p53 overexpression and K-ras gene mutations in primary sclerosing cholangitis-associated biliary tract cancer

Cholangiocarcinoma occurs frequently in patients with primary sclerosing cholangitis (PSC). We evaluated the incidence and prognostic significance of p53 protein overexpression and K-ras gene mutations in patients with biliary tract cancer and PSC. p53 protein expression was determined in specimens from 12 patients with biliary tract cancer, using the antibody, D07. K-ras mutations were detected using DNA sequencing and a mutation ligation assay. Accumulation of p53 protein was detected in 6 of 12 tumors (50%). K-ras mutations were detected in 4 of 12 tumors (33%). Overall survival in patients with p53-negative tumors was significantly longer (P < 0.05) than that in patients with p53-positive (mutant) tumors. Similarly, overall survival was significantly longer (P < 0.05) in the absence of a K-ras mutation than in patients with a tumor containing a K-ras mutation. Mean interval from the time of diagnosis of PSC until the diagnosis of biliary tract cancer was significantly shorter (P < 0.05) in patients with p53 overexpression than in those patients without p53 overexpression (2 versus 47 months). p53 overexpression and K-ras mutations occur commonly in patients with PSC and biliary tract cancer and are associated with a shortened survival. Patients with longstanding PSC are less likely to have these genetic alterations and may have a better prognosis. Received: September 24, 1999 / Accepted: June 6, 2000     

p16 and p53 gene alterations and accumulations in the malignant evolution of intraductal papillary-mucinous tumors of the pancreas

Abstract. Background/Purpose: In general, intraductal papillary-mucinous tumors of the pancreas have a favorable prognosis. However, some invasive carcinomas show a rapid progression and a poor prognosis, such as invasive ductal carcinomas of the pancreas. The aim of this study was to clarify the genetic backgrounds underlying the evolution necessary for these tumors to become invasive carcinomas. Methods: Twenty-three patients with intraductal papillary-mucinous tumors, including 9 adenomas, 5 borderline tumors, 3 noninvasive carcinomas, and 6 invasive carcinomas, along with 24 patients with invasive ductal carcinomas were studied. After microdissection, K-ras mutation and loss of heterozygosity (LOH) of the p16 and p53 genes were investigated. Results: In the intraductal papillary-mucinous tumors, K-ras mutations were frequently seen, without a relationship to histological grade. LOH of the p16 gene was observed increasingly with increasing degrees of histological atypia. LOH of the p53 gene was seen only in invasive carcinomas. The frequency of each genetic alteration in invasive carcinomas was the same as that in invasive ductal carcinomas. The accumulation of genetic alterations was as common in invasive carcinomas, as they were in invasive ductal carcinomas. Conclusions: The p16 and p53 gene alterations and accumulations observed are crucial events during the carcinogenesis and malignant progression of intraductal papillary-mucinous tumors of the pancreas. Received: October 4, 2001 / Accepted: November 16, 2001     

p53 tumor suppressor gene mutation and prognosis in 105 cases of bladder cancer--the relationship between mutation of the p53 gene with clinicopathological features and smoking]

PURPOSE: Alterations of the p53 tumor suppressor gene are the most common genetic change detected in human cancers. The incidence of p53 gene mutation in bladder tumor patients were studied and were compared with clinicopathological findings, smoking history and prognosis. MATERIALS AND METHODS: Polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) was used for analysis from exon 4 to 9 of p53 gene in 105 cases of primary bladder tumors. RESULT: p53 matations were detected in 38 or 105 patients (36.2%). Kaplan-Meier Survival curves fo groups wit or without p53 gene mutation show a statistically significant difference (p = 0.0098). The mutation of p53 gene in stages pT 1 pT 1. pT 2, pT 3, pT 4 was found in 2 of 12 (16.7%), 8 of 32 (25.0%), 10 of 25 (40.0%), 12 of 20 (60.0%), 6 of 16 (37.5%) and in grades I, II, III, was noted in 1 of 17 (5.9%), 16 of 49 (32.7%), 21 of 39 (53.8%) cases, respectively. Significant differences were found for groups with grade I and grade II-III (p = 0.0045) cancers and in cases of superficial (stage pTa-1) and muscle-invasive (pT 2-4) tumors (p = 0.0148). However, mutation of p53 was not related to either age or sex in 105 patients. Recurrence rates in stage pTa-1 superficial tumor group with or without p53 mutation showed a statistically significant difference (p = 0.0419). No statistically significant difference was noted between p53 mutation and habitual smoking as well as durations of smoking. CONCLUSIONS: p53 mutations occur more commonly in higher grades and later stages of bladder tumors. Our results suggest that the prognostic factor is linked to not only histological findings but also to the presence of p53 mutation. The mutations of the p53 gene may be involved in the late events of tumorigenesis and might be used as good molecular markers for prognosis in bladder tumor.     

The significance of p53 mutations as an indicator of the biological behavior of recurrent hepatocellular carcinomas

The significance of p53 mutations in the primary lesion for recurrent hepatocellular carcinoma (HCC) was evaluated. Mutations of p53 were examined using non-radioisotopic (nonRI)-polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) in 98 resected HCCs. Of the 98 cases, 25 (26%) had a p53 mutation. In 83 patients who survived surgery, the presence of a p53 mutation was associated with a shortened overall survival (P<0.001) and a shortened cancer-free survival (P<0.05). In 43 patients who developed recurrence, there was no statistically significant correlation between the status of p53 in the primary lesion and the clinical features of recurrent HCCs examined, i.e., extrahepatic metastasis, the number of recurrent tumors, extent of recurrent tumors, and treatment for recurrent tumors. However, postrecurrence survival was significantly lower in patients in whom a p53 mutation had been detected in the primary lesion (P<0.01). A multivariate analysis for prognostic value after recurrence revealed that the p53 mutation was a useful independent prognostic factor affecting survival after recurrence (P<0.01). In conclusion, our findings suggest that HCCs with p53 mutations have a high malignant potential based on their poor prognosis. Therefore, a p53 mutation in the primary lesion is useful as an indicator of the biological behavior of recurrent HCCs.     

Prognostic implications of p53 gene mutations in bladder tumors

PURPOSE: Alterations in the p53 gene related to neoplastic progression were studied in tumor tissue samples from patients with transitional cell carcinoma and correlated with classic staging parameters. On this basis, biological characterization of the tumor was performed to establish subgroups of patients at high risk and those with a more favorable prognosis. MATERIALS AND METHODS: This observational, analytical and cross-sectional study included 115 patients divided into 4 homogeneous groups of 1-control, 2-primary superficial transitional cell carcinoma, 3-recurrent superficial transitional cell carcinoma, and 4-infiltrative transitional cell carcinoma. DNA was obtained from tumor tissue samples and polymerase chain reaction-single strand conformational polymorphism analysis was performed on exons 5 to 9 of the p53 gene. Samples showing mutations were submitted to automatic sequencing. Statistics included bivariate analysis and logistic regression. RESULTS: Of the tumors the 63.8% were superficial and 37.2% were infiltrative transitional cell carcinoma. Of the infiltrative tumors 23.5% (8 of 34) resulted from recurrent transitional cell carcinoma. Mutations were found in samples from 46.8% of patients, all with bladder tumors. There was a trend toward increasing appearance of mutations as the size of the tumor, number of tumor implants, degree of dedifferentiation and stage of local infiltration increased. The presence of mutations in p53 was 2.5 times greater in infiltrative tumors than in low stage and 4.3 times greater in moderate to high grade than in low grade tumors. All mutations found were point mutations and 79.25% provoked severe alterations in protein structure. CONCLUSIONS: Mutations in the p53 gene are mainly point mutations that aggregate in hot spots, and provoke genetic instability and substantial changes that alter p53 function, implying a trend to tumor progression and dissemination (with a greater proportion of mutations in high stage high grade tumors). Since a large percentage of bladder tumors are under staged, analysis of p53 gene mutations could be useful as a factor for prognosis and therapeutic decisions.     

Relevance of p53 gene alterations for tumor recurrence in patients with superficial transitional cell carcinoma of the bladder

PURPOSE: The prognostic relevance of p53 protein accumulation in muscle-invasive bladder carcinoma is well documented, but the prognostic relevance of p53 alterations in superficial bladder tumors remains uncertain. Immunohistochemical data are divergent, possibly because of the use of nonstandardized techniques. We therefore investigated the relevance of p53 gene point mutations and loss of heterozygosity (LOH) for tumor recurrence. The results of this molecular analysis were compared with accumulation of the p53 protein as shown by immunohistochemistry. MATERIAL AND METHODS: Representative tumor tissue was selected and microdissected from 40 patients (pTa, 18 patients; pT1, 22 patients; grade I, 7 patients; grade II, 28 patients; grade III, 5 patients). Polymerase chain reaction (PCR) was carried out with exons 5-8. All PCR products were screened for p53 mutations with temperature-gradient gel electrophoresis (TGGE). When mobility shift was observed, direct nucleotide sequencing was performed. Detection of LOH was performed with nonradioactive microsatellite analysis using three markers (TP 53, D17S513 and D17S786) on chromosome 17p. Immunohistochemistry was performed with the DO 7 antibody. Tumor samples with p53 accumulation of 5% or more positive nuclei were classified as positive. Univariate analysis for disease-free survival was performed using Kaplan-Meier analysis and the log-rank test. RESULTS: TGGE and direct sequencing detected mutations in 10 of 40 patients (2 of 18 pTa and 8 of 22 pT1 patients). LOH was detected in 11 patients. Both a mutation and LOH were detected in 3 patients. p53 immunohistochemistry detected at least 5% positive nuclei in 28 of 40 patients (70%). After a median follow-up of 26 months 14 patients suffered disease recurrence. Whereas disease-free survival did not correlate with a mutation (p = 0.77, log-rank test), LOH (p = 0.2) or a mutation in combination with LOH (p = 0.23), a positive p 53 immunoreaction was significantly associated with short disease-free survival (p = 0.009). CONCLUSION: Despite the relatively high percentage of patients with p53 gene alteration in this population no significant correlation between the detection of molecular alteration and disease recurrence could be found. We conclude that, in contrast to immunohistochemical accumulation, gene alterations play only a minor role in tumor recurrence of p53 in patients with superficial transitional cell carcinoma of the bladder, and that immunohistochemical accumulation of the p53 protein has to be explained by mechanisms other than gene mutations.     

Synchronous colon carcinomas: Molecular-genetic evidence for multicentricity

Background: The synchronous presentation of multiple colonic adenocarcinomas is an unusual, but well-recognized event accounting for 2–11% of these neoplasms. Synchronous tumors may have a different biology and prognosis than solitary tumors. Evidence based on measurement of DNA ploidy suggests that a significant percentage of synchronous tumors have a common clonal origin, probably resulting from translumenal metastasis. Methods: Fifteen synchronous colorectal cancers (30 tumors) were examined for histologic differences as well as genetic mutations. p53 gene abnormalities were detected by polymerase chain reaction (PCR) followed by single-strand conformation polymorphism analysis. Ki-ras mutations were detected by PCR followed by oligonucleotide-specific hybridization. Results: p53 gene mutations were detected in 12 of 30 tumors. In only one case was the same p53 mutation present in both tumors from one patient. Similarly, Ki-ras mutations were observed in 9 of 30 tumors. Concordant Ki-ras mutations were observed in only one case, which was also concordant for p53 mutation. Conclusion: Because p53 and Ki-ras mutations tend to occur fairly early in tumor development, it seems likely that cases discordant for p53 and Ki-ras mutations represent independently developing tumor foci. Taken together, these findings strongly suggest that the great majority of synchronous colonic adenocarcinomas arise as independent neoplasms and their worsened prognosis is not a result of unusually early metastatic spread.Results of this study were presented in part at the 48th Annual Cancer Symposium of The Society of Surgical Oncology, Boston, Massachusetts, March 23–26, 1995.     

Significance of P53 in human thyroid tumors

Mutational changes in the p53 tumor suppressor gene are the most frequent genetic alterations in human malignant tumors. Studies have shown a correlation of p53 expression in breast cancer with tumor prognosis. In contrast to mutational activation of ras and GSP in thyroid tumors, little is known about the role of p53 in thyroid tumor development. Therefore thyroid tumors and thyroid tumor cell lines were studied for the presence of p53 mutations. Snap-frozen tissues from 57 differentiated thyroid carcinomas (DTCs) and 5 goiters were studied by immunohistochemical methods. A panel of six antibodies (pAb 240, 421, 1620, 1801, DO7, and CM1) was employed by using the ABC technique. Five cell lines from DTCs (FTC133, 236, 238, PTC337, MTC164) were examined by the same technique. Additionally, genomic DNA from the cells was amplified by the polymerase chain reaction (PCR) and the PCR product studied for p53 mutations (R273H) by mutation-specific oligonucleotide hybridization (MOH) and temperature gradient gel electrophoresis (TGGE) for the p53 exon 8. None of the benign thyroid tumors and 7 of 57 (12%) DTCs strongly express p53 with a heterogeneous distribution in the tumor tissue. All seven patients have metastatic disease or dedifferentiated tumors G3 (three of seven). CM1 was positive in two cell lines (FTC-133, PTC-337), questionable in FTC-238, and negative in FTC-236 and MTC-164. All three follicular cell lines, however, and the original tumor tissue showed the same p53 mutation (R273H) in MOH analysis and TGGE. P53 mutations are rare in thyroid tumors, but the presence of p53 mutations indicates a poor prognosis. TGGE seems to be a sensitive method for detecting p53 mutations (1% sensitivity) and might play a role in tumor screening in the future.     

Potential Increase in the Prognostic Value of p53 Mutation by Pro72 Allele in Stage I Non-Small-Cell Lung Cancer

Background  Accumulated evidence suggests that p53 function altered by its gene mutation or genetic polymorphism contributes to tumor malignancy. Association of p53 mutation and its codon 72 polymorphism with lung cancer prognosis has been extensively studied. However, the joint effect of p53 mutation and p53 codon 72 polymorphism on lung cancer prognosis remains uncertain. Methods  In the present study, 266 primary lung cancer patients were included and overall survival was calculated. Genomic DNA prepared from adjacent normal lung and lung tumor tissues was used to determine p53 codon 72 genotype and p53 mutation by polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) and direct sequencing, respectively. Results  For all stages, neither p53 codon 72 genotype nor p53 mutation is associated with lung cancer prognosis. However, stage I patients with p53 mutation had a 1.79-fold hazard ratio [95% confidence interval (CI) 1.04–3.10] for overall survival when compared with p53 wild-type patients. Notably, stage I patients with p53 mutation and p53 codon 72 Pro/Pro genotype experienced a 2.66-fold hazard ratio (95% CI 1.21–5.85) for overall survival when compared with those with p53 wild-type and Arg/Arg genotype. An increased prognostic value was not observed in stage I patients with p53 wild-type and p53 Pro72 allele or in those with p53 mutation and p53 codon 72 Arg/Arg genotype. Conclusions  We therefore suggest that p53 codon 72 Pro allele potentially increases the prognostic value of p53 mutation in stage I non-small-cell lung cancer. Wen-Pin Chien and Ruey-Hong Wong equally contributed to this work.     

Clinical significance of p53 mutations in adenocarcinoma of the esophagus and cardia

OBJECTIVE: To compare the frequency and spectrum of p53 gene mutations in adenocarcinomas of the esophagus and cardia and to compare clinical and pathologic features in patients with p53 mutant and nonmutant cancers. SUMMARY BACKGROUND DATA: The p53 gene is commonly mutated in human cancers, and a p53 mutation is reported to be present in more than 50% of esophageal adenocarcinomas. Although many studies have investigated the frequency of p53 protein overexpression in adenocarcinomas of the esophagus or esophagogastric junction, few studies have assessed the frequency and clinical significance of p53 mutations in these tumors. In particular, the prognostic importance of p53 mutation is uncertain. Adenocarcinomas of the esophagus and cardia share many epidemiologic and pathologic features, but it is controversial whether they represent the same tumor. A comparison of the frequency and spectrum of mutations in adenocarcinomas of the esophagus and cardia would test whether these tumors are also similar at the molecular level. METHODS: DNA was isolated from microdissected paraffin-embedded tumor tissues of patients who underwent esophagogastrectomy for adenocarcinoma of the esophagus (n = 19), cardia (esophagogastric junction, n = 12), or subcardia (n = 6). Exons 5 to 8 of the p53 gene were analyzed for the presence of mutations using the polymerase chain reaction with single-strand conformation polymorphism and DNA sequencing of bands showing abnormal mobility. The presence of mutation was confirmed by selective hybridization of a mutant-specific oligonucleotide to DNA isolated from the tumor. RESULTS: p53 mutations were identified in 18 of 37 (48.6%) tumors. Patients with p53 mutant tumors were significantly younger and had a significantly poorer prognosis. There was a similar prevalence of p53 mutations in adenocarcinomas of the esophagus (53%) and cardia (58%). In contrast, mutations were relatively uncommon in subcardia adenocarcinomas (one mutant tumor [17%]). The types of mutations found in the esophageal and the cardia cancers were also similar. CONCLUSIONS: Adenocarcinomas of the esophagus and cardia have a similar frequency and spectrum of p53 gene mutations, suggesting that these tumors have a common pathogenesis. Patients with mutations are younger, have signs of more advanced disease, and have a poorer prognosis than patients without mutations.     

p53 Gene mutations and overexpression of p53 product in cancerous and noncancerous biliary epithelium in patients with pancreaticobiliary maljunction

To investigate the molecular mechanisms of the high incidence of carcinogenesis in the biliary epithelium of patients with pancreaticobiliary maljunction, we examined p53 gene mutations, loss of heterozygosity of p53, and overexpression of p53 gene product in the cancerous and noncancerous biliary epithelium of 27 patients with pancreaticobiliary maljunction. Mutations of the p53 gene were examined by polymerase chain reaction-single strand conformation polymorphism and a direct sequencing method. Loss of heterozygosity of the p53 gene was determined using a double-targeted fluorescence in situ hybridization method. Expression of p53 gene product was examined using immunohistochemical staining. Mutations of the p53 gene were found in 4 of 5 biliary carcinomas (80%) and in 10 of 26 noncancerous biliary lesions (38.5%). Point mutations of the p53 gene were detected at codons 207, 212, and 217 on exons 5 through 8. The incidence of p53 gene mutations on exons 5, 6, 7, and 8 was 12.9%, 36.4%, 0.0%, and 13.8%, respectively. Loss of heterozygosity of p53 was shown in 72% of the cells obtained from the cancerous lesion, and in an average of 14% obtained from the noncancerous lesions. Overexpression of p53 protein was found in 57.1% of carcinoma, and in 31.3% of the noncancerous lesions. These results suggest that p53 gene mutations are involved in the carcinogenesis of biliary epithelium in patients with pancreaticobiliary maljunction. Received for publication on Feb. 5, 1999; accepted on March 29, 1999     

Impact of KRAS and EGFR Gene Mutations on Recurrence and Survival in Patients with Surgically Resected Lung Adenocarcinomas

Background

Oncogenic gene mutations observed in lung adenocarcinomas, such as epidermal growth factor receptor (EGFR) and KRAS, have some predictive value for chemotherapeutic drugs or EGFR–tyrosine kinase inhibitors. However, the influence of these gene alterations on patients’ prognosis remains controversial.

Methods

We retrospectively analyzed the tumors of 180 patients with completely resected pathological stage I–III lung adenocarcinoma which harbored either KRAS codon 12 mutation or EGFR gene mutations within exons 18–21 to investigate the impact of these gene mutations on the patients’ survival. Gene mutations were detected by established methods.

Results

Of 180 patients, 32 had KRAS codon 12 mutations (KRAS group), 148 had EGFR mutations within exon 18–21 (EGFR group). Pathological stage and operation mode were independent factors for disease-free survival. However, the EGFR group had better overall survival than the KRAS group (P = 0.0271). Cox proportional hazard model revealed pathological stage (P = 0.0001) and presence of EGFR gene mutations (P = 0.0408) were independent factors for overall survival. In survival after tumor recurrence, the EGFR group had a better median survival time (46.7 months) after recurrence than the KRAS group (26.0 months).

Conclusions

In patients with completely resected lung adenocarcinomas, KRAS and EGFR gene mutation status of tumors was not associated with disease-free survival. However, the presence of an EGFR gene mutation boded well for the patient’s overall survival, and thus patients with EGFR mutations have a better prognosis than those with KRAS mutations.

     

Detection of human papillomavirus DNA and p53 gene mutations in human prostate cancer

The relationship between integration with human papillomavirus (HPV) and p53 gene mutations in tissues of prostate cancer was examined. Tissue samples analyzed were obtained by total prostatectomy (29 stage B cancer cases) and from autopsy (22 endocrine therapy-resistant metastatic disease cases). HPV DNA was detected in 8 of 51 (16%, 5 in stage B and 3 in autopsy cases) by polymerase chain reaction (PCR) using consensus primers on L1 region. Genotypes of HPV were entirely type 16. Structural abnormalities of p53 gene were detected in 7 of the 22 autopsy cases (32%) by PCR-single-strand conformation polymorphism analysis and direct sequencing. No p53 gene mutation was found in stage B cancer cases. Analysis of mutation spectra revealed clear differences between Japanese and Westerners. There was a significant difference in the mutation frequency between stage B and autopsy cases (P < 0.01, Fisher's exact test). One case showed both integration of HPV and p53 gene mutation in different cancer foci. However, the other cases revealed an inverse correlation between the presence of HPV DNA and p53 gene mutations. These data show that p53 genetic alteration is correlated with the progression of prostate cancer, in contrast to the integration of HPV that may occur in a relatively early stage. In conclusion, this study may indicate that either p53 gene mutation or the presence of HPV's oncogenic protein E6 is involved in the development of prostate cancer. © 1996 Wiley-Liss, Inc.     

K-ras and p53 gene mutations in noncancerous biliary lesions of patients with pancreaticobiliary maljunction

Background/Purpose: We investigated the molecular mechanisms of carcinogenesis in the biliary epithelium in patients with pancreaticobiliary maljunction. Methods: Point mutations of the K-ras gene and the p53 gene, and the overexpression of p53 gene products were examined in the cancerous and noncancerous biliary epithelium of 37 patients with pancreaticobiliary maljunction, with or without biliary dilatation. Results: In the gallbladder epithelium of 5 patients with pancreaticobiliary maljunction associated with biliary carcinoma, K-ras gene mutations were detected in 3 (60%), p53 gene mutations in 3 (60%), and the overexpression of p53 gene products in 4 (80%), while in the bile duct epithelium of these patients, these features were found in 2 of 3 (66.7%), in all of 3 (100%), and none of 3 (0%) specimens, respectively. In the gallbladder epithelium of patients with pancreaticobiliary maljunction without biliary carcinoma, K-ras gene mutations were detected in 8 of 24 (33.3%) specimens, p53 gene mutations were detected in 16 of 27 specimens (59.3%), and the overexpression of p53 protein was detected in 5 of 27 (18.5%) specimens, while in the bile duct epithelium of these patients, these features were found in 10 of 25 (40%) specimens, 14 of 25 (56%) specimens, and 6 of 24 (25%) specimens, respectively. Conclusions: These results suggest that noncancerous lesions of the biliary epithelium in patients with pancreaticobiliary maljunction have mutations of the K-ras gene and/or the p53 gene, which provides genetic evidence that biliary epithelium has high carcinogenic potential. Received: June 7, 2001 / Accepted: August 21, 2001     

胆管癌患者胆汁脱落细胞端粒酶活性和p53基因突变检测的临床意义

目的探讨胆汁中端粒酶活性和P53基因突变在胆管癌早期诊断中的价值。方法应用重复片段扩增法(TRAP)和PCR-SSCP-银染法检测、分析30例胆管癌患者及10例良性胆道疾病患者胆汁脱落细胞的端粒酶活性及P53基因突变情况。结果胆管癌组端粒酶阳性表达率60%,P53基因突变率50%,良性胆道疾病组无端粒酶活性表达及P53基因突变,两者比较有显著性差异(P<0.05)。胆管癌组胆汁细胞学检查分级和端粒酶活性及p53基因突变差异均有显著性(P<0.05)。胆管癌组胆汁端粒酶活性和p53基因突变之间有正相关关系(P<0.05)。结论胆汁中端粒酶活性和P53基因联合检测有助于胆管癌的诊断。     

Mutations in TP53 or DNA damage repair genes define poor prognostic subgroups in primary prostate cancer

BackgroundMutations in DNA damage repair genes, in particular genes involved in homology-directed repair, define a subgroup of men with prostate cancer with a more unfavorable prognosis but a therapeutic vulnerability to PARP inhibition. In current practice, mutational testing of prostate cancer patients is commonly done late i.e., when the tumor is castration resistant. In addition, most sequencing panels do not include TP53, one of the most crucial tumor suppressor genes in human cancer. In this proof-of-concept study, we sought to extend the clinical use of these molecular markers by exploring the early prognostic impact of mutations in TP53 and DNA damage repair genes in men with primary, nonmetastatic prostate cancer undergoing radical prostatectomy (RPX).MethodsTumor specimens from a cohort of 68 RPX patients with intermediate (n = 11, 16.2%) or high-risk (n = 57, 83.8%) disease were analyzed by targeted next generation sequencing using a 37 DNA damage repair and checkpoint gene panel including TP53. Sequencing results were correlated to clinicopathologic variables as well as PSA persistence or time to PSA failure. In addition, the distribution of TP53 and DNA damage repair gene mutations was analyzed in three large publicly available datasets (TCGA, MSKCC and SU2C).ResultsOf 68 primary prostate cancers analyzed, 23 (33.8%) were found to harbor a mutation in either TP53 (n = 12, 17.6%) or a DNA damage repair gene (n = 11, 16.2%). The vast majority of these mutations (22 of 23, 95.7%) were detected in primary tumors from patients with high-risk features. These mutations were mutually exclusive in our cohort and additional data mining suggests an enrichment of DNA damage repair gene mutations in TP53 wild-type tumors. Mutations in either TP53 or a DNA damage repair gene were associated with a significantly worse prognosis after RPX. Importantly, the presence of TP53/DNA damage repair gene mutations was an independent risk factor for PSA failure or PSA persistence in multivariate Cox regression models.ConclusionTP53 or DNA damage repair gene mutations are frequently detected in primary prostate cancer with high-risk features and define a subgroup of patients with an increased risk for PSA failure or persistence after RPX. The significant adverse impact of these alterations on patient prognosis may be exploited to identify men with prostate cancer who may benefit from a more intensified treatment.     

p53 Protein and Gene Alterations in Pathological Stage C Prostate Carcinoma

Purpose

We determined the extent of p53 immunoreactivity in pathological stage C prostate cancer as well as its correlation to tumor grade, substage, recurrence and proliferation rate. To define better the temporal relationship of p53 nuclear reactivity in prostate cancer p53 immunoreactivity was evaluated in all associated prostatic intraepithelial neoplasia lesions.

Materials and Methods

Using immunohistochemistry p53 status and proliferation rate were determined in 96 tumors from patients with pathological stage C prostate cancer. Single strand conformational polymorphism in exons 5 to 8 was used in a subset of specimens to assess the association of p53 nuclear accumulation with mutations in the p53 gene.

Results

p53 Nuclear reactivity was demonstrated in 10 tumors (10.4%), including 6 with high and 4 with low level nuclear reactivity. Of the tumors 86 (89.6%) had no evidence of p53 immunoreactivity. Each of the 6 tumors with high level p53 reactivity had associated areas of prostatic intraepithelial neoplasia that also showed p53 nuclear reactivity. Furthermore, pathological stage C substage (C1, 2 or 3) was significantly associated with p53 nuclear reactivity (p = 0.04). Proliferation rates were correlated with p53 nuclear reactivity (p = 0.09), while there was no association with tumor grade or recurrence. p53 Gene alterations were noted in 2 of the 3 p53 positive tumors versus no alterations in the p53 gene of 3 p53 negative tumors.

Conclusions

p53 Nuclear accumulation is uncommon in pathological stage C prostate cancer and its presence in premalignant prostatic intraepithelial neoplasia lesions suggests that it may be an early event in a subset of prostate cancers.     

Characterization of K-ras gene mutations in association with mucinous hypersecretion in intraductal papillary-mucinous neoplasms

Background/Purpose Intraductal papillary-mucinous neoplasms (IPMNs) of the pancreas have a favorable prognosis. However, invasive ductal carcinomas of the pancreas show a rapid progression. The aim of this study was to investigate gene mutations in pure pancreatic juice from IPMN patients and to define these genetic mutations in relation to the histopathological and clinical features of IPMNs. Methods Twenty-two patients with IPMN, 21 patients with ductal carcinoma, and 20 patients with normal pancreas or chronic pancreatitis were recruited for this study. We measured the main pancreatic duct’s largest diameter and the maximum size of a dilated branch was assessed by ultrasonography or endoscopic ultrasonography. Pure pancreatic juice was collected and was investigated for K-ras, p16, and p53 mutations. Results Mutant K-ras gene was detected in 13 of the 22 patients (59.1%) with IPMNs. Different kinds of mutations were detected in the same patient in 4 cases. In the 13 patients with mutant K-ras gene, the diameter of the most dilated part of the main pancreatic duct was 2–8 mm (average, 4.5 mm) and in 7 patients with wild-type K-ras gene, the diameter was 2–5 mm (average, 2.7 mm). There was a significant difference in the diameter of the main pancreatic duct between patients with and without the mutant K-ras gene (P = 0.0323). Conclusions The incidence of K-ras mutation may be associated with the hypersecretion of mucin.     

Tumor Progression Through Epigenetic Gene Silencing of O<Superscript>6</Superscript>−Methylguanine-DNA Methyltransferase in Human Biliary Tract Cancers

Background We previously demonstrated in an immunohistochemical study that reduced expression of O⁶–methylguanine-DNA methyltransferase (MGMT) correlated with a poorer prognosis in patients with biliary tract cancers. The purpose of this study was to clarify how MGMT deficiency leads to a poor outcome in biliary tract cancer. Thus, we examined epigenetic (promoter methylation) and genetic (gene mutation) alterations in biliary tract cancer.Methods We examined 37 biliary tract cancer specimens from patients who underwent surgical resection. Promoter methylation was determined by one-step or two-step methylation-specific polymerase chain reaction. Gene mutation was identified by direct sequencing. The expression of MGMT protein in paraffin-embedded tissue was examined by immunohistochemistry.Results Frequencies of promoter methylation were 70% for p16/INK4a, 49% for MGMT, 46% for hMLH1, 41% for E-cadherin, and 32% for DAPK genes. MGMT methylation status was closely correlated with the MGMT protein expression determined by immunohistochemistry (P < .001). Although this was not statistically significant, biliary tract cancer tumors with MGMT methylation expressed multigene methylation more frequently than tumors without MGMT methylation (P = .071). A total of 33 mutations were identified in 4 cancer-related genes: p53, K-ras, -catenin, and p16/INK4a genes. The most common mutation was GC to AT transitions (58%), which were significantly associated with MGMT promoter methylation (P = .011). These findings suggest that loss of MGMT expression by promoter methylation results in accumulation of GC to AT gene mutations.Conclusions Reduced MGMT expression may increase the malignant potential of biliary tract cancer through both epigenetic and genetic mechanisms.     

IMMUNOHISTOCHEMICAL DETECTION OF p53 PROTEIN OVEREXPRESSION VERSUS GENE SEQUENCING IN URINARY BLADDER CARCINOMAS

PURPOSE: Mutations of p53 tumor suppressor gene and nuclear accumulation of p53 protein are common in bladder tumors. The prognostic significance of p53 alterations in bladder tumors has not been established. The aim of the present study was to evaluate an immunohistochemical (IHC) method for the routine determination of p53 protein overexpression in human bladder tumors and to determine the relation between nuclear accumulation of p53 with the traditional prognostic indicators and patient survival. MATERIALS AND METHODS: 104 transitional cell carcinomas of the bladder were analyzed simultaneously by immunohistochemistry for p53 protein overexpression and direct DNA sequencing for p53 gene mutations. RESULTS: The overexpression of p53 protein was reported in 30.8% of the cases and mutations of p53 gene in 23.0%. A significant association was observed between p53 alterations established either by IHC or direct DNA sequencing and stage (p<0.0001), grade (p<0.001), vascular invasion (p = 0.0005), DNA ploidy (p = 0.0002) and carcinoma in situ (p<0.0001). The correlation between the p53 gene mutations and p53 nuclear reactivity as detected by IHC was highly significant (p<0.0001). Univariate statistical analysis showed that the expression of p53 was significantly correlated to poor prognosis (p<0.0001). However, in multivariate analysis, only stage was significantly correlated to prognosis (p<0.0001). CONCLUSIONS: The IHC method was highly sensitive and specific and simple to apply for the routine examination of p53 overexpression in bladder tumors. However, overexpression of p53 as determined immunohistochemically, does not appear to have a better predictive prognostic value than stage in bladder tumors.