Analysis of loss of heterozygosity (LOH) at the p53 and Rb suppressor genes in urinary bladder carcinoma

Loss of heterozygosity (LOH) at the p53 and Rb genes, and its clinical correlations were examined in 58 urinary bladder carcinomas. DNA was extracted from formalin-fixed, paraffin-embedded tissues, and amplified by polymerase chain reaction (PCR). The LOH at p53 was examined by restriction fragment length polymorphism (RFLP), and the LOH at Rb by single-strand conformation polymorphism (SSCP). Among patients with urinary bladder carcinoma, 60.3% (35/58 patients) showed heterozygosity at p53 and LOH was detected in carcinoma in 60.0% (21/35), i.e., LOH in 61.9% (13/21) of superficial (< or = pT1) and in 57.1% (8/14) of muscular invasive carcinomas. Therefore, LOH at p53 was considered to occur before the beginning of muscular invasion. Except for the patients who died of other causes or were missed during the follow-up, 67.4% (29/43) showed 43.8% (7/16) died of carcinomatosis, but, only 15.4% (2/13) of the patients without p53-LOH died. The 5-year survival rate calculated by the Kaplan-Meier method was 76.9% in patients with heterozygosity at p53, and 25.1% in those with LOH at p53, being significantly lower at p < 0.05 in the former than in the latter. From an analysis of multiple bladder carcinomas, LOH at p53 occurred on the same allele in different tumors of the same bladder, suggesting the monoclonal origin of each tumor of multiple bladder carcinomas. On the other hand, 51.7% (30/58) of patients showed heterozygosity at Rb, and LOH was detected in 16.7% (5/30) of them, i.e., 6.3% (1/16) in superficial carcinoma and 28.6% (4/14) in muscular invasive carcinoma.     

Genotypic analysis of tumor suppressor genes PTEN/MMAC1 and p53 in head and neck squamous cell carcinomas

OBJECTIVES: Tumor suppressor gene mutations in both p53 and PTEN/MMAC1 genomic DNA have been detected in many types of cancer. The purpose of this study was to investigate the presence and importance of PTEN/MMAC1 mutations in squamous cell carcinomas. METHODS: Exons of each gene were amplified after polymerase chain reaction (PCR) using genomic DNA derived from cell lines of squamous cell carcinoma of the head and neck (SCCHN) and snap-frozen biopsy specimens from primary established head and neck tumors. The amplified and purified DNA was then sequenced directly. RESULT: As anticipated, point mutations of the p53 gene were found in 80% of cell lines examined. A single base mutation in codon 151 was found in six of 10 cell lines studied. PTEN/MMAC1 gene mutations were found in neither the cell lines tested nor the tumor biopsy samples. CONCLUSION: This study, as well as a large volume of data, confirms that mutations of the p53 gene are frequent events in head and neck cancer cell lines. Although PTEN/MMAC1 gene mutations have been found in a variety of carcinomas, this gene was not found to be mutated in SCCHN cell lines or in primary squamous cell carcinomas of the head and neck. This information is useful for further studies of mutations in these cell lines.     

Chromosome 3p loss of heterozygosity and mutation analysis of the FHIT and beta-cat genes in squamous cell carcinoma of the head and neck

BACKGROUND: Patients treated with clozapine have been reported to gain weight. We hypothesized that patients would also experience an increase in body mass, which can be more directly related to cardiovascular morbidity. METHODS: Forty-two patients who had been treated with clozapine for at least 1 year were weighed and measured, and waist-hip ratios (WHR) and body mass index (BMI), measured as kg/m2, were calculated. Patients were also asked about a series of factors potentially related to change in body mass. RESULTS: Female patients gained both weight and body mass. Their WHR after 37 months of clozapine therapy was .83, with a significant increase in BMI from 23.2 to 29.1 kg/m2 (p = .001). Male subjects also gained weight and body mass. Their WHR after 39 months of clozapine therapy was .93, with a significant increase in BMI from 26.4 to 29.7 kg/m2 (p < .001). Stepwise multiple-regression analysis showed that factors related to final body mass were initial body mass, dose of clozapine, and decrease in smoking. Baseline BMI contributed most to the final BMI, but the addition of dose and decrease in smoking made significant contributions to the model. CONCLUSIONS: Both female and male patients treated with clozapine gain body mass. This may place them at greater risk for cardiovascular morbidity.     

Late p53 mutation in head and neck oncogenesis

p53 mutation are currently recognized as the commonest genetic event in human tumors. In this paper are studied, through immunochemistry, the p53 protein expression of 25 carriers from squamous cell carcinoma of the larynx and hypopharynx as well in the non tumoral mucosa of the larynx removed and corresponding metastases. Because of the great overexpression of this protein among tumors and its metastases, aside with the low expression in normal far-off mucous membranes of the studied growths, the AA. draw out the conclusion that p53 mutation is a late event by neck and head oncogenesis.     

Loss of heterozygosity and mutation analysis of the p16 (9p21) and p53 (17p13) genes in squamous cell carcinoma of the head and neck

We analyzed allelic loss at the p53 gene (17p13) and at chromosome region 9p21 in 35 primary head and neck squamous cell carcinomas. Loss of heterozygosity (LOH) at p53 and 9p21 was found in 50 and 75% of informative cases, respectively. LOH at the p53 gene did not increase significantly with tumor stage, but was more frequent in moderately and poorly differentiated tumors than in well-differentiated tumors. LOH plus mutation or homozygous deletion of p53 was limited to advanced stage and poorly differentiated tumors. Allelic loss at 9p21 is frequent in early stage head and neck squamous cell carcinoma and is not significantly associated with LOH at p53. The second exon of the p16/MTS1/CDKN2 gene was found to be homozygously deleted in 1 of 19 cases showing LOH at 9p21, but direct sequencing did not show mutations in the remaining 18 cases. This suggests that p16 plays a limited role in the development of head and neck squamous cell carcinoma.     

Rare loss of heterozygosity of the MTS1 and MTS2 tumor suppressor genes in differentiated human thyroid cancer

Loss of heterozygosity (LOH) of the MTS1 (p16) tumor suppressor gene has been reported to occur frequently in thyroid cancer cell lines. In order to determine the frequency of LOH for these multiple tumor suppressor genes, we used microsatellite markers IFNA and D9S171 to perform differential quantitative polymerase chain reaction. Tumor DNA was isolated from native sections of tumor tissue. Control DNA was isolated from blood. PCR products were separated on 6% polyacrylamide sequencing gels and quantified according to peak height and area. Analysis was informative in 70% of cases for both markers, and in 88% for at least one out of both. LOH was found in 3 out of 35 informative patients (8.6%) with papillary thyroid cancer, in 1 out of 7 patients with follicular thyroid cancer (14.2%), and in 0 out of 18 medullary cancers (0%). No LOH was found in 11 informative patients with multinodular goitre, 7 with follicular adenoma, 4 with Graves' disease, and 6 with other thyroid disease. 75% of LOH was found in T1 and T2 stages, it was not more frequent in patients with lymphonodular metastasis. The low frequency of LOH in these types of thyroid cancer argues against a role of loss of heterozygosity at the MTS 1 and 2 gene locus in the development of differentiated thyroid neoplasia.     

K-ras and p53 mutations are an independent unfavourable prognostic indicator in patients with non-small-cell lung cancer

We examined 159 consecutive cases of non-small-cell lung cancer (NSCLC) for a mutation at codon 12 of the K-ras gene and for a mutation of the p53 gene occurring in exons 5-8. Eleven (6.9%) had mutations of the K-ras (ras+) and 57 (35.8%) had mutations of the p53 (p53+). There were 95 cases (59.7%) with ras- p53-, seven cases (4.4%) with ras+/p53-, 53 cases (33.3%) with ras-/p53+ and four cases (2.5%) with ras+/p53+. The ras+ group had a worse prognosis than the ras group in all cases and in 107 early-stage cases (stage I-II, P<0.05). The p53+ group had a worse prognosis in 107 early-stage cases (P<0.01), but there was no statistically significant difference when 52 advanced-stage cases (stage III-IV) or all patients were considered. Both ras and p53 mutations were unfavourable prognostic factors in 94 cases with adenocarcinoma, but there was no statistical significance in 57 cases with squamous cell carcinoma. According to Cox's model, the pathological stage, ras mutation and p53 mutation were found to be independent prognostic factors. Our results suggest that ras and p53 mutations were independent unfavourable prognostic markers especially in the early stage of NSCLC or in adenocarcinoma.     

Use of rectal cancer position as a prognostic indicator

The hypothesis of this study was that the position of rectal cancer within the circumference of the rectum influences mortality. Tumor position was prospectively documented in 181 patients with rectal carcinoma by two examiners. The results were analyzed for correlation to survival using the Lifetest model and for multivariate correlation using the Cox regression model. An anterior tumor location was present in 43 patients and was found to have a significantly higher survival rate than other positions. Two-thirds of anterior tumors were of pathologically favorable Dukes' stages. Fifty-five patients had posterior tumors with decreased survival rates, two-thirds of which were of unfavorable stages. Circumferential position in 61 patients was most predictive of poorer outcome, with a relative risk of death being increased by 4.6 times (P = 0.014) and a 5-year survival rate of 68.8 per cent; 85 per cent of these tumors were of pathologically unfavorable stages. The 5-year survival rate for the whole group, which included 181 patients with all histopathological stages except those with distant metastases at presentation, was 78.5 per cent. This ranking of survival rates was found to be consistent in each category with the postoperatively determined Dukes' stage, which carried a prognostic significance of P = 0.0001. We conclude that tumor position is a significant indicator of prognosis available before surgery for rectal cancer.     

Human papilloma virus and p53 in head and neck cancer: clinical correlates and survival

Recent studies have shown that p53 mutations are frequently found in cancer of the head and neck, whereas others have indicated that human papilloma virus (HPV) infection may be involved. Thus far, no studies have examined both p53 and HPV in the same patient population and correlated the results with clinical characteristics and outcome. The purpose of this study was to examine any interrelationship between p53 and HPV in patients with squamous cell carcinoma (SCC) of the head and neck. We also planned to correlate the experimental findings with clinical characteristics, known risk factors, and treatment outcome to determine whether any prognostic factors could be detected. Archival material from 66 patients with SCC of the head and neck were selected for study based on the availability of tissue from the primary tumors prior to treatment. A data base was constructed containing all clinical parameters at the time of diagnosis and risk factors. Genomic DNA was isolated and amplified using PCR, followed by SSCP analysis and direct genomic sequencing of all variants to detect p53 mutations. Two independent methods were used for HPV detection: (a) PCR amplification using primers homologous to the E6 region of HPV 16, 18, and 33, followed by RFLP analysis; and (b) PCR amplification with HPV L1 consensus primers, followed by triple restriction enzyme digestion. The results were entered into the data base for statistical analysis. Twenty-four percent of patients were found to have p53 mutations, and 18% were positive for HPV infection. Only one patient was positive for both. Tonsilar cancer was strongly correlated with HPV (P = 0.0001) and inversely correlated with p53 (P = 0.03). The only clinical parameter associated with p53 mutation was a trend toward a heavier smoking history. A subset analysis of the patients with tonsilar cancer revealed inverse correlations with smoking (P = 0. 015) and alcohol use (P = 0.05). Also, white patients with SCC of the tonsil were more likely to be HPV positive (P = 0.015). No significant relationships with outcome were detected with either p53 or HPV in the entire population. A subset analysis of patients with stage IV disease revealed that HPV infection was correlated with overall survival. This is the largest study to date to examine both p53 and HPV in patients with SCC of the head and neck. Our results suggest that HPV may be involved in the development of these cancers in patients without traditional risk factors and that HPV-related cancers are more prevalent in the white race.     

Thymidine uptake in vitro as a prognostic indicator for primary gastric cancer

BACKGROUND: Prognostic significance of in vitro thymidine uptake by cancer cells remains unclear in patients with gastric cancer. METHODS: In 173 patients with operable gastric cancer, the relations between thymidine uptake by gastric cancer cells in semi-solid media and their clinicopathologic features as well as their survival lengths were studied. RESULTS: There were significant correlations between in vitro thymidine uptake and such clinicopathologic features as lymph node metastasis (P = 0.00002), lymphatic invasion (P = 0.003), vessel invasion (P = 0.006), peritoneal metastasis (P = 0.010), depth of invasion (P = 0.011), and hepatic metastasis (P = 0.032). Ninety-five of 173 cancers (54.9%) that incorporated 1000 or more cpm in a single well were designated as being a high uptake group. Other gastric cancers (78 of 173; 45.1%) were designated as being a low uptake group. The overall survival rate of the patients was demonstrated to be significantly longer in the group with a low thymidine uptake than with a high uptake (P < 0.00001). The multivariate analysis showed that thymidine is one of the two variables that are the most highly correlated with the probability of patient death (P = 0.00044). CONCLUSIONS: These results indicated that in vitro thymidine uptake is an independent prognostic parameter for gastric cancer and may be useful for selecting patients who would benefit from more intensive therapy.     

MDM2 amplification and loss of heterozygosity at Rb and p53 genes: no simultaneous alterations in the oncogenesis of liposarcomas

PURPOSE: The present study aimed to investigate the status of alterations of the MDM2, Rb and p53 genes in a series of 45 liposarcomas. Furthermore, the possible correlation with histological and clinical parameters was studied. METHODS: MDM2 amplification was examined by non-radioactive Southern blot hybridization with a human MDM2 cDNA probe. Mutations in the p53 gene were screened by polymerase chain reaction/single-strand conformation polymorphism analysis and direct sequencing. To study loss of heterozygosity (LOH) at the tumor-suppressor genes Rb and p53, we used four polymorphic intragenic Rb markers (introns 1, 17, 20, and 25) and two p53 markers (intron 1 and exon 4). RESULTS: MDM2 amplification was found in 19 of 45 liposarcomas (42.2%). The frequency of LOH in Rb and p53 was nearly identical (22%). In 4 of 9 tumors (44.4%) with LOH, allelic loss was a concurrent event in both genes. Of 45 liposarcomas, 6 (13.3%) showed p53 mutations. Overall, alterations of the p53/MDM2/Rb pathway occurred in 30 of 45 liposarcomas (66.6%). In contrast to myxoid and pleomorphic variants, well-differentiated liposarcomas were characterized by a high frequency of MDM2 amplification, a lack of LOH of Rb and p53, and p53 mutations. CONCLUSIONS: Obviously MDM2 amplification and LOH at the Rh and p53 genes do not occur simultaneously in the oncogenesis of liposarcomas, as is the case for MDM2 amplification and p53 gene mutations (with one exception). We suggest that well-differentiated, myxoid and pleomorphic liposarcomas are characterized by a different pattern of molecular alterations.     

Overexpression of the p53 gene in primary and metastatic head and neck carcinomas

The p53 gene has been correlated with disease progression in a number of human malignancies, and p53 abnormalities are found in a high percentage of head and neck squamous cell carcinomas. The objectives of this study were 1. to correlate p53 expression with disease progression in squamous cell carcinoma of the head and neck (SCCHN), and 2. to determine whether there are site-specific differences in p53 expression. Primary lesions and/or lymph node metastases from 147 patients with invasive SCCHN were immunostained for p53 overexpression. Expression of p53 was similar (42% versus 43%) in primary lesions and lymph node metastases. Expression also did not vary significantly by site in the head and neck. In conclusion, increased p53 expression did not correlate with disease progression in our series of patients with invasive SCCHN. The finding of a lack of increased expression with disease spread to lymph nodes supports the belief that p53 alterations occur early in head and neck carcinogenesis.     

p53 expression in breast cancer related to prognostic factors

In this article the results of molecular marker p53 examinations were presented in relation to the following established breast cancer prognostic factors: age, histologic type, histologic grade, lymph node involvement, tumor size as well as estrogen a progesterone receptor status. Twenty one percent of these primary breast cancer specimens exhibited the overexpression of p53 protein. Significant associations were found between p53 overexpression and younger age, high histologic grade and low content of estrogen and progesterone receptors. Identification of p53-positive breast carcinomas potentially represents a clinically useful indicator of breast cancer aggressiveness.     

p53 mutation is a poor prognostic indicator for survival in patients with hepatocellular carcinoma undergoing surgical tumour ablation

Forty-two patients with hepatocellular carcinoma (HCC) were resected and their tumours were analysed for p53 mutations by GC-clamped denaturing gradient gel electrophoresis (DGGE), single-strand conformation polymorphism (SSCP) and gene sequencing. All the exons have been analysed in this study. Eight of 12 HCCs with cirrhosis due to viral hepatitis and the two patients with sarcomatoid changes displayed p53 mutations. In contrast, no mutation was observed in the fibrolamellar variant (n = 9), non-cirrhotics (n = 13) and alcoholic cirrhosis (n = 6). The mutations observed were in exons 5-8. Two mutations were observed in codons 136 and 213 as well as a T insertion between residues 156 and 157 (exon 5) and these are reported for the first time in HCC. Likewise, the silent mutation polymorphism in codon 213 was noticed in 3 of the 42 patients. Survival analysis of these patients after surgery showed the mean and median survival in patients with wild-type p53 to be 60 and 43 months respectively. In the group with p53 mutations, the mean and median survival was 15 and 12 months. The difference was statistically significant (P= 0.003).     

TP53 DNA contact mutations are selectively associated with allelic loss and have a strong clinical impact in head and neck cancer

Recent studies have suggested that different mutation types within the core domain of the tumour suppressor protein p53, i.e. DNA contact mutations and structural mutations, confer different biological properties. We have analysed in 86 head and neck squamous cell carcinomas (HNSCC), whether these p53 mutation types have a differential clinical impact. Thirty-seven missense mutations were identified. Thirteen of these (36%) were DNA contact mutations, occurring in the L3 loop, in the H2 loop sheet helix motif, in the S10 beta strand and in Zinc binding residues. Microsatellite marker analysis revealed a selective association between these mutations and the loss of wild-type alleles (100% LOH vs 50% LOH in tumours with structural mutations; P=0.0034, Fisher's exact, 2-tailed). In comparison to structural mutations or to the absence of mutations in the core domain, DNA contact mutations were associated with higher tumour stages (84.6% vs 62%), a higher incidence of lymph node metastasis (91.7% vs 56%; P=0.014, Fisher's exact, 2-tailed), a shortened recurrence-free survival (8.1 months vs 23.7 months, P=0.047, log rank test) and overall survival (11 months vs 29.2 months; P=0.003, log rank test). The latter was also the case when only stage IV tumours were analysed (P=0.0055, log rank test). These data indicate that in HNSCC, TP53 DNA contact mutations confer a strong selection pressure to eliminate wild-type alleles, and that they result in an accelerated tumour progression and reduced therapeutic responsiveness.     

Biphenotypic blast crisis of chronic myelogenous leukemia: abnormalities of p53 and retinoblastoma genes

The molecular mechanisms responsible for progression of chronic myelogenous leukemia (CML) to blast crisis have not been well defined. Blast crisis may be partially related to inactivation of tumor suppressor genes/such as p53 or retinoblastoma (Rb) gene. There is evidence for an association of blast cell phenotypes in CML with alterations of these genes: a strong association of myeloid phenotypes with abnormalities of the p53 gene and a weaker association of lymphoid phenotypes with abnormalities of the Rb system. We found a marked decrease in Rb gene product and rearrangements of the p53 gene simultaneously in two cases of biphenotypic blast crisis of CML (myeloid and B-lymphoid). These results support the association of blast cell phenotypes with alterations in tumor suppressor genes in CML blast crisis.     

Loss of p53 gene mutation after irradiation is associated with increased aggressiveness in recurring head and neck cancer

The p53 gene plays a pivotal role in the control of a checkpoint during G1 and in the apoptotic program. It has been postulated that alterations of p53 may influence radio-sensitivity and prognosis in several malignancies. We studied the p53 gene status of 35 consecutive head and neck cancer patients who failed primary radiotherapy (RT) in preirradiated and postirradiated tumor samples using DNA single-strand conformational polymorphism analysis. Sixteen of 35 (46%) preirradiated samples presented with band shifts suggestive of point mutations in one or two exons. Eleven of these tumors (69%) showed the same shift even in the postirradiated samples. Exons 5 and 8 were prevalently affected in this group. Five tumors (31%) lost the mutation following RT. The missed mutations clustered in exon 7. All mutations were confirmed by sequencing. Actuarial analysis demonstrated increased survival in patients with tumors bearing a p53 gene mutation in both preirradiated and postirradiated samples (P = 0.05 and P = 0.01, respectively). We also found that loss of p53 gene mutation in postirradiated cancers is associated with a significantly shorter disease-free interval (P < 0.02) and a worse prognosis (P < 0.05). A possible explanation in such cases is clonal selection by RT of more aggressive and radioresistant cell subpopulations, which are wild-type for the p53 gene. Taken together, our data suggest that not only p53 gene status but also the pattern of mutations could modulate the response of tumor cell to RT in vivo.