P53 Point Mutations Research Articles

P53 alterations in human leukemia-lymphoma cell lines: in vitroartifact or prerequisite for cell immortalization?

Alteration of the p53 gene is one of the most frequent events in human tumorigenesis. The inactivation of p53 tumor suppressor function can be caused by chromosome deletion, gene deletion, or mainly by point mutations. p53 mutations occur moderately often in hematopoietic malignancies. A significantly higher frequency of p53 alterations in cell lines vs primary samples has been observed for all types of malignant hematopoietic cell lines. It has been postulated that p53 gene abnormalities arise in cell lines during in vitro establishment of the culture or prolonged culture; but it is also conceivable that those cases that carry p53 mutations may be more suitable for in vitro establishment as permanent cell lines. We analyzed data on the p53 gene status in a panel of matched primary hematopoietic tumor cells and the respective cell lines derived from this original material. In 85% (53/62) of the pairs of matched primary cells and cell lines, the in vivo and in vitro data were identical (both with p53 wild-type or both with the same p53 mutation). In some instances, serial clinical samples (eg at presentation and relapse) and serial sister cell lines were available. These cases showed that a clinical sample at presentation often had a p53 wild-type configuration whereas the derived cell line and a relapse specimen carried an identical p53 point mutation. These findings suggest that a minor clone, at first undetectable by standard analysis, represents a reservoir for the outgrowth of resistant cells in vivo and also a pool of cells with a growth advantage in vitro, providing a significantly higher chance of immortalization in culture. This was further supported by studies employing mutant allele-specific gene amplifications, a technique which is significantly more sensitive (100- to 1000-fold) than the commonly applied SSCP assay with a sensitivity threshold of about 10% mutated cells within a pool of wild-type cells. Taken together, this analysis confirms the usefulness of human hematopoietic cell lines as in vitro model systems for the study of the biology of hematopoietic malignancies. It further underlines the notion that p53 gene alterations confer a survival advantage to, at least some, malignant cells in vitro and presumably also in vivo; however, it is highly unlikely that a p53 mutation alone would suffice for the immortalization of a cell line in vitro or tumor development in vivo. Leukemia (2000) 14, 198-206.

Effect of mutation and conformation on the function of p53 in colorectal cancer.

In vitro studies have shown that immunoprecipitation with conformation-specific antibodies allows discrimination between different forms of p53; however, the significance of this has not been determined for human tumours. This study therefore examined p53 conformation in colorectal tumours and correlated this with mutational status and evidence of in vivo p53 downstream activity. Moreover, it was shown that for in vitro cell lines, DNA-damaging agents induce wild-type p53 to form a mutant conformation (PAb240+), with a concomitant rise in p21(WAF-1) expression. Induction of p53-mediated apoptosis, on the other hand, is associated with a wild-type conformation (PAb1620+). These results were confirmed for wild-type p53 in colorectal tumours. A range of p53 point mutations were found in exons 5-8 in colorectal tumours. Mutants with a wild-type conformation gave weak immunohistochemical staining, whereas mutations with flexible conformation (240+/1620+) gave intense positivity. Interestingly, this latter group of flexible mutants was also associated with features of poor prognosis. These studies show that not all p53 mutants are equal; thus, knowledge of the p53 status of a tumour may be required for a more precise prediction of prognosis and response to treatment for cancer patients.

Frequent Occurrence of Loss of Heterozygosity among Tumor Suppressor Genes in Uterine Leiomyosarcoma

Objective. Leiomyosarcoma of the uterus is a rare smooth muscle tumor; it is extremely malignant and the rates of local recurrence and metastasis are high. Since tumor suppressor genes are commonly altered in malignant tumors, it is possible that mutations in such genes are involved in the development of uterine leiomyosarcoma.Methods. Fifty-five patients (37–70 years of age) diagnosed as having smooth muscle tumors of the uterus were selected. DNA was extracted from four or five 8-μm-thick consecutive tissue sections of each smooth muscle tumor from the paraffin-embedded blocks. Loss of heterozygosity (LOH) was investigated at nine loci within or close to tumor suppressor genes (TP53, RB1, DCC, NM23, WT1, D14S267, P16, DPC4, PTCH).Results. Nineteen of twenty leiomyosarcomas revealed at least one instance of LOH among eight of the nine markers tested (one locus showed no LOH at all). In fact, 11 of the 20 cases exhibited two or more instances of LOH and, of the remaining 9 cases, 4 showed a point mutation of p53 in addition to an alteration in one of the 9 markers, while one exhibited a p53 mutation only.Conclusion. An accumulation of genetic alterations among tumor suppressor genes may play a key role in the tumorigenesis and progression of uterine leiomyosarcoma.

A point mutation of human p53, which was not detected as a mutation by a yeast functional assay, led to apoptosis but not p21 Waf1/Cip1/Sdi1 expression in response to ionizing radiation in a human osteosarcoma cell line, Saos-2

Purpose: The 123A point mutation of p53 showed increased radiosensitivity, whereas other mutations (143A, 175H, and 273H) were not affected. To determine the reason for increased radiosensitivity of the 123A mutation, the response of the transformant of 123A mutation to ionizing radiation (IR) was examined and compared to those of transformants with the wild type p53 or other point mutations (143A, 175H, and 273H). Methods and Materials: Stable transformants with a mutant or wild type p53 made by introducing cDNA into the human osteosarcoma cell line, Saos-2, which lacks an endogenous p53 were used. The transcriptional activity of mutant p53 was examined using a yeast functional assay. The transformants were examined for the accumulation of p53, the induction of p21 Waf1/Cip1/Sdi1 (hereafter referred to as p21), and the other response of p53-responsive genes (MDM2, Bax, and Bcl-2) by Western blotting. Apoptosis was analyzed by detection of DNA fragmentation. Results: The 123A point mutation of p53 was detected as a wild type in the yeast functional assay. The 123A mutant accumulated p53 in response to IR. The 123A mutant did not induce p21, but normally responded to MDM2, Bax, and Bcl-2. The 123A mutant entered apoptosis earlier than the wild type p53 transformant, and induced Fas at earlier in response to IR. Conclusion: The 123A mutant led to apoptosis, but not p21 expression in response to IR. The occurrence of apoptosis, but not induction of p21, corresponded to the radiosensitivity in the transformant. The early occurrence of apoptosis in 123A transformants may depend on the early induction of Fas.

Ionizing radiation enhances immunogenicity of cells expressing a tumor-specific T-cell epitope

Background: p53 point mutations represent potential tumor-specific cytolytic T lymphocyte (CTL) epitopes. Whether ionizing radiation (IR) alters the immunological properties of cells expressing mutant p53 in respect of the CTL epitope generated by a defined point mutation has not been evaluated. Methods: Mutant p53-expressing syngeneic, nontumor forming BALB/c 3T3 fibroblasts, tumor forming ras-transfected BALB/c 3T3 sarcomas, and DBA/2-derived P815 mastocytoma cells, which differ at the level of minor histocompatibility antigens, were used as cellular vaccines. Cells were either injected with or without prior IR into naive BALB/c mice. Cellular cytotoxicity was assessed after secondary restimulation of effector spleen cells in vitro. Results: Injection of P815 mastocytoma cells expressing the mutant p53 induced mutation-specific CTL in BALB/c mice irrespective of prior irradiation. However, syngeneic fibroblasts or fibrosarcomas endogenously expressing mutant p53 were able to induce significant mutation-specific CTL only when irradiated prior to injection into BALB/c mice. IR of fibroblasts did not detectably alter the expression of cell surface molecules involved in immune response induction, nor did it alter the short-term in vitro viability of the fibroblasts. Interestingly, radioactively-labeled fibroblasts injected into mice after irradiation showed altered organ distribution, suggesting that the in vivo fate of these cells may play a crucial role in their immunogenicity. Conclusions: These findings indicate that IR can alter the immunogenicity of syngeneic normal as well as tumor forming fibroblasts in vivo, and support the view that ionizing radiation enhances immunogenicity of cellular tumor vaccines.

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

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

P53 point mutation and survival in colorectal cancer patients: effect of disease dissemination and tumour location.

The relationship between p53 point mutation and patient survival was examined in 328 colorectal cancer patients. Point mutation was detected in 51% (166/328) of cases and was associated with a poorer prognosis in univariate and multivariate analysis. However, subcohort analysis showed that this relationship was restricted to patients with lymphatic dissemination, patients without evident distant metastatic lesions at the time of presentation and in tumours confined to the distal colorectum. These results suggest that the utility of p53 point mutation as a specific, patient based prognostic marker may be restricted to certain classes of patients.

Morphological-Histochemical Study of Intestinal Carcinoids and K-ras Mutation Analysis in Appendiceal Carcinoids

Intestinal carcinoids are potentially malignant neoplasms. Their histogenesis and pathogenesis are currently uncertain. The morphological and histochemical characteristics of twenty intestinal carcinoids are studied. The primary sites of three mucin-producing tumors were examined by electron microscope. Furthermore 11 appendiceal carcinoids were analysed by the polymerase chain reaction (PCR) for the detection of ras and p53 point mutations. Microscopically all carcinoids were of mixed type. Focal mucin production was evident in three carcinoids that metastasised to regional lymph nodes. HID-Alcian blue staining proved that mucin in both primary and secondary foci did not belong to the sulphated group. The secretory granules and mucin droplets found in a single neoplastic cell suggest that carcinoids of the small intestine and some of the appendix arise from the endoderm. Neither ras nor p53 mutations were detected. It seems that ras oncogenes are probably not involved in the pathogenesis of appendiceal carcinoids. Copyright 1999 W.B. Saunders Company Ltd on behalf of the Arányi Lajos Foundation

P53 mutations in tumor and non-tumor tissues of thorotrast recipients: a model for cellular selection during radiation carcinogenesis in the liver.

Concerns over cancer development from exposure to environmental sources of densely ionizing, high linear energy transfer (LET) radiation, such as alpha-particles from radon, is a current public health issue. The study of tumors attributable to high LET irradiation would greatly augment our insights into the biological mechanisms of carcinogenesis. Chronic low-dose-rate internal exposure to alpha-radiation from thorium dioxide deposits following intravascular administration of the radiographic contrast agent Thorotrast is known to markedly increase the risk of cancer development, especially that of hepatic angiosarcomas and cholangiocarcinomas. Although the mechanism is hypothesized to be via cellular damage, DNA being a major target, wrought by the high LET alpha-particles, the specific genes and the actual sequence of events involved in the process of transforming a normal cell into a malignant one are largely unknown. To shed some light on the molecular mechanisms of cancer development during a lifetime exposure to alpha-radiation, we analyzed the most commonly affected tumor suppressor gene in humans, p53, in 20 Thorotrast recipients who developed cancer, mostly of hepatic bile duct and blood vessel origin. Of the 20 cases, 19 were found to harbor p53 point mutations. Moreover, the accompanying non-tumor tissues from these patients also had p53 mutations, albeit at lower frequency. The distribution pattern of the point mutations was significantly different between the non-tumor and tumor tissues, with most mutations in malignant tissues located in the highly conserved domains of the p53 gene. Our results support the idea that p53 mutations are important in the genesis of Thorotrast-induced tumors but that these point mutations are a secondary outcome of genomic instability induced by the irradiation. Additionally, non-tumor cells harboring p53 mutations may gain some survival advantage in situ but mutations in the domains responsible for the formation of structural elements critical in binding DNA may be necessary for a cell to reach full malignancy.

Apoptosis-related genes and proteins in Hodgkin's disease.

During recent years it has become increasingly evident that L&H cells in nodular lymphocytic predominance (LP) Hodgkin's disease (HD) and Hodgkin and Reed-Sternberg (H-RS) cells in approximately half the cases of classical HD originate from B-lymphocytes, and that H-RS cells in most of the remaining cases of classical HD express a null phenotype. The pathogenesis of HD is unknown. An association with Epstein-Barr virus (EBV) has been suggested and there are also indications that genes involved in programmed cell death (apoptosis) may be implicated. In this study, the expression of four apoptosis-related proteins (bcl-2, bcl-x, bax and p53) in 53 cases of HD was examined and the data were correlated with the genotype, the EBV status and the phenotype (B, T or null) of the neoplastic cells. The H-RS cells expressed a B-cell phenotype in 3/3 cases of nodular LP and in 19/ 50 (38%) cases of classical HD. The remaining cases showed a null-cell phenotype in 29/50 (58%) and a T-cell phenotype in 2/50 (4%). EBV was more often positive in B (14/19, 74%) than in null (7/29, 24%) type HD. The H-RS cells were bcl-2-positive in 19/53 (36%), bcl-x-positive in 17/53 (32%), bax-positive in 1/53, and p53-positive in 41/53 (77%) cases. No relationship was found between bcl-2 expression and EBV status, or between bcl-2 and bcl-x expression. A t(14;18) translocation was seen in 2 of 34 cases. P53 point mutations were not detected. Our findings indicate that nodular LP and classical HD originate from B-cells in a high proportion of cases. They also suggest a role for bcl-2, bcl-x and p53 in tumorigenesis. The pathogenesis is not known at this stage.

Semiquantitative chemiluminescent detection of UV-B-induced point mutations in the p53 tumor-suppressor gene.

The technique of allele-specific PCR (AS-PCR) enables the detection of a small number of mutant alleles in a large number of wild-type (WT) alleles. We used the AS-PCR technique and Southern blotting, using a nonradioactive labeled probe to analyze the formation of point mutations in the tumor-suppressor gene p53 of primary keratinocytes after UV-B irradiation. These permanent mutations resulting from CC dimers occur at distinct "hot-spots", one of which is affected in the human keratinocyte cell line HaCaT. This enabled us to establish the method with a defined positive control template, which also allowed semiquantitative determination of the mutation frequency. This, and the determination of the detection limit, was done with the use of serial dilutions of WT genomic DNA from primary keratinocytes with mutant genomic HaCaT DNA in the AS-PCR assay.

The human p53 gene mutated at position 249 per se is not sufficient to immortalize human liver cells.

A particular point mutation of the tumor suppressor gene p53, namely a G-->T transversion at the third base of codon 249, is frequently detected in primary hepatocellular carcinomas from patients living in areas where the levels of dietary exposure to aflatoxin B1 and the rates of infection with the hepatitis B virus are very high. Very recently, a nontumorigenic liver epithelial cell line (HACL-1) with a finite life-span and expressing a number of hepatocyte-specific markers was established from a human hepatocellular adenoma in our laboratory. To analyze the role of mutated p53 in the immortalization of human liver cells, we transfected HACL-1 cells with an expression vector containing a human p53 complementary DNA mutated at the third base of codon 249 and analyzed the consequences of this gene transfer on the growth properties of this cell line. HACL-1 cells transfected with mutant p53 showed no increase in their life-span (when compared with HACL-1 cells transfected with the antibiotic resistance gene alone) and did not grow in soft agar, whereas transfection of wild-type p53 into HACL-1 cells led to a proliferation stop. Thus, these results strongly support the view that the mutation at codon 249 of the p53 gene may serve as a fingerprint for aflatoxin B1-induced hepatocellular carcinomas, but is not, by itself, sufficient to immortalize human liver cells.

Association of p19(ARF) with Mdm2 inhibits ubiquitin ligase activity of Mdm2 for tumor suppressor p53.

We have demonstrated previously that the oncoprotein Mdm2 has a ubiquitin ligase activity for the tumor suppressor p53 protein. In the present study, we characterize this ubiquitin ligase activity of Mdm2. We first demonstrate the ubiquitination of several p53 point mutants and deletion mutants by Mdm2. The point mutants, which cannot bind to Mdm2, are not ubiquitinated by Mdm2. The ubiquitination of the C-terminal deletion mutants, which contain so-called Mdm2-binding sites, is markedly decreased, compared with that of wild-type p53. The binding of Mdm2 to p53 is essential for ubiquitination, but p53's tertiary structure and/or C-terminal region may also be important for this reaction. DNA-dependent protein kinase is known to phosphorylate p53 on Mdm2-binding sites, where DNA damage induces phosphorylation, and p53 phosphorylated by this kinase is not a good substrate for Mdm2. This suggests that DNA damage-induced phosphorylation stabilizes p53 by inhibiting its ubiquitination by Mdm2. We further investigated whether the tumor suppressor p19(ARF) affects the ubiquitin ligase activity of Mdm2 for p53. The activity of p19(ARF)-bound Mdm2 was found to be lower than that of free Mdm2, suggesting that p19(ARF) promotes the stabilization of p53 by inactivating Mdm2.

Detection of point mutations by non-isotopic single strand conformation polymorphism.

We have developed a procedure for nonradioactive single strand conformation polymorphism analysis and applied it to the detection of point mutations in the human tumor suppressor gene p53. The protocol does not require any particular facilities or equipment, such as radioactive handling, large gel units for sequencing, or a semiautomated electrophoresis system. This technique consists of amplification of DNA fragments by PCR with specific oligonucleotide primers, denaturation, and electrophoresis on small neutral polyacrylamide gels, followed by silver staining. The sensitivity of this procedure is comparable to other described techniques and the method is easy to perform and applicable to a variety of tissue specimens.

Analysis of p53 Gene Mutations in Helicobacter pylori-Associated Gastritis Mucosa in Endoscopic Biopsy Specimens

Background: No previous report has shown the relationship between Helicobacter pylori infection and a direct sequence analysis of p53 gene mutation in a biopsy sample of human gastric mucosa. Methods: A total of 60 endoscopic biopsies samples (21 patients with H. pylori-positive gastritis and 9 patients with H. pylori-negative gastritis), including antral mucosa and corpus mucosa, were used in this study. Direct DNA sequencing of exons 5, 6, 7, and 8 of the p53 gene was performed by the dyedeoxy terminator method. Results: Mutations in the p53 gene were identified in non-hot spot codons in exon 7 and 8 in 11 of 21 samples (52.4%) from H. pylori-positive gastritis patients. There was no mutation in H. pylori-negative gastritis patients. Conclusions: This finding shows that H. pylori infection can induce p53 point mutations and appears to be involved in the pathway leading to dysplasia or carcinoma.

P21/WAF1, p53 and PCNA expression and p53 mutation status in hepatocellular carcinoma.

The cyclin-dependent kinase inhibitor p21/WAF1 is regulated by p5S3-dependent and p53-independent pathways. In addition, p21/WAF1 binds with proliferating cell nuclear antigen (PCNA) and inhibits the action of PCNA. To investigate the possible role of p21/WAF1 in human hepatocellular carcinomas (HCCs), we examined the expression of p21/WAF1 and its relation with PCNA and p53 expression in 97 surgically resected HCCs by immunohistochemistry and with the mutation status of p53 in 26 HCCs. p53 mutation status was examined by direct DNA sequencing using 3 sets of primers covering exons 5-9. Six of the 26 tumors showed p53 point mutations and only 33% of these HCCs demonstrated p21/WAF1 expression. In contrast, 75% of HCCs without p53 mutations showed p21/WAF1 expression. Of all 97 HCCs, p21/WAF1 expression was significantly higher in the tumors than in corresponding non-tumorous liver. When the tumors were stratified into 2 groups by the median tumor p21/WAF1 score, those with higher expression were found to have a lower incidence of multiple tumor nodules (p = 0.008) and tumor microsatellite formation (p = 0.050). The tumor p21/WAF1 score was positively associated with tumor PCNA expression (p = 0.036) but not with tumor p53 expression. Thus, in HCC, expression of p21/WAF1 is in part dependent on p53 status, but a p53-independent pathway also plays a significant role in the regulation of p21/WAF1 expression. High p21/WAF1 expression is significantly associated with solitary tumor nodules and, to a lesser extent, tumor microsatellites but may not be enough to suppress tumor progression.

Concurrent disruption of cell cycle associated genes in mantle cell lymphoma: a genotypic and phenotypic study of cyclin D1, p16, p15, p53 and pRb.

Mantle cell lymphomas (MCL) are morphologically and immunophenotypically distinctive lymphoid neoplasms characterised by overexpression of cyclin D1. Recent studies have suggested that co-operating aberrations of cell cycle associated genes may provide a growth advantage to a tumour. To address this issue further, we investigated five typical and three aggressive (blastoid) MCL for alterations in the cell cycle regulating genes p15, p16, CDK4, Rb and p53. In 3/3 aggressive cases with cyclin D1 overexpression we found aberration of at least one additional gene. One case showed diminished expression of the retinoblastoma protein (pRb); one case harboured deletion of both p15 and p16; and one case exhibited both deletion of p16 and point mutation of p53. However, we also identified two typical cases which in addition to cyclin D1 overexpression exhibited diminished pRb expression and p15 and p16 hypermethylation, respectively. Our findings confirm and extend other recent investigations and indicate that co-operating genetic alterations of cell cycle-associated genes may contribute to the pathogenesis of MCL.

P53 mutation in patients with ulcerative colitis in rectal biopsy.

ObjectivesLong standing ulcerative colitis (UC) has been known to be one of the precancerous diseases of colorectal cancer. Although the frequent loss of p53 allele (LOH) and aneuploidy were reported as the molecular events in carcinoma and dysplasia known as the precursor of UC, p53 genetic alteration was not reported in indefinite dysplasia and UC involved mucosa in long standing UC. Therefore, we investigated the mutational inactivation of the p53 gene in UC patients who showed dysplastic mucosa, as well as non-dysplastic mucosa on H & E stain and, secondly, if there is p53 mutation, we examined the relationship between p53 alteration and clinical data.MethodSixteen patients with UC who had different duration of colitis were studied by endoscopic examination with rectal mucosal biospies. p53 gene alterations were detected by PCR-SSCP for exon 4–8 and immunohistochemical staining with p53 monoclonal antibody.ResultsAmong 16 patients, 2 patients (12%) showed dysplasia on H-E stain. The p53 point mutations were detected in 4 (two dysplasia and 2 normal looking mucosa) on PCR-SSCP. 4 patients who had p53 gene mutation were positive in immunohistochemical staining. With regard to clinical characteristics, these patients with p53 point mutation showed poor resoponse to medical treatment.ConclusionThese results suggest that the p53 mutation may be an early molecular event of cancerous change in UC.

P53 sequence analysis predicts treatment response and outcome of patients with esophageal carcinoma

BACKGROUND. The ability to predict biologic behavior and treatment responsiveness would be a valuable asset in the multimodality approach to esophageal carcinoma. The authors examined whether alterations of the p53 gene correlate with clinicopathologic parameters, response to preoperative chemotherapy/radiotherapy, and outcome in patients with esophageal carcinoma. METHODS. Histopathologic/genetic analysis of p53 was performed on formalin fixed, paraffin embedded tissues. Tissue sections were stained immunohistochemically for p53 protein followed by topographic genotyping comprised of polymerase chain reaction amplification and direct sequencing of p53 exons 5-8. All patients received induction chemotherapy (5-fluorouracil, cisplatin, and α-interferon) and concurrent external beam radiotherapy (4500 centigrays) followed by resection. RESULTS. p53 analysis performed on 42 tumors from patients with potentially resectable esophageal carcinoma revealed 25 of the 42 tumors (59.5%) to be p53 immunopositive; however, only 17 of the 42 tumors (40.5%) were proven to contain p53 point mutational damage in exons 8 (n =5), 5 (n =5), 7 (n = 4), and 6 (n = 3). Eight cases were weakly immunopositive and had no genotype mutation suggesting hyperexpression of normal wild-type p53. Genotyping also identified two immunonegative cases with deletion-type mutations (exons 5 and 6). Tissue samples collected before and after chemotherapy/radiotherapy exhibited fidelity in p53 mutational genotype in all cases. The presence of a p53 point mutation positively correlated with pTNM stage (P = 0.003) and residual disease in the resected specimen (P = 0.01). Moreover, survival of patients with p53 mutations was significantly lower than that of patients without mutations (overall survival of 21.6 months vs. 40 months; P = 0.0038; and disease free survival of 14.1 months vs. 38 months; P = 0.0004). CONCLUSIONS. Histopathologic/genetic analysis is a better determinant of p53 mutational damage than immunohistochemistry alone and can be used as a prognostic marker for esophageal carcinoma. p53 genotyping may define a subset of patients who respond to chemotherapy/radiotherapy and may predict who potentially benefits from multimodality therapy.

P53 sequence analysis predicts treatment response and outcome of patients with esophageal carcinoma

The ability to predict biologic behavior and treatment responsiveness would be a valuable asset in the multimodality approach to esophageal carcinoma. The authors examined whether alterations of the p53 gene correlate with clinicopathologic parameters, response to preoperative chemotherapy/radiotherapy, and outcome in patients with esophageal carcinoma. METHODS. Histopathologic/genetic analysis of p53 was performed on formalin fixed, paraffin embedded tissues. Tissue sections were stained immunohistochemically for p53 protein followed by topographic genotyping comprised of polymerase chain reaction amplification and direct sequencing of p53 exons 5-8. All patients received induction chemotherapy (5-fluorouracil, cisplatin, and alpha-interferon) and concurrent external beam radiotherapy (4500 centigrays) followed by resection. p53 analysis performed on 42 tumors from patients with potentially resectable esophageal carcinoma revealed 25 of the 42 tumors (59.5%) to be p53 immunopositive; however, only 17 of the 42 tumors (40.5%) were proven to contain p53 point mutational damage in exons 8 (n=5), 5 (n=5), 7 (n=4), and 6 (n=3). Eight cases were weakly immunopositive and had no genotype mutation suggesting hyperexpression of normal wild-type p53. Genotyping also identified two immunonegative cases with deletion-type mutations (exons 5 and 6). Tissue samples collected before and after chemotherapy/radiotherapy exhibited fidelity in p53 mutational genotype in all cases. The presence of a p53 point mutation positively correlated with pTNM stage (P=0.003) and residual disease in the resected specimen (P=0.01). Moreover, survival of patients with p53 mutations was significantly lower than that of patients without mutations (overall survival of 21.6 months vs. 40 months; P=0.0038; and disease free survival of 14.1 months vs. 38 months; P=0.0004). Histopathologic/genetic analysis is a better determinant of p53 mutational damage than immunohistochemistry alone and can be used as a prognostic marker for esophageal carcinoma. p53 genotyping may define a subset of patients who respond to chemotherapy/radiotherapy and may predict who potentially benefits from multimodality therapy.