Detection by denaturant gradient gel electrophoresis of tumor-specific mutations in biopsies and relative bronchoalveolar lavage fluid from resectable non-small cell lung cancer.

A PCR-denaturant gradient gel electrophoresis (DGGE) method was developed for the detection of p53 and K-ras mutations in primary operable tumors and paired BAL samples of non-small cell lung cancer. Among 36 patients, 9 showed p53 exon V mutations in biopsies and in three paired bronchoalveolar lavage (BAL) specimens with a 33% concordance. Five patients presented p53 exon VI mutations in biopsies and in two paired BALs with a 40% concordance. No mutations were found in p53 exon VII either in biopsies or in paired BAL samples with 100% concordance. Exon VIII mutations were found in six primary tumors and in two BALs with a 33% concordance. Of 36 patients, we detected 7 (19.4%) with K-ras exon I mutations on tumor samples. DGGE analysis of DNA from BAL samples revealed three mutations distributed on K-ras exon I with a 42% overall concordance with respect to tumor tissue. Molecular screening by DGGE of p53-amplified DNA from BAL had cumulative 46.6% sensitivity, 100% specificity, and 77.7% accuracy. DGGE K-ras detection showed 43% sensitivity, 100% specificity, and 88.8% test accuracy. The method proposed demonstrated to be specific, accurate, and at relatively low cost but limited by low sensitivity in detecting the presence of neoplastic cells in patients with resectable non-small cell lung cancer.     

Molecular detection of tumor cells in bronchoalveolar lavage fluid from patients with early stage lung cancer

BACKGROUND: Conventional cytologic analysis of sputum is an insensitive test for the diagnosis of non-small-cell lung cancer (NSCLC). We have recently demonstrated that polymerase chain reaction (PCR)-based molecular methods are more sensitive than cytologic analysis in diagnosing bladder cancer. In this study, we examined whether molecular assays could identify cancer cells in bronchoalveolar lavage (BAL) fluid. METHODS: Tumor-specific oncogene mutations, CpG-island methylation status, and microsatellite alterations in the DNA of cells in BAL fluid from 50 consecutive patients with resectable (stages I through IIIa) NSCLC were assessed by use of four PCR-based techniques. RESULTS: Of 50 tumors, 28 contained a p53 mutation, and the identical mutation was detected with a plaque hybridization assay in the BAL fluid of 39% (11 of 28) of the corresponding patients. Eight of 19 adenocarcinomas contained a K-ras mutation, and the identical mutation was detected with a mutation ligation assay in the BAL fluid of 50% (four of eight) of the corresponding patients. The p16 gene was methylated in 19 of 50 tumors, and methylated p16 alleles were detected in the BAL fluid of 63% (12 of 19) of the corresponding patients. Microsatellite instability in at least one marker was detected with a panel of 15 markers frequently altered in NSCLC in 23 of 50 tumors; the identical alteration was detected in the BAL fluid of 14% (three of 22) of the corresponding patients. When all four techniques were used, mutations or microsatellite instability was detected in the paired BAL fluid of 23 (53%) of the 43 patients with tumors carrying a genetic alteration. CONCLUSION: Although still limited by sensitivity, molecular diagnostic strategies can detect the presence of neoplastic cells in the proximal airway of patients with surgically resectable NSCLC.     

Correlation between the status of the p53 gene and survival in patients with stage I non-small cell lung carcinoma

The association of p53 abnormalities with the prognosis of patients with non-small cell lung carcinoma (NSCLC) has been extensively investigated to date, however, this association is still controversial. Therefore, we investigated the prognostic significance of p53 mutations through exons 2 to 11 and p53 protein expression in 103 cases of stage I NSCLC. p53 mutations were detected in 49 of 103 (48%) tumors. Two separate mutations were detected in four tumors giving a total of 53 unique mutations in 49 tumors. Ten (19%) of mutations occurred outside exons 5-8. Positive immunohistochemical staining of p53 protein was detected in 41 of 103 (40%) tumors. The concordance rate between mutations and protein overexpression was only 69%. p53 mutations, but not expression, were significantly associated with a shortened survival of patients (P<0.001). Furthermore, we investigated the correlation between the types of p53 mutations and prognosis. p53 missense mutations rather than null mutations were associated with poor prognosis (P < 0.001 in missense mutations and P=0.243 in null mutations). These results indicated that p53 mutations, in particular missense mutations, rather than p53 expression could be a useful molecular marker for the prognosis of patients with surgically resected stage I NSCLC.     

Lack of prognostic significance of p53 and K-ras mutations in primary resected non-small-cell lung cancer on E4592: a Laboratory Ancillary Study on an Eastern Cooperative Oncology Group Prospective Randomized Trial of Postoperative Adjuvant Therapy.

PURPOSE: To determine the prognostic and predictive significance of p53 and K-ras mutations in patients with completely resected non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: Patients were randomized preoperatively to receive adjuvant postoperative radiotherapy (Arm A) or radiotherapy plus concurrent chemotherapy (Arm B). p53 protein expression was studied by immunohistochemistry (IHC) and p53 mutations in exons 5 to 8 were evaluated by single-strand conformational analysis. K-ras mutations in codons 12, 13, and 61 were determined using engineered restriction fragment length polymorphisms. RESULTS: Four hundred eighty-eight patients were entered onto E3590; 197 tumors were assessable for analysis. Neither presence nor absence of p53 mutations, p53 protein expression, or K-ras mutations correlated with survival or progression-free survival. There was a trend toward improved survival for patients with wildtype K-ras (median, 42 months) compared with survival of patients with mutant K-ras who were randomized to chemotherapy plus radiotherapy (median, 25 months; P = .09). Multivariate analysis revealed only age and tumor stage to be significant prognostic factors, although there was a trend bordering on statistical significance for K-ras (P = .066). Analysis of survival difference by p53 by single-stranded conformational polymorphism and IHC, interaction of p53 and K-ras, interaction of p53 and treatment arm, nodal station, extent of surgery, weight loss, and histology did not reach statistical significance. CONCLUSION: p53 mutations and protein overexpression are not significant prognostic or predictive factors in resected stage II or IIIA NSCLC. K-ras mutations may be a weak prognostic marker. p53 or K-ras should not be routinely used in the clinical management of these patients.     

Determination of microsatellite instability, p53 and K-RAS mutations in hepatic metastases from patients with colorectal cancer: relationship with response to 5-fluorouracil and survival

In vitro and clinical studies have suggested that high-frequency microsatellite instability (MSI-H) phenotype, p53 and K-ras mutations might influence the response to chemotherapy in a variety of tumors, including primary colorectal cancers (CRC). Unresectable hepatic metastases from CRC are commonly treated with 5-fluorouracil (5FU) and folinic acid. Since several new active drugs are now used for treating CRC, molecular determinants predictive to response to 5FU would thus be crucial for optimizing indications of chemotherapy to those patients. MSI-H phenotype, p53 and K-ras status were characterized in a prospective study of 56 patients with CRC metastatic to the liver and treated with 5FU-based chemotherapy. The objective response rate after a 3-month treatment was 32.1%. The prevalence of p53 mutations, K-ras mutations and MSI-H phenotype was 62.5%, 30.3% and 1.8%, respectively. No significant association was found between response to chemotherapy and p53 mutations (78% mutated tumors in responders vs. 55% in nonresponders; p = 0.10) and K-ras mutations (39% mutated tumors in responders vs. 26% in nonresponders; p = 0.34). Survival was longer for patients with p53-mutated metastases than for patients with unresected wild-type p53 metastases (median survival 15 months vs. 17 months; p = 0.06). The determination of the MSI-H phenotype, p53 and K-ras status in hepatic metastases from CRC does not discriminate a group of patients that should preferentially benefit from 5FU-based chemotherapy. The prognosis of patients with treated liver metastases is better when p53 is mutated.     

O(6)-Methylguanine-DNA methyltransferase promoter hypermethylation shifts the p53 mutational spectrum in non-small cell lung cancer.

The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) removes mutagenic adducts from the O6 position of guanine, thereby protecting the genome against G to A transition mutations. MGMT is inactivated by promoter hypermethylation in many human cancers and has been associated with G to A mutations in K-ras in colorectal cancer. We hypothesized that MGMT promoter hypermethylation would be associated with an increase in G to A transitions in the p53 gene in non-small cell lung cancer (NSCLC). p53 mutations were detected by both dideoxy sequencing and p53 GeneChip analysis in 92 patients with primary NSCLC. Methylation of the promoter region of the MGMT gene was determined using methylation-specific PCR and was present in 27 of 92 (29%) tumors. Hypermethylation of the MGMT promoter was more common in adenocarcinoma than in other histological types of NSCLC and was also more common in poorly differentiated tumors. MGMT promoter hypermethylation was present significantly more often in tumors with a G to A mutation in p53 (9 of 14; 64%) than in tumors with other types of p53 mutations (11 of 41; 27%; P = 0.02) or in tumors with wild-type p53 (7 of 37; 18%; P = 0.006). MGMT promoter hypermethylation was also strongly associated with G to A transitions at CpG sites. Inactivation of the MGMT gene by promoter hypermethylation alters the pattern of p53 mutation in NSCLC.     

P53 and K-ras mutations are frequent events in microscopically negative surgical margins from patients with nonsmall cell lung carcinoma

BACKGROUND: The objective of the current study was to determine whether tumor cells harboring P53 and K-ras mutations could be detected in histopathologically tumor-free surgical margins in patients with nonsmall cell lung carcinoma who underwent complete pulmonary resection. METHODS: In 118 consecutive patients, DNA obtained from primary tumors and from surgical margins was extracted for molecular analysis. A fragment of P53 gene encompassing exons 5-8 and codon 12 of the K-ras gene were amplified with the polymerase chain reaction technique and were assayed for the presence of mutations. RESULTS: P53 and K-ras mutations were found in 30% and 39% of primary tumors, respectively, and in 11 (9%) and 22 (18%) apparently tumor-free surgical margins, respectively. At least 1 of those mutations was found in surgical margins in 29 patients (25%), and both mutations were found in 2 patients (1.7%). P53 mutations in surgical margins accompanied mutations in primary tumors in 9 of 35 patients (26%), and K-ras mutations accompanied mutations in primary tumors in 20 of 46 patients (44%). Among patients with either mutation in primary tumors, the incidence of at least 1 mutation in surgical margins was 43% (28 of 65 patients). In four patients, mutations (two K-ras mutations and two P53 mutations) were found in surgical margins despite the absence of the corresponding mutations in primary tumors. The presence of mutations in primary tumors and in surgical margins was not related significantly to clinical characteristics or to patient outcomes. CONCLUSIONS: P53 and K-ras mutations are frequent events in surgical margins determined to be tumor free on light microscopy. The clinical relevance of these findings remains to be established.     

K-ras and p16(INK4A)alterations in sputum of NSCLC patients and in heavy asymptomatic chronic smokers

NSCLC rates among the most frequent and lethal neoplasm world-wide and a significant decrease in morbidity and mortality relies only upon effective early diagnostic strategies. We investigated K-ras mutations and p16(INK4A) hypermethylation in tumor tissue and sputum of 50 patients with NSCLC and correlated them with sputum cytology and with tumor staging, grading and location, to ascertain, in sputum, their potential diagnostic impact. The same genetic/epigenetic abnormalities and cytological features were also evaluated in sputum from 100 chronic heavy smokers. Genetic analysis identified molecular abnormalities in 64% tumors (14/50 K-ras mutations and 24/50 p16(INK4A) hypermethylation) and in 48% sputum (11/50 K-ras mutations and 16/50 p16(INK4A) hypermethylation). In tumors K-ras mutations and p16(INK4A) hypermethylation were mostly mutually exclusive, being found in the same patients in 3 cases only. Genetic abnormalities in sputum were detected only in molecular abnormal tumors. Molecular changes in sputum had rates of detection similar to cytology (42%) but the cyto-molecular combination increased the diagnostic yield up to 60%. Interestingly, the rate of detection of genetic changes in sputum of tumors at early stage (T1) was not significantly different from that of tumors at more advanced stage (T2-T4). In fact K-ras point mutations were frequently recognised in tumors at early stage while p16(INK4A) inactivation prevailed in tumors at advanced stage ( P=0.0063). As expected, diagnostic cytological findings were more frequently found in tumors at advanced stage (P=0.004). No correlation was found between tumor grading and location (central versus peripheral) and molecular changes. p16(INK4A) hypermethylation, but not K-ras mutations, was documented in sporadic cases of asymptomatic heavy smokers (4%) where it was uncoupled from cytological abnormalities. In conclusion the cyto-molecular diagnostic strategy adopted in this study was able to detect the majority of tumors but in order to be proposed as effective and early diagnostic tool, this molecular panel needs to be tested in prospective studies with adequate follow-up.     

Genetic alterations in p53 and k-ras in lung-cancer in relation to histopathology of the tumor and smoking history of the patient

The inactivation of the p53 suppressor gene and the activation of the ras proto-oncogenes are frequent events in non-small cell lung cancer as well as in many other solid neoplasms in man. Somatic mutations in exons 5-8 of the p53 gene were detected in 59% (30/51) of the squamous cell carcinoma and in 38% (14/37) of the adenocarcinoma tumors using GC-clamped, non-radioactive denaturing gradient gel electrophoresis (DGGE). The mutations in the exons 5 and 8 represented larger proportion of the alterations in squamous cell carcinoma tumors (p=0.04; Fisher's exact test, two-tailed); in the adenocarcinoma tumors, mutations were most common in the exon 7 of p53. Most of the identified mutations (25/39; 64%) are predicted to cause an amino acid substitution. Mutations leading to the premature termination of translation were more frequent in adenocarcinoma (6/14) than in squamous cell carcinoma (3/30) tumors (p=0.02). In adenocarcinoma, also base substitutions in the K-ras gene were detected more often (18/37; 49%) than in squamous cell carcinoma (p<0.01). However, a mutation both in p53 and Kras was detected in only 4% of the lung tumors which does not support importance of co-operation between the genes in vivo. Mutations in p53 and K-ras did not correlate with tumor differentiation in either histological type. In squamous cell carcinoma, mutations in p53 showed relation to pack years smoked whereas in adenocarcinoma, mutations in the K-ras gene were associated with cigarette consumption. G to T transversion was the most common type of base substitution in both genes (31% in p53 and 53% in K-ras).     

Differences in K-ras and p53 gene mutations among pancreatic adenocarcinomas associated with regional environmental pollution

BACKGROUND: Variations in genetic mutations in pancreatic carcinoma between different geographical regions have not been studied extensively, especially in developing countries where pancreatic cancer is relatively rare. METHODS: We studied the molecular pathology of 54 pancreatic adenocarcinomas from Egyptian patients residing in a heavily polluted region of the eastern Nile River delta and compared the findings with 45 tumors from patients residing in low-pollution regions. RESULTS: Rates of K-ras mutation in codon 12 and of p53 mutation in exons 5-8 were higher in tumors of patients from the high-pollution region as compared with the low-pollution regions (61.5 versus 34.2%, respectively, for K-ras, P = 0.01; 25.9 versus 11.6%, respectively, for p53, P = 0.08). There were also distinct differences in the specific types of K-ras and p53 mutations between the two regions. The ratio of G-to-T k-ras transversion mutation (codon 12) relative to wild-type was significantly higher in tumors from the high-pollution region (0.90) than tumors from the non-pollution site (0.28) (P = 0.03). Relative to tumors with wild-type, the ratio of p53 mutations in exons 5, 7 or 8 to wild-type in tumors from the high-pollution region was significantly higher than the ratio from the non-pollution site (0.28 versus 0.03, P = 0.01). Logistic regression showed that G-to-T transversion mutation in K-ras was predicted by the region of residence of the patients. CONCLUSIONS: Our study reveals that there are differences in the frequencies and types of K-ras and p53 mutations found in pancreatic adenocarcinomas of patients in high-pollution and low-pollution regions in Egypt and suggests that environmental factors may explain these differences. We speculate that gene-environment interactions in pancreatic carcinogenesis also occur in other populations.     

Differing molecular pathology of pancreatic adenocarcinoma in Egyptian and United States patients

Variations in genetic mutations in pancreatic carcinoma between different populations have not been studied extensively, especially in developing countries where pancreatic cancer is rare. We studied the molecular pathology of 44 pancreatic carcinomas from patients residing in a heavily polluted region in the Nile River delta and compared the findings with tumors from 44 United States (US) patients. We evaluated K-ras mutations in codon 12, p53 mutations in exons 5-8, and Gadd45a mutations in exons 1 and 4. Overall, rates of K-ras, p53 and Gadd45 mutations were not statistically different in tumors of patients from Egypt and the US (67.4 vs. 63.4%; 27.3 vs. 36.4% and 9.1 vs. 4.5%, respectively). However, there were distinct differences in the specific types of K-ras and p53 mutations between the 2 groups. In K-ras, G --> T transversion mutation was more frequent in the tumors from Egypt than from the US (58.6 vs. 26.9%), whereas G --> C transversion was detected in 26.9% of US tumors but none from Egypt (p = 0.003). We also found a trend toward differences in the p53 exons in which mutations occurred, with higher frequency of exon 5 mutation and lower frequency of exon 6 mutation in Egyptian tumors. Logistic regression showed that K-ras G --> T transversion mutations and p53 exon 6 mutations were predicted by the country of residence of the patients. Our study identifies that there are differences in the types of mutations found in tumors from pancreatic carcinoma patients in Egypt and the US, and suggests that environmental factors may explain these differences.     

The K-ras gene regulates vascular endothelial growth factor gene expression in non-small cell lung cancers

Tumor angiogenesis is an essential step for tumor cell growth, progression and metastasis. Vascular endothelial growth factor (VEGF) is mitogen specific for endothelial cells, and therefore is believed to play a key role in tumor angiogenesis. However, the mechanisms underlying the regulation of VEGF expression remain virtually unknown and the only major regulator of VEGF expression has been reported to be hypoxia. Recently, it was reported that a mutant p53 in#duced the expression of VEGF mRNA, and that wild-type p53 down-regulated endogenous VEGF mRNA levels. In contrast, it has also been reported that mutant ras oncogenes were associated with the marked up-regulation of VEGF in transformed epithelial cells. Based on these results, we performed a retrospective study of the p53 and K-ras genes status and VEGF gene expression in the tumor tissues from 181 patients with non-small cell lung cancer using SSCP, sequencing, RT-PCR and immunohistochemical techniques. Forty-six carcinomas (25.4%) were evaluated as having high VEGF expression, and 135 tumors (74.6%) had low VEGF expression. Of the 181 primary NSCLC studied, 63 carcinomas (34.8%) contained mutations of p53, whereas only 14 carcinomas (7.7%) had mutations of K-ras. There were no significant relationships between VEGF expression and p53 status or each mutant exon of p53. In contrast, a significant difference was found between VEGF expression and K-ras status. Of the 14 tumors with mutant K-ras genes, 7 cases (50.0%) had high VEGF expression whereas only 39 of the 167 tumors with wild-type K-ras (23.4%) had high VEGF expression (p=0.0278). The mean VEGF conservation rate for the 14 tumors with mutant K-ras genes was 0.77+/-0.58 and the rate of the 167 tumors with wild-type K-ras genes was 0.49+/-0.46 (p=0. 0350). Moreover, the overall survival rate of patients with high VEGF expression was lower than patients with low VEGF expression (45.7% vs 60.7%, p=0.0419). On the other hand, there was no significant difference in the overall survival rate between patients with a mutant p53 and those with a wild-type p53; there was also no difference in the overall survival between patients with a mutant K-ras and those with a wild-type K-ras. The Cox regression model analysis indicated that three variables, VEGF status, K-ras status and nodal status, were found to be significant indicators for prognosis (p=0.0236, p=0.0172 and p<0.0001, respectively). Our data suggest that a high expression of VEGF in lung cancer may be associated with a poor prognosis. This may be a clue to improving lung cancer diagnoses and therapies aimed at inhibiting tumor angiogenesis due to VEGF.     

A novel molecular staging protocol for non-small cell lung cancer.

A molecular staging protocol using reliable markers is of importance in predicting the prognosis of patients with non-small cell lung cancer (NSCLC) and for instituting their appropriate post-surgical treatment. We analysed tumor tissues from 187 NSCLC patients. The DNA and mRNA were extracted from frozen specimens, and then polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and direct sequencing were performed to investigate mutations of p53 from exons 5-8, and mutations of K-ras at exon 1. To determine MRP-1/CD9 gene and KA11/CD82 gene expression, which have been postulated to be metastasis suppressor genes, we have applied quantitative RT-PCR. A Cox multivariate regression analysis showed that nodal status, MRP-1/CD9 and K-ras status were significant factors for prognosis (P<0.0001, P=0.0083 and P=0.0004, respectively). Based on these results, we classified the patients into three groups according to their MRP-1/ CD9 and K-ras status. Patients with both MRP-1/CD9 positive and wild K-ras tumors were defined as group A, patients with either reduced MRP-1/CD9 or mutant K-ras tumors were defined as group B and patients with both reduced MRP-1/CD9 and mutant K-ras tumors were designated as group C. This new classification was significantly correlated with the tumor status and pathological stage (P=0.0098 and P=0.0017, respectively). The overall survival rate of the group A patients was significantly better than the group B patients (59.6% vs 27.9%, P=0.0001) and also that of group B patients was better than the group C patients (27.9% vs 20.0%, P=0.0378). This tendency was also found in patients with 110 node-negative NSCLCs (A vs B vs C=75.8% vs 34.9% vs 0.0%, P<0.0001). A Cox multivariate regression analysis in NSCLC patients demonstrated that an evaluation for both MRP-1/CD9 expression and K-ras mutations had a significant prognostic effect as well as nodal status (P<0.0001).     

Prognostic significance of K-ras codon 12 mutations in patients with resected stage I and II non-small-cell lung cancer.

PURPOSE: The aim of this study was to investigate the prognostic importance of codon 12 K-ras mutations in patients with early-stage non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: We identified 260 patients with surgically resected stage I (n = 193) and stage II (n = 67) NSCLC with at least a 5-year follow-up. We performed polymerase chain reaction analysis of DNA obtained from paraffin-embedded NSCLC tissue, using mutation-specific probes for codon 12 K-ras. RESULTS: K-ras mutations were detected in 35 of 213 assessable specimens (16.4%). K-ras mutations were detected in 27 of 93 adenocarcinomas (29.0%), one of 61 squamous cell carcinomas (1.6%), five of 39 large-cell carcinomas (12.8%), and two of 20 adenosquamous carcinomas (10%) (P = .001). G to T transversions accounted for 71% of the mutations. There was no statistically significant difference in overall survival for all patients with K-ras mutations (median survival, 39 months) compared with patients without K-ras mutations (median survival, 53 months; P = .33). There was no statistically significant difference in overall or disease-free survival for subgroups with stage I disease, adenocarcinoma, or non-squamous cell carcinoma or for specific amino acid substitutions. The median survival time for stage II patients with K-ras mutations was 13 months, compared with 38 months for patients without K-ras mutations (P = .03). CONCLUSION: Codon 12 K-ras mutations were more common in adenocarcinomas than in squamous cell carcinomas. For the subgroup with stage II NSCLC, there was a statistically significant adverse effect on survival for the presence of K-ras mutations. However, when the entire group was considered, the presence of K-ras mutations was not of prognostic significance in this cohort of patients with resected early-stage NSCLC.     

p53 and K-ras mutations in lung cancers from former and never-smoking women

Somatic p53 mutations are common in lung cancer. Active cigarette smoking is positively correlated with the total frequency of p53 mutations and G:C to T:A transversions on the nontranscribed (DNA coding) strand. Mutational hotspots within the p53 gene, e.g., codon 157, have been identified for tobacco-related lung cancer, whereas these same mutations are found rarely in other cancers. Such data implicate specific p53 mutations as molecular markers of smoking. Because limited data exist concerning the p53 mutation frequency and spectra in ex-smokers and nonsmokers, we have analyzed p53 and K-ras mutations in 126 lung cancers from a population-based case-control study of nonsmoking (n = 117) or ex-smoking (n = 9) women from Missouri with quantitative assessments of exposure to environmental tobacco smoke. Mutations in the p53 gene were found in lung cancers from lifetime nonsmokers (19%) and ex-smokers (67%; odds ratio, 9.08; 95% confidence interval, 2.06-39.98). All deletions were found in tumors from patients who were either ex-smokers or nonsmokers exposed to passive smoking. The G:C to A:T transitions (11 of 28; 39%) were the most frequent p53 mutations found and clustered in tumors from lifetime nonsmokers without passive smoke exposure. The incidence of K-ras codon 12 or 13 mutations was 11% (14 of 115 analyzed) with no difference between long-term ex-smokers and nonsmokers. These and other results indicate that p53 mutations occur more commonly in smokers and ex-smokers than in never-smokers. Such comparisons provide additional evidence of genetic damage caused by tobacco smoke during lung carcinogenesis.     

Mucinous carcinomas of the colorectum have distinct molecular genetic characteristics

We compared the frequency of CpG island methylation phenotype (CIMP), inactivation of APC, p53 and DCC genes and K-ras and BRAF mutations in 39 mucinous carcinomas (MC) and 34 non-mucinous carcinomas (NMC) of the colorectum with different microsatellite instability (MSI) status. The higher incidence of MSI (36% vs. 18%) was observed in MC compared with NMC. APC inactivation and K-ras mutations occurred more frequently in NMC (APC, 88%, p<0.001; K-ras, 58%, p=0.01) than in MC (APC, 24%; K-ras, 28%) regardless of MSI status. BRAF mutation occurred at a higher frequency in MC (18%, p=0.01) than in NMC (0%). However, with respect to inactivation of p53 and DCC, MSI status did matter and in both NMC and MC, more frequent inactivation of p53 and DCC was observed in MSS tumors than in MSI tumors. MSS tumors of NMC had a higher frequency of p53 (58% by IHC, p=0.03 and 83% by LOH, p=0.02) and DCC inactivation (83%, p=0.02) compared to MSI tumors of NMC (p53, 33% by IHC and 20% by LOH; DCC, 20%). MSS tumors of MC also showed a higher frequency of p53 and DCC inactivation (p53, 45% by IHC, p=0.02 and 53% by LOH, p=0.005; DCC, 82%, p=0.001) compared to MSI tumors of MC (p53, 0% by IHC and 0% for LOH; DCC, 17%). MC showed a higher frequency of CIMP compared with NMC (41% vs. 11%, p=0.01). These results indicate that mucinous carcinomas of the colorectum exhibit distinct molecular genetic characteristics and may arise from distinct pathogenic pathways.     

p53 and K-ras gene mutations correlate with tumor aggressiveness but are not of routine prognostic value in colorectal cancer.

PURPOSE: p53 gene and K-ras mutations are among the most common genetic alterations present in colorectal cancer. The prognostic utility of such mutations remains controversial. The purpose of this study was to prospectively evaluate the prognostic significance of p53 and K-ras gene mutations in colorectal cancer. PATIENTS AND METHODS: One hundred forty patients were analyzed. Tumors belonging to the microsatellite mutator phenotype were excluded (n = 8). Mutations at the K-ras and p53 genes were detected and characterized by restriction fragment length polymorphism, single-strand conformation polymorphism, and sequencing, as appropriate. RESULTS: p53 mutations were detected in 66 (50%) and K-ras mutations were detected in 54 (41%) of the 132 patients. In 26 cases (20%), ras and p53 mutations coexisted; in 38 cases (29%), neither mutation was found. Multivariate analysis of the whole population analyzed (n = 132) showed that survival was strongly correlated with the presence of p53 mutations alone or in combination with K-ras mutations (P = .002; log-rank test). When only patients undergoing a radical resection were considered (R0; n = 101), p53 mutations were no longer of prognostic significance. CONCLUSION: p53 mutations alone or in combination with K-ras mutations are correlated with a worse outcome. However, the routine use of these mutations as prognostic markers in the clinical setting is not recommended.     

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.     

p53 mutations do not predict response to paclitaxel in metastatic nonsmall cell lung carcinoma

BACKGROUND: In vitro data and animal studies suggest that paclitaxel may have a unique ability to activate tumor cell apoptosis in the absence of wild-type p53 function. The authors previously demonstrated that response to paclitaxel and concurrent radiation was not affected by p53 mutations in nonsmall cell lung carcinoma (NSCLC). We sought to determine whether p53 mutations affect response to paclitaxel alone in patients with metastatic NSCLC. METHODS: Twenty-five patients with metastatic NSCLC who participated in Brown University Oncology Group protocols utilizing single-agent weekly paclitaxel had tumor tissue that was adequate for p53 analysis. Tumor tissue was evaluated for p53 gene mutations in exons 5 through 8 by single-strand conformation polymorphism analysis. Mutations were confirmed by direct sequencing of altered mobility polymerase chain reaction products. RESULTS: Mutations in p53 were found in 8 of 25 patients (32%). The response rates of 75% for patients with tumors with p53 mutations and 47% for patients with wild-type p53 do not differ significantly (P = 0.12). The 1-year survival rates for patients with and without p53 mutation after treatment with weekly paclitaxel were 63% (95% confidence interval [CI], 31-100%) and 53% (95% CI, 33-86%), respectively. CONCLUSIONS: p53 mutations do not adversely affect response to paclitaxel as a single agent in metastatic NSCLC. These results provide clinical support for in vitro observations that paclitaxel can bypass mutant p53 and lead to tumor cell death by alternate pathway(s). Paclitaxel should be considered as a component of treatment for patients with metastatic NSCLC with tumors that have p53 mutations.     

NAD(P)H:quinone oxidoreductase-dependent risk for colorectal cancer and its association with the presence of K-ras mutations in tumors

NAD(P)H:quinone oxidoreductase (NQO1) is a polymorphic enzyme involved in the detoxification of potentially mutagenic and carcinogenic quinones. The homozygous C609T NQO1 genotype resulting in loss of reductase activity is found in 2-20% of individuals. In the present study, the NQO1-dependent risk for sporadic colorectal cancer (CRC) was studied in 247 incident CRC cases and 296 hospital-based controls recruited during 1996-1997. Four subgroups of cases were studied: (i) all CRCs; (ii) a molecular CRC subgroup (n = 117, cases with molecular tumor analyses); (iii) within the molecular subgroup those tumors with K-ras mutations in codon 12 (CRC K12); (iv) within the molecular subgroup those tumors with K-ras mutations in codon 13 (CRC K13). The C609T NQO1 genotype was found to be twice as prevalent in all CRC patients (6.8%) compared with controls (3%) and six times more common in the subset CRC K12 (20%). Multivariant analyses in the overall population of 247 cases and 296 controls showed a significant age and gender adjusted risk for CRC associated with the C609T NQO1 genotype (OR 2.9, 95% CI 1.19-6.97; P = 0.01) or with any variant genotype (the low activity allele frequency, i.e. heterozygotes plus homozygotes) (OR 1.41, 95% CI 1.02-1.92; P = 0.03). Within cases of the molecular subgroup (n = 117) the C609T NQO1 genotype was associated with the presence of K-ras codon 12 mutation (OR 6.5 95%, CI 1.39-34.9; P = 0.003). Logistic regression showed an age and gender adjusted risk for K-ras codon 12 mutant CRC associated with the C609T NQO1 genotype (OR 10.5, 95% CI 2.99-36.7; P: = 0.0002) or with any variant NQO1 genotype (OR 2.23, 95% CI 1.23-4.00; P = 0.007) compared with the control group. Genetically determined variations in NQO1 may modify the risk for CRC and these risks may be greatest for tumors containing K-ras codon 12 mutations. CRC with K-ras codon 12 mutations may represent a distinct and etiologically more homogeneous subtype of the disease, which may be associated with toxicants that are metabolized via a NQO1-dependent pathway.