin (log-rank P = 0.002). Conversely, KRAS-variant patients appeared to experience some improvement in disease control when cetuximab was added to their platinum-based regimen (log-rank P = 0.04). CONCLUSIONS: The TG/GG rs61764370 KRAS-variant is a potential predictive biomarker for poor platinum response in R/M HNSCC patients. CLINICAL TRIAL REGISTRATION NUMBERS: NCT00503997, NCT00425750, NCT00003809.

Nuclear factor-kappa B pathway and response in a phase II trial of bortezomib and docetaxel in patients with recurrent and/or metastatic head and neck squamous cell carcinoma.

BACKGROUND: Our previous study has shown that nuclear factor-kappa B (NF-kappaB)-signaling pathway was associated with a higher rate of recurrence in head and neck squamous cell carcinoma (HNSCC). The combination of bortezomib, an NF-kappaB inhibitor by inhibition of proteasomes, plus docetaxel was assessed for efficacy and toxicity. MATERIALS AND METHODS: Patients with recurrent and/or metastatic HNSCC were enrolled on a phase II bortezomib/docetaxel trial (bortezomib 1.6 mg/m(2) and docetaxel 40 mg/m(2) on days 1 and 8 of a 21-day cycle). Response was assessed using RECIST. Tissue specimens were evaluated for the presence of human papillomavirus (HPV) and expression of NF-kappaB-associated genes. RESULTS: Twenty-one of 25 enrolled patients were assessable for response; one partial response (PR, 5%), 10 stable disease (SD, 48%) and 10 progressive disease (PD, 48%). Patients with PR/SD had significantly longer survival compared with patients with PD and the regimen was well tolerated. Only one of 20 tumors was positive for HPV. Patients with PD had higher expression of NF-kappaB and epidermal growth factor receptor-associated genes in their tumors by gene expression analysis. CONCLUSION: Further understanding of treatment resistance and interactions between bortezomib and docetaxel may provide novel approaches in managing HNSCC.

Microsatellite mutations in buccal cells are associated with aging and head and neck carcinoma.

Carcinogen exposure from tobacco smoking is the major cause of upper aerodigestive tract cancer, yet heavy smokers only have about a 10% life-time risk of developing one of these cancers. Current technologies allow only limited prediction of cancer risk and there are no approved screening methods applicable to the general population. We developed a method to assess somatic mutational load using small-pool PCR (SP-PCR) and analysed mutations in DNA isolated from cells obtained by mouth rinse. Mutation levels in the hypermutable tetranucleotide marker D7S1482 were analysed in specimens from 25 head and neck squamous carcinoma (HNSCC) cases and 31 controls and tested for associations with age, smoking history and cancer status. We found a significant association between mutation frequency and age (P=0.021, Generalized Linear Model (GLM), N=56), but no influence of smoking history. Cases had higher mutation frequencies than controls when corrected for the effects of age, a difference that was statistically significant in the subgroup of 10 HNSCC patients who were treated with surgery only (P=0.017, GLM, N=41). We also present evidence that cancer status is linked to levels of nonunique, and presumably clonally derived, mutations in D7S1482. Insertion mutations were observed in 833 (79%) of 1058 alleles, of which 457 (43%) could be explained by insertion of a single repeat unit; deletion mutations were found in 225 (21%) of tested alleles. In conclusion, we demonstrate that the sensitive detection of single molecule mutations in clinical specimens is feasible by SP-PCR. Our study confirms an earlier report that microsatellite mutations increase with age and is the first to provide evidence that these mutations may be associated with cancer status in individual subjects.

A novel host cell reactivation assay to assess homologous recombination capacity in human cancer cell lines.

Repair of DNA double-strand breaks (DSB) is essential for cell viability and genome stability. Homologous recombination repair plays an important role in DSB repair and impairment of this repair mechanism may lead to loss of genomic integrity, which is one of the hallmarks of cancer. Recent research has shown that the tumor suppressor genes p53 and BRCA1 and -2 are involved in the proper control of homologous recombination, suggesting a role of this type of repair in human cancer. We developed a novel assay based on recombination between two Green Fluorescent Protein (GFP) sequences in transiently transfected plasmid DNA. The plasmid construct contains an intact, emission-shifted, "blue" variant of GFP (BFP), with a 300 nucleotide stretch of homology to a nonfunctional copy of GFP. In the absence of homologous recombination only BFP is present, but homologous recombination can create a functional GFP. The homologous regions in the plasmid were constructed in both the direct and the inverted orientation of transcription to detect possible differences in the recombination mechanisms involved. A panel of human tumor cell lines was chosen on the basis of genetic background and chromosome integrity and tested for homologous recombination using this assay. The panel included cell lines with varying levels of karyotypic abnormalities, isogenic cell lines with normal and mutant p53, isogenic cell lines with or without DNA mismatch repair, BRCA1 and -2 mutant cell lines, and the lymphoma cell line DT40. With this assay, the observed differences between cell lines with the lowest and highest levels of recombination were about 100-fold. Increased levels of recombination were associated with mutant p53, whereas a low level of recombination was found in the BRCA1 mutant cell line. In the cell line HT1080TG, a mutagenized derivative of HT1080 with two mutant alleles of p53, high levels of recombination were found with the direct orientation but not with the inverted orientation plasmid. No difference in recombination was detected between two isogenic cell lines that only differed in DNA mismatch repair capability. We conclude that this assay can detect differences in homologous recombination capacity in cultured cell lines and that these differences follow the patterns that would be expected from the different genotypes of these cell lines. Future application in normal cells may be useful to identify genetic determinants controlling genomic integrity or to detect differences in DNA repair capacity in individuals.

K-ras and p53 in pancreatic cancer: association with medical history, histopathology, and environmental exposures in a population-based study.

Pancreatic cancer is a highly fatal cancer with few identified risk factors. Increased risk of pancreatic cancer in tobacco smokers and among diabetic patients is well established, and some reports have suggested associations with coffee consumption and occupational exposure to organochlorines. At present, there is little information regarding the possible association of these risk factors with the known genetic alterations found in pancreatic cancers, such as activation of the K-ras oncogene and inactivation of the p53 tumor suppressor gene. Knowledge of such relationships may help to understand the molecular pathways of pancreatic tumorigenesis. We investigated the association between these molecular defects and risk factors for pancreatic cancer in 61 newly diagnosed patients identified through an ongoing study of pancreatic cancer in the San Francisco Bay Area. Interview information was obtained regarding environmental exposures, medical history, and demographic factors. Serum levels of dichlorodiphenyltrichloroethylene (DDE) and polychlorinated biphenyls were available on a subset of 24 patients. Tumor blocks were located from local hospitals and used for K-ras mutational analysis at codon 12 and for p53 protein immunohistochemistry. The molecular analyses were facilitated through the use of laser capture microdissection, which provides a reliable method to obtain almost pure populations of tumor cells. Mutations in K-ras codon 12 were found in 46 (75%) of 61 pancreatic cancers. A prior diagnosis of diabetes was significantly associated with K-ras negative tumors (P = 0.002, Fisher's exact test). The absence of this mutation was also associated with increased serum levels of DDE, although this association was not statistically significant (P = 0.16, Wilcoxon's test). There was no difference in polychlorinated biphenyl levels between the K-ras wild-type and mutant groups. Immunohistochemical staining for p53 protein did not differ by patient characteristics or clinical history, but significant associations were found with poor glandular differentiation (P = 0.002, chi2 trend test), severe nuclear atypia (P = 0.0007, chi2 trend test), and high tumor grade (P = 0.004, chi2 trend test). Our results are suggestive of the presence of K-ras codon 12 mutation-independent tumorigenesis pathways in patients with prior diabetes and possibly in patients with higher serum levels of DDE. Our results also support a role for the p53 tumor suppressor protein in the maintenance of genomic integrity.

The frequency of p53, K-ras mutations, and microsatellite instability differs in uterine endometrioid and serous carcinoma: evidence of distinct molecular genetic pathways.

BACKGROUND: The two most common types of uterine endometrial carcinoma, endometrioid (UEC) and serous (USC), differ in their histopathologic appearance and biologic behavior. Recent studies suggest that these differences may be associated with distinct molecular genetic alterations. METHODS: In the current study, the authors compared the frequencies of K-ras and p53 mutations and microsatellite instability (MI) between UEC and USC by analyzing all 3 molecular genetic changes in one set of tumors. Furthermore, the distribution of these molecular genetic alterations was determined among UECs of different histopathologic grade. The authors analyzed 58 UECs with known MI status for K-ras and p53 mutations. The K-ras and p53 genes were analyzed in 45 and 6 cases of USC, respectively. These results were combined with previous data on p53 mutations (21 cases) and MI (34 cases) in USC. RESULTS: MI was present in 16 of 57 UECs (28%) but in none of 34 USCs. p53 mutations were found in 7 of 42 UECs (17%) and 25 of 27 USCs (93%) by direct sequencing of exons 5-8. UECs and USCs with p53 mutations showed strong immunoreactivity for p53 in about 85% of the cases, whereas about 15% of the cases were immunonegative. K-ras mutations at codon 12 were found in 15 of 58 UECs (26%) and in only 1 of 45 USC (2%) by dot blot oligohybridization after polymerase chain reaction amplification of exon 1. Notably, the frequency of both K-ras and p53 mutations and MI was significantly different between UEC and USC (P < 0.001). In UECs, MI and K-ras mutations occurred in low grade as well as in high grade tumors, whereas p53 mutations were present almost exclusively in high grade tumors. CONCLUSIONS: The results of this study suggest that different molecular genetic pathways are involved in the pathogenesis of UEC and USC and that low grade UEC may progress to high grade UEC. These findings support the hypothesis that UEC and USC are separate entities and suggest that different molecular genetic alterations may be responsible for their distinct morphology and biologic behavior.

Inactivation of the p53 tumor suppressor gene via a novel Alu rearrangement.

Inactivation of the p53 tumor suppressor gene is a common finding in human cancer. In most cases, inactivation is due to a point mutation in the gene, but rearrangement of the p53 gene is sometimes observed. We analyzed the inactivation of p53 in the human pancreas cancer cell line Hs766T, which harbors a structural alteration in the p53 gene. This inactivation was found to be the result of a complex deletion/insertion event involving at least two different Alu elements. The rearrangement eliminated exons 2-4 from the p53 gene, whereas a 175-bp Alu fragment was inserted between the breakpoints of the deletion. DNA sequence analysis of this Alu fragment revealed that it is identical to an Alu element in intron 1 of the p53 gene. This is the first report of p53 inactivation due to a rearrangement involving Alu elements. This type of inactivation may go unnoticed when only traditional methods to detect p53 alterations are used.

p53 alterations in atypical alveolar hyperplasia of the human lung.

Atypical alveolar hyperplasia (AAH) is a potential precursor lesion from which lung adenocarcinomas arise and may be a good target for studying the early events of lung tumorigenesis. We have previously shown that AAHs are neoplastic epithelial proliferations that often harbor activating mutations of the K-ras oncogene. In the current study, we examined a spectrum of AAHs to determine the frequency and timing of p53 alterations in lung tumorigenesis. We analyzed 37 AAHs and their paired overt lung neoplasms for p53 protein accumulation using the monoclonal antibody DO7. DNA sequence analysis of the p53 gene was performed on those cases demonstrating p53 protein accumulation. AAHs were classified as low-grade, high-grade, or AAH-like carcinoma based on cytoarchitectural features. Accumulation of the p53 protein was found in none (0%) of 20 low-grade AAHs, in 1 (9%) of 11 high-grade AAHs, and in three (50%) of six AAH-like carcinomas. There was a statistically significant trend toward p53 accumulation with increasing grade of the AAHs. A missense mutation in exon 7 of the p53 gene was found in 1 AAH-like carcinoma, whereas mutations in exons 5 through 8 could not be detected in the other three AAHs with p53 protein accumulation. Three of the paired overt carcinomas harbored p53 mutations that were not present in the AAHs. Alterations of p53 do not appear to be common events in AAHs, especially when these lesions exhibit low-grade cytoarchitectural features. Alterations of p53, however, are more frequent at the level of AAH-like carcinoma and may be associated with the transition from a benign to a malignant proliferation of pneumocytes.

Several noncontiguous domains of CDK4 are involved in binding to the P16 tumor suppressor protein.

Cyclin-dependent kinase 4 (CDK4) is a key molecule in the regulation of cell cycle progression at the G1-S phase restriction point. Its activity is specifically regulated by p16 (also known as p16/CDKN2A, p16(INK4a), and MTS1), a tumor suppressor frequently altered in human cancers. A specific mutation in CDK4 codon 24 (Arginine to Cysteine) prevents p16 binding and thus inhibition by p16. This mutated CDK4 acts as a dominant oncogene and has been found in both sporadic and familial melanoma. To study the effects of other mutations in CDK4, we generated a panel of 18 CDK4 mutants using Charged-to-Alanine scanning mutagenesis, and investigated the p16-binding capacity of these mutants to identify novel sites involved in p16 binding. The mutant CDK4 proteins were generated by direct coupled transcription-translation in vitro and tested for binding to p16 using a p16-GST fusion protein. Several mutants demonstrated loss of p16 binding. In addition to the previously identified codon 24 mutants, alterations in and around codon 22, 25, 97, and 281 all showed loss of p16 binding capacity. These results indicate that several noncontiguous amino acid sequences on CDK4 are required for binding to p16, which suggests the existence of multiple sites of interaction with p16. Since p16-binding deficient CDK4 has oncogenic potential, these mutations may be present in melanomas or other human neoplasms.

Mutational analysis of the P16-binding domain of cyclin-dependent kinase 4 in tumors in the head region of the pancreas.

Alterations in genes involved in cell cycle regulation are common in many tumor types. In pancreatic adenocarcinomas, inactivating mutations in the CDKN2 gene, encoding the cyclin-dependent kinase inhibitor p16, are frequently observed. CDKN2 mutations have also been identified in the germline of 50% of patients with hereditary melanoma. Interestingly, such patients also have an increased risk for pancreatic cancers. In melanoma-prone kindreds with CDKN2 wild-type status, a mutation in one of the targets of p16, cyclin-dependent kinase 4 (CDK4) was reported, which abolishes p16 inhibition. To test the possible involvement of CDK4 mutations in pancreatic carcinoma, we analyzed sequence alterations in the p16-binding domain of CDK4 in DNA isolated from 32 tumors in the head region of the pancreas. Alterations in the CDK4 region between codon 1 and codon 56 were not observed in any of the tumors. Our results do not support disruption of the p16 pathway through CDK4 mutation as an oncogenic mechanism in pancreatic head tumorigenesis.

K-ras mutations in the duodenal fluid of patients with pancreatic carcinoma.

BACKGROUND: Many patients with carcinoma of the pancreas die because their disease is not detected until late in its course. Methods that detect these cancers earlier will improve patient outcome. Over 80% of pancreatic carcinomas contain mutations in codon 12 of the K-ras gene. Screening duodenal fluid for these mutations may lead to early detection of these cancers and assist in establishing a diagnosis of pancreatic carcinoma. METHODS: Polymerase chain reaction (PCR), with and without restriction enzyme-mediated mutant enrichment, was performed on DNA isolated from duodenal fluid specimens from 61 patients who underwent pancreaticoduodenectomy (Whipple's operation) for either periampullary cancer or a benign condition of the pancreas. Representative sections of pancreas pathology (primary carcinoma, benign tumor, or chronic pancreatitis) from the patients with duodenal fluid specimens containing amplifiable DNA were also analyzed for K-ras mutations. Wild-type and mutant K-ras were detected by hybridization of the PCR products with K-ras codon 12 mutant and wild-type specific probes. RESULTS: Seven of the 61 duodenal fluid specimens contained DNA that did not amplify. Thirteen (24% of the 54 duodenal fluid specimens with amplifiable DNA and 21% of the total of 61 specimens) contained activating point mutations at codon 12 of the K-ras gene. Mutations were detected in 13 of the 51 duodenal fluid specimens from patients with cancer (sensitivity, 25%), whereas mutations were not detected in any of the 9 amplifiable duodenal fluid specimens from patients with benign conditions of the pancreas (specificity, 100%). One duodenal fluid specimen from a patient with adenocarcinoma of the pancreas had two different K-ras mutations. DNA from three of the primary carcinomas did not amplify or was not available. Twenty-nine (69%) of the 42 primary tumors with amplifiable DNA contained K-ras mutations, whereas 3 (30%) of the 10 pancreata with benign conditions harbored mutations. Twenty-two (65%) of 34 ductal adenocarcinomas of the pancreas with amplifiable DNA had K-ras mutations. It is noteworthy that the same mutation was present in both the duodenal fluid and the primary carcinomas of 11 (92%) of the 12 patients who had primary tumors with amplifiable DNA as well as K-ras mutations in their duodenal fluid specimens. CONCLUSIONS: The identification of genetic alterations in cancer-causing genes in duodenal fluid may form the basis for the development of new approaches to the detection of carcinoma of the pancreas. Some pancreata without cancer, however, may also harbor K-ras mutations, potentially limiting the specificity of K-ras-based tests.

Can K-ras codon 12 mutations be used to distinguish benign bile duct proliferations from metastases in the liver? A molecular analysis of 101 liver lesions from 93 patients.

It can be difficult to distinguish benign bile duct proliferations (BDPs) from well-differentiated metastatic peripancreatic adenocarcinomas on histological grounds alone. Most peripancreatic carcinomas harbor activating point mutations in codon 12 of the K-ras oncogene, suggesting that K-ras mutational status may provide a molecular basis for distinguishing BDPs from liver metastases. The ability of tests for mutations in codon 12 of K-ras to make this distinction was examined in a two-part study. In the first part we determined the K-ras mutational status of 56 liver lesions and 48 primary peripancreatic adenocarcinomas obtained from 48 patients. In the second part of this study an additional 45 liver lesions were studied. In the first 48 patients, activating point mutations in codon 12 of K-ras were detected in 28 (61%) of the 46 primary carcinomas, in 8 (100%) of 8 liver metastases, in 2 (6.5%) of 31 BDPs, and in none (0%) of 14 liver granulomas. Three BDPs and two primary carcinomas did not amplify. To further estimate the prevalence of K-ras mutations in BDPs we analyzed an additional series of 45 mostly incidental BDPs for K-ras mutations. Three (6.7%) of these 45 harbored K-ras mutations. These results suggest that K-ras mutations may be useful in distinguishing BDPs from metastases in the liver; however, there is some overlap in the mutational spectra of BDPs and pancreatic carcinomas.

Multiple hyperplastic polyps in the stomach: evidence for clonality and neoplastic potential.

The origin and neoplastic potential of gastric epithelial polyps remains an area of great interest, and treatment choices are a topic of controversy. This report describes a patient diagnosed with three concurrent hyperplastic gastric polyps that were studied for genetic alterations. The polyps were investigated for alterations in the K-ras oncogene and the p53 tumor suppressor gene and for p21WAF1/Cip1 and MDM2 protein overexpression. In addition, loss of heterozygosity at several loci that are frequently involved in human cancer was analyzed, microsatellite instability, a hallmark of the "mutator" phenotype, was determined, and Epstein-Barr virus infection was investigated. All separate areas from the three independent polyps harbored the same activating point mutation in codon 12 of the K-ras oncogene, indicating a clonal origin. DNA sequence alterations in p53 were not found, although high p53 protein levels could be shown by immunohistochemistry in areas of carcinoma within the largest polyp. No alterations in any of the other molecular markers were observed. The results strongly favor a clonal origin of the three independent gastric polyps and support the notion that these hyperplastic polyps may carry a risk for malignancy.

Mutational activation of the K-ras oncogene and the effect of chemotherapy in advanced adenocarcinoma of the lung: a prospective study.

PURPOSE: To determine whether the clinical course and the response to chemotherapy of patients with advanced adenocarcinoma of the lung depends on the presence or absence of a ras mutation in the tumor. Mutational activation of K-ras is a strong adverse prognostic factor in stage I or II lung cancer and laboratory studies have suggested that ras mutations lead to resistance against ionizing radiation and chemotherapy. PATIENTS AND METHODS: Patients with advanced adenocarcinoma of the lung with measurable or assessable disease received chemotherapy with mesna, ifosfamide, carboplatin, and etoposide (MICE). Archival biopsies, fresh biopsies, or fine-needle aspirations were tested for the presence of ras gene mutations. Associations of ras mutations with clinical characteristics, response to chemotherapy, and survival were studied. RESULTS: The presence or absence of ras gene mutations could be established in 69 of 83 patients (83%). A total of 261 courses of MICE were administered to 62 informative patients, 16 of whom were shown to have a K-ras mutation-positive tumor. The frequency of mutations (26%) and the type of mutations closely matched the pattern we have previously reported in operable disease. Patients with a ras mutation in their tumor were more likely to have a close relative with lung cancer, but other clinical characteristics, such as pattern of metastases, response, and survival, were similar between the ras mutation-positive and ras mutation-negative groups. CONCLUSION: Patients with advanced lung adenocarcinoma who harbor a ras mutation may have major responses to chemotherapy and have similar progression-free and overall survival as patients with ras mutation-negative tumors. K-ras mutations may represent one of several ways in which early tumors are enabled to metastasize to distant sites.

Peutz-Jeghers polyps, dysplasia, and K-ras codon 12 mutations.

BACKGROUND: Peutz-Jeghers syndrome (PJS) is a rare, autosomal dominant, polyposis syndrome, associated with an increased risk of gastrointestinal and extragastrointestinal malignancy. Occasionally dysplasia occurs in PJS polyps. AIMS: In colorectal carcinomas, mutations in codon 12 of the K-ras oncogene are common and are found at similar frequency in precursor adenomas. Therefore, K-ras codon 12 point mutations in PJS polyps, were evaluated. MATERIALS AND METHODS: Fifty two PJS polyps, including four with dysplasia, collected from 19 patients with PJS, were analysed for mutations in the K-ras codon 12 by a mutant enriched polymerase chain reaction procedure, followed by allele specific oligodeoxynucleotide hybridisation. RESULTS: A K-ras codon 12 mutation was identified, in one colonic polyp with dysplasia. The mutation was found in the non-neoplasmic epithelial cells and not in the dysplastic component of the polyp. CONCLUSIONS: K-ras codon 12 point mutations are very rare in PJS polyps, by contrast with colorectal adenomas. The findings support previous evidence that there seems to be no intrinsic relation between K-ras codon 12 mutation and dysplasia.

Clinical and pathological associations with p53 tumour-suppressor gene mutations and expression of p21WAF1/Cip1 in colorectal carcinoma.

Inactivation of the p53 tumour-suppressor gene is common in a wide variety of human neoplasms. In the majority of cases, single point mutations in the protein-encoding sequence of p53 lead to positive immunohistochemistry (IHC) for the p53 protein, and are accompanied by loss of the wild-type allele. Recently, the WAF1/Cip1 gene was identified as one of the genes induced by wild-type p53, and increased expression of p21WAF1/Cip1 has been found to reflect the status of the p53 tumour-suppressor pathway. We investigated the inactivation of p53 in a relatively small, but well-characterised, group of 46 colorectal carcinomas that were previously studied for allelic alterations, ras oncogene mutations and DNA aneuploidy. Alterations in p53 were identified by IHC, loss of 17p and DNA sequence analysis of exons 5-8, whereas p21WAF1/Cip1 protein expression was determined by IHC. p53 mutations were identified in 19 of the 46 tumours (41%), whereas positive IHC for p53 was found in 21 of the 46 tumours (46%). Positive IHC for p21WAF1/Cip1 was detected in 16 of 42 cases (38%). We found no relationship between p21WAF1/Cip1 staining and p53 protein expression or p53 mutational status. Inactivating mutations in the p53 gene correlated with LOH at 17p but not with LOH at 5q or 18q, Dukes' stage, tumour grade or DNA ploidy. There was a higher survival rate independent of Dukes' stage in the group with no alterations in p53 compared with those with evidence of dysfunction of p53, but the difference was not statistically significant. We conclude that inactivation of p53 and altered expression of p21WAF1/Cip1 are common in colorectal carcinoma but do not correlate with each other or with the clinical or pathological parameters investigated.

Clinical significance of K-ras oncogene activation in ampullary neoplasms.

AIMS: To investigate the prevalence of K-ras codon 12 point mutations in ampullary neoplasms, to explore their clinical usefulness, and to test whether the detection of these mutations could be used to identify ampullary malignancies at an early stage. METHODS: Forty one tumour specimens from 28 patients with ampullary neoplasms were analysed for activating point mutations in K-ras codon 12 using a sensitive polymerase chain reaction (PCR) based assay. RESULTS: Eleven (39%) of the 28 primary tumours harboured point mutations in K-ras. Mutations were identified in seven (41%) of the 17 carcinomas and four (36%) of the 11 adenomas. Four of the possible six permutations in codon 12 were found in these 11 samples. This spectrum of mutations is different from pancreatic carcinoma but resembles that of colorectal neoplasms. Cytological brush specimens were available in 11 cases, and in all of these specimens, the K-ras status in the primary tumour and brush specimens was identical. CONCLUSIONS: K-ras codon 12 point mutations occur in about 40% of ampullary neoplasms at a relatively early stage in tumorigenesis. The pattern of mutations in these tumours resembles that of the adenoma-carcinoma sequence in the colorectum. These results indicate that ampullary neoplasms can be detected at an early stage by searching for genetic alterations in the K-ras oncogene in cytological brush specimens.

K-ras oncogene activation in atypical alveolar hyperplasias of the human lung.

Atypical alveolar hyperplasia (AAH) is a potential precursor lesion from which lung adenocarcinomas arise and may be a good target for studying the early events of lung tumorigenesis. A common genetic alteration in lung adenocarcinomas is mutational activation of K-ras. To determine the timing of K-ras activation, we evaluated formalin-fixed and paraffin-embedded tissue samples of 41 AAHs and their paired lung neoplasms from 28 patients for codon 12 point mutations of the K-ras oncogene. K-ras codon 12 mutations were detected using PCR followed by allele-specific oligonucleotide hybridization. Mutations were found in 16 (39%) of the 41 AAHs, 8 (42%) of the 18 adenocarcinomas, and none (0%) of the 5 lung neoplasms that were not adenocarcinomas. Of the 18 patients with both an AAH and a synchronous lung adenocarcinoma, 6 had K-ras mutation in the adenocarcinoma but not in the AAH, 6 had mutations in the AAH but not in the adenocarcinoma, 4 did not harbor mutations in either the AAH or the adenocarcinoma, and 2 had mutations in both their AAH and their synchronous adenocarcinoma. In just 1 of the 18 patients was the same K-ras mutation present in the AAHs and adenocarcinoma of the patient. The detection of independent activating point mutations in a cancer-causing gene points to the neoplastic nature of AAH and suggests that glandular neoplasms of the lung arise from a background of field cancerization.

Potential false-positive results with antigen enhancement for immunohistochemistry of the p53 gene product in colorectal neoplasms.

Immunohistochemistry (IHC) for intranuclear p53 gene product is a simple, routine alternative to molecular genetic analysis of the p53 gene. Several methods for antigen enhancement are currently in use for IHC. This study evaluates the effect of extreme antigen enhancement for p53, using a monoclonal antibody (DO7) and a polyclonal antibody (CM1). The cases studied were five colorectal carcinomas, two specimens of normal colorectal mucosa, and four colorectal carcinomas with genetic alterations which are expected to preclude p53 gene product expression, namely mutation to a STOP codon in the p53 gene detected by denaturing gradient gel electrophoresis with subsequent sequencing and allelic loss of 17p in the region where p53 is located, detected by restriction fragment length polymorphism analysis. The findings suggest that extreme antigen enhancement may cause false-positive results with a distinct nuclear staining pattern when MAb DO7 is used as a primary antibody. It is concluded that all antigen enhancement methods should be thoroughly tested to evaluate their validity and that there may be a limit to the extent to which antigen enhancement can be applied in IHC for p53 protein.