p53 gene mutations associated with anaplastic transformation of human thyroid carcinomas.

Anaplastic carcinoma of the thyroid gland, which is one of the most aggressive, malignant tumors in humans, is considered to originate from preexisting differentiated thyroid cancer. To define the genetic alterations associated with such progression, we examined nine cases of anaplastic thyroid carcinoma for mutation in exons 4-9 of the p53 tumor suppressor gene. Preliminary screening for mutation by RNase protection analysis demonstrated that two out of nine anaplastic carcinomas contained sequence alterations in the p53 gene. Subsequent DNA sequencing identified the mutated nucleotides in these two cases; one was a nonsense mutation at codon 165, and the other was a single-base deletion at codon 176 resulting in the creation of a stop codon downstream due to frameshift. The fact that no mutations were detected in coexisting foci of papillary carcinomas from the same patients shows that these mutations of the p53 gene occurred after development of papillary carcinomas. These results suggest that p53 gene mutation triggers the progression from differentiated into anaplastic carcinoma in the human thyroid gland.     

p53 Gene Mutations Associated with Anaplastic Transformation of Human Thyroid Carcinomas

Anaplastic carcinoma of the thyroid gland, which is one of the most aggressive, malignant tumors in humans, is considered to originate from preexisting differentiated thyroid cancer. To define the genetic alterations associated with such progression, we examined nine cases of anaplastic thyroid carcinoma for mutation in exons 4–9 of the p53 tumor suppressor gene. Preliminary screening for mutation by RNase protection analysis demonstrated that two out of nine anaplastic carcinomas contained sequence alterations in the p53 gene. Subsequent DNA sequencing identified the mutated nucleotides in these two cases; one was a nonsense mutation at codon 165, and the other was a single-base deletion at codon 176 resulting in the creation of a stop codon downstream due to frameshift. The fact that no mutations were detected in coexisting foci of papillary carcinomas from the same patients shows that these mutations of the p53 gene occurred after development of papillary carcinomas. These results suggest that p53 gene mutation triggers the progression from differentiated into anaplastic carcinoma in the human thyroid gland.     

p53 gene mutations occur in combination with 17p allelic deletions as late events in colorectal tumorigenesis

Coordinate loss of one copy of the p53 gene and mutation of the remaining copy occur in colorectal carcinomas and in many other human malignancies. However, the prevalence of p53 gene mutations in carcinomas which maintain both parental copies of p53 has not previously been evaluated. Moreover, it is not known whether p53 gene mutations are limited to malignant tumors or whether they can also occur in benign neoplasms. To answer these questions, a total of 58 colorectal tumors have been examined; in each tumor, allelic losses were assessed using restriction fragment length polymorphisms and p53 gene mutations were assessed by sequencing cloned polymerase chain reaction products. The following conclusions emerged: (a) p53 gene mutations occurred but were relatively rare in adenomas, regardless of size and whether the adenomas were derived from patients with familial adenomatous polyposis; (b) In carcinomas as well as in adenomas, p53 gene mutations were infrequently observed in tumors which contain both copies of chromosome 17p (17% of 30 tumors), while tumors which lost one copy of chromosome 17p usually had a mutation in the remaining p53 allele (86% of 28 tumors); (c) p53 gene mutations were found at similar frequencies in primary tumor samples and in cell lines derived from tumors. These and other data suggest that the rate limiting step in p53 inactivation is point mutation and that once a mutation occurs, loss of the remaining wild-type allele rapidly follows. Both mutations and allelic losses generally occur near the transition from benign to malignant growth, and the p53 gene may play a causal role in this progression.     

Mutation profile of the p53, fhit, p16INK4a/p19ARF and H-ras genes in Indian breast carcinomas

Breast cancer is the second most prevalent cancer affecting Indian women. Genetic alterations of oncogenes and tumor suppressor genes were attributed to the development of breast carcinomas. In the present study, human breast tumor DNAs from untreated, non-familial, Indian patients were analysed for the presence of mutations in p53, fhit, p16INK4a/p19ARF and H-ras genes. Polymerase chain reaction-single strand conformation polymorphism and sequencing analysis were used to detect point mutations. Exons 5-8 of p53, exons 1-2 of p16INK4a, exon 2 of p19ARF, exons 5-9 of fhit gene and exons 1-2 of H-ras genes were amplified and analysed individually using exon-flanking primers. Only 12% of the tumors had mutation in p53, 8% had mutation in fhit gene and none of the tumors showed evidence for mutation in p16INK4a/p19ARF and H-ras genes. Tumor B18 exhibited two novel mutations in the p53 gene, ATGright curved arrow GTG (Metright curved arrow Val) at codon 237 and AATright curved arrow GAT (Asnright curved arrow Asp) at codon 263. Both of these mutations are hitherto unreported in breast carcinomas. Tumor B20 had a non-sense mutation CGAright curved arrow TGA (Argright curved arrow Stop) at codon 306 of p53 gene. In fhit gene, tumor B1 exhibited TTCTright curved arrow TACT mutation at intron 8 and tumor B15 had a silent mutation GAGright curved arrow GAA (Gluright curved arrow Glu) at codon 123. Our results indicate that, among the genes analysed, the p53 gene was more frequently mutated than fhit, p16INK4a/p19ARF and H-ras genes in Indian mammary tumors. Transcribable point mutations of fhit gene were found to be extremely uncommon in these tumors. Mutations in the above genes are mutually exclusive and are infrequent in fhit, p16INK4a/p19ARF and H-ras genes suggesting that these genes may not play a major role in Indian breast carcinomas. However, the significant frequency of mutations in the p53 gene suggest that p53 could be one of the genes involved in the genesis of sporadic breast carcinomas in Indian women.     

Role of p53 mutations in endocrine tumorigenesis: mutation detection by polymerase chain reaction-single strand conformation polymorphism.

To elucidate the molecular basis for endocrine tumorigenesis, p53 mutations in human endocrine tumors were analyzed by using polymerase chain reaction-single strand conformation polymorphism. Exons 5 through 10 of the p53 gene were studied in genomic DNAs from 134 primary endocrine tumors and 6 human endocrine cancer-derived cell lines. Mutations were detected and identified in 4 endocrine tumors, including one parathyroid adenoma and three thyroid carcinoma cell lines. The sites of these mutations were in exons 5 (codon 151 and 152) and 7 (codon 248 and 255). In all of three tumor cell lines, but not in a parathyroid adenoma, the normal allele encoding the p53 gene was lost. However, p53 mutations were not found in any other endocrine tumors or cell lines. Based upon these results, we concluded that the p53 gene may play a role in the tumorigenesis of a limited number of parathyroid adenoma and thyroid cancers, and that the p53 mutation with an allelic loss of the p53 gene is an important factor in malignant tumorigenesis of the thyroid gland.     

p53 gene mutation spectrum in hepatocellular carcinoma.

In order to clarify the significance of mutation of the p53 tumor suppressor gene in the genesis and development of human hepatocellular carcinoma (HCC) in an aflatoxin B1 low-exposure area, the spectrum, i.e., incidence, type, and site, of p53 gene mutations was examined in 169 tissue samples resected mainly from Japanese patients using single-strand conformation polymorphism analysis and direct sequencing. Forty-nine tumors (29%) showed a p53 mutation (39 point mutations and 10 frameshifts). The point mutations comprised 18 transitions, only 4 of which occurred at CpG sites, and 21 transversions. Two evolutionarily conserved domains, IV and V, contained 65% of all mutations and codon 249 was the most frequent mutation site (7/49). The spectrum of p53 mutation did not differ among HCCs in relation to the type of hepatitis virus infection, sex, age, and background liver disease of patients, tumor size, or presence of metastasis, but incidence and site were significantly associated with the degree of differentiation of cancer cells. In poorly differentiated HCC, p53 mutation was frequent (54%) and clustered on domains IV and V, whereas in well or moderately differentiated HCC, the mutation was less frequent (21%) and equally distributed on domains II to V. Restriction fragment length polymorphism analysis revealed loss of heterozygosity on chromosome 17p in 55 (69%) of 80 informative cases and in 34 (95%) of 36 cases with p53 mutation. Therefore, p53 gene mutation is suggested to occur independently of the type of viral infection or status of preexisting liver disease and to occur preferentially in moderately and poorly differentiated HCCs in association with or after loss of another p53 allele as a late event of HCC progression.     

Chromosomal deletions and K-ras gene mutations in human endometrial carcinomas.

Forty-two endometrial carcinomas of various stages of progression were analyzed to search for loss of chromosomal regions and for point mutations of ras genes and amplification of Int-2 gene. This approach is particularly favorable for observation of genetic events and their significance in the process of neoplastic conversion by considering the clinico-pathological characteristics of each tumor. At least 3 genetic events, including 18q, 17p deletions, and point mutations at codon 12 of the K-ras gene, are implicated in the development of endometrial carcinomas. Likely targets for allelic losses on chromosomes 18q and 17p are the DCC gene and the p53 gene sequences, respectively. Overall numbers of allelic losses in individual tumors appeared to increase in case of advanced stage tumors, thereby indicating the association of allelic loss accumulation with tumor progression. The genetic features seen in 2 juvenile-type adenocarcinomas and 2 clear-cell carcinomas suggested the possibility that etiological factors providing selective pressure for particular mutation sub-sets during carcinogenesis are probably heterogeneous.     

Low frequency of p53 gene mutation in tumors induced by aflatoxin B1 in nonhuman primates.

Aflatoxin B1 has been suggested as a causative agent for a G to T mutation at codon 249 in the p53 gene in human hepatocellular carcinomas from southern Africa and Qidong in China. To test this hypothesis, nine tumors induced by aflatoxin B1 in nonhuman primates were analyzed for mutations in the p53 gene. These included four hepatocellular carcinomas, two cholangiocarcinomas, a spindle cell carcinoma of the bile duct, a hemangioendothelial sarcoma of the liver, and an osteogenic sarcoma of the tibia. None of the tumors showed changes at the third position of codon 249 by cleavage analysis of the HaeIII enzyme site at codon 249. A point mutation was identified in one hepatocellular carcinoma at the second position of codon 175 (G to T transversion) by sequencing analysis of the four conserved domains (II to V) in the p53 gene. These data suggest that mutations in the p53 gene are not necessary in aflatoxin B1 induced hepatocarcinogenesis in nonhuman primates. The occurrence of mutation in codon 249 of the p53 gene in selective samples of human hepatocellular cancers may indicate involvement of environmental carcinogens other than aflatoxin B1 or that hepatitis B virus-related hepatitis is a prerequisite for aflatoxin B1 induction of G to T transversion in codon 249.     

Mutations in the p53 gene in primary human breast cancers.

Twenty-six primary breast tumors were examined for mutations in the p53 tumor suppressor gene by an RNase protection assay and nucleotide sequence analysis of PCR-amplified p53 complementary DNAs. Each method detected p53 mutations in the same three tumors (12%). One tumor contained two mutations in the same allele. Single strand conformation polymorphism analysis of genomic DNA and complementary DNA proved more sensitive in the detection of mutations. Combining this technique with the other two a total of 12 mutations in the p53 gene were demonstrated in 11 tumors (46%), and a polymorphism at codon 213 was detected in another tumor. Loss of heterozygosity on chromosome 17p was detected by Southern blot analysis in 30% of the tumor DNAs. Not all of the tumors containing a point mutation in p53 also had loss of heterozygosity of the remaining allele, suggesting that loss of heterozygosity may represent a later event.     

Infrequent mutation of p53 gene in human renal cell carcinoma detected by polymerase chain reaction single-strand conformation polymorphism analysis.

Mutation of the p53 gene, which plays an important role in the genesis of diverse human cancers, was investigated in 23 surgical specimens of human renal cell carcinoma using the polymerase chain reaction single-strand conformation polymorphism method of analysis. Only one of the 23 tumors (4.3%) carried a mutated p53 gene, which was present in exons 7-8. Direct DNA sequencing confirmed a point mutation at codon 276 (GCC to CCC) resulting in a substitution of alanine for proline. No specific clinicopathological characteristics were observed in the case with the p53 gene mutation in human renal cell carcinoma. These observations suggest that mutation of the p53 gene is rare and thus does not contribute significantly to the genesis of this tumor.     

p53 Mutations in human hepatocellular carcinomas from Germany.

Mutations in the p53 gene are frequent genetic alterations in human hepatocellular carcinomas. We have examined 13 cases of human hepatocellular carcinomas from Germany for the presence of p53 aberrations in exons 4 to 8 of the gene by single-strand conformation polymorphism and restriction fragment-length polymorphism analyses and by sequencing of polymerase chain reaction products. Single base substitutions occurred in two human hepatocellular carcinomas: a C:G----T:A transition at a CpG site in codon 257, and a T:A----A:T transversion at codon 273. One of these point-mutated tumors and two additional tumors without point mutations demonstrated a loss of one p53 allele. None of the tumors was mutated in codons 12 or 61 of the c-Ha-ras gene.     

Murine p53 intron sequences 5-8 and their use in polymerase chain reaction/direct sequencing analysis of p53 mutations in CD-1 mouse liver and lung tumors.

Inactivating point mutations and small deletions in the p53 tumor suppressor gene have been found in human liver and lung tumor--derived cell lines and tumors. However, little evidence has been reported concerning inactivation or mutation of the p53 gene in mouse primary tumors. To examine CD-1 mouse liver and lung tumors for mutations in the p53 gene, we first sequenced p53 introns 5-8 so that polymerase chain reaction amplification and sequencing primers located within the introns could be prepared. Use of these primers prevented amplification of the mouse p53 pseudogene and allowed sequencing of exons 5-8 in their entirety as well as their intron-exon junctions. DNA isolated from CD-1 mouse tumors was amplified and directly sequenced using nested primers. Nine spontaneous hepatocellular carcinomas (HCCs) and 34 chemically induced HCCs (induced by single intraperitoneal injections of N-nitrosodiethylamine [DEN] [8 HCCs], 7,12-dimethylbenz[a]anthracene [DMBA] [8 HCCs], 4-aminoazobenzene [8 HCCs], and N-OH-2-acetylaminofluorene [10 HCCs]) were examined for mutations in exons 5-8 of the p53 gene. In addition, 12 spontaneous, 10 DMBA-induced, and 13 DEN-induced lung adenocarcinomas or adenomas were analyzed for mutations. No mutations were found in any of the tumors examined. However, a mutation was demonstrated at codon 135 in the positive-control plasmid LTRp53cG(val). The results of this study suggest that inactivation of p53 is unlikely to play a major role in murine lung or liver carcinogenesis. However, inactivation of p53 may occur at a very low frequency, or it may occur as a late event and therefore be present in only a very small number of the tumor cells, rendering it undetectable by this method. Lastly, although few p53-inactivating mutations are found outside of exons 5-8 in human tumors, it is possible that these murine tumors contained mutations outside of this region and were therefore missed by our approach.     

Analysis of mdm2 and p53 Gene Alterations in Glioblastomas and its Correlation with Clinical Factors

Malignant gliomas are the most frequent primary brain tumors. Recent studies defined several genetic markers, which might characterize molecular-biological subsets of glioblastomas with probably prognostic implications. To elucidate the involvement of murine-double-minute (mdm)2 gene amplifications and mutations of the tumor suppressor gene p53 in the tumorigenesis of malignant gliomas we analyzed a series of 75 glioblastomas. The p53 mutations occur in one-third of glioblastomas, mdm2 amplifications were found in 13% of cases. Our analysis revealed a hot spot in the p53 gene locus in codon 156, the same point mutation was detected in 4 tumor samples. None of the mdm2 amplified tumors had p53 mutations, supporting the hypothesis, that mdm2 amplifications are alternative mechanisms for p53 inactivation. Patients with p53 mutated tumors were significantly younger characterized by a mean age of 44 years. Additionally association with longer overall survival could be detected for this subgroup of patients. In our study, survival estimation revealed a significant correlation of mdm2 gene amplification with shorter survival time, and support the hypothesis, that mdm2 oncogene activation appears to occur late in tumor progression and may be characteristic as negative prognostic marker.     

Involvement of p53 gene mutations in human endometrial carcinomas

Mutations in the p53 gene are associated with a wide variety of human malignancies. Point mutation in one allele and loss of the remaining one generally lead to inactivation of p53 protein. A high frequency of allelic losses corresponding to the 17p13.3 region that contained the p53 gene sequence was also noted in human endometrial carcinoma. Thus, in order to confirm involvement of the p53 gene in endometrial carcinogenesis, we searched for nucleotide sequence change in this gene in 42 endometrial carcinomas that had been subjected to previous LOH analyses. Using the polymerase-chain-reaction-single-strand conformation polymorphism (PCR-SSCP) method, we detected p53 gene mutations in 4 specimens. Two adenocarcinomas with allelic loss on 17p contained a mutant p53 gene in the allele that was retained. One specimen with a p53 gene mutation contained a 17q deletion but was uninformative for LOH on 17p. p53 gene mutation was also noted in the remaining stage-1 carcinoma, though the 17p deletion was not detected in the previous LOH examination. However, 5 specimens with the LOH on 17p retained the wild-type p53 gene. In the remaining 33 specimens, both alleles of p53 gene seemed to be normal. The mutations observed in 2 specimens (cases 10 and 24), involving C-to-T and T-to-G substitutions, were located in a highly conserved region. However, the mutations identified in the remaining 2 cases (29 and 35) were at regions positioned outside conserved stretches.     

Detection of p53 gene mutations in human ovarian and endometrial cancers by polymerase chain reaction-single strand conformation polymorphism analysis.

The presence of mutations in the p53 gene was examined in ovarian cancers by a polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. The primers were designed to amplify exons 5 through 9 that contain phylogenetically conserved domains of the p53 gene. Mutations were detected in 5 out of 10 cases, one of which contained a deletion in the second allele. A single base substitution was detected in 4 cases at codons 162, 175, 205 and 273 and a single base insertion in one case within codon 315. A high frequency of p53 mutations in ovarian cancers and lack of mutation in 6 benign ovarian tumors and 2 normal ovaries suggested that the mutation of the p53 gene was associated with the genesis and/or progression of ovarian cancer. In 1 of 7 endometrial cancers, two mutations at codons 239 and 254 were detected.     

Detection of p53 Gene Mutations in Human Ovarian and Endometrial Cancers by Polymerase Chain Reaction-Single Strand Conformation Polymorphism Analysis

The presence of mutations in the p53 gene was examined in ovarian cancers by a polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. The primers were designed to amplify exons 5 through 9 that contain phylogenetically conserved domains of the p53 gene. Mutations were detected in 5 out of 10 cases, one of which contained a deletion in the second allele. A single base substitution was detected in 4 cases at codons 162,175, 205 and 273 and a single base insertion in one case within codon 315. A high frequency of p53 mutations in ovarian cancers and lack of mutation in 6 benign ovarian tumors and 2 normal ovaries suggested that the mutation of the p53 gene was associated with the genesis and/or progression of ovarian cancer. In 1 of 7 endometrial cancers, two mutations at codons 239 and 254 were detected.     

p53 gene mutations in colorectal tumors from patients with familial polyposis coli.

The p53 gene has been elucidated as a tumor suppressor gene, and inactivation of this gene caused by deletion or point mutations may play a crucial role in the development of human malignancies. In colorectal carcinomas with an allelic deletion of the p53 gene, the remaining p53 gene was mutated with considerable frequency. It is most difficult to detect point mutations or small deletions of the gene because the mutations occur in diverse regions, although four hot spots have been observed [J.M. Nigro et al., Nature (Lond.), 342: 705-708, 1989]. The polymerase chain reaction and denaturing gradient gel electrophoresis facilitate detection of mutations in the hot spots of the p53 gene. Using these methods, we detected mutations in three adenomatous polyps and one carcinoma from familial polyposis coli patients and three carcinomas of sporadic cases. The DNA sequence analysis confirmed mutations of the p53 gene in 2 adenomas (13 base-pair deletions in one and a point mutation in the other) and 1 carcinoma (point mutation) from familial polyposis coli patients. These results suggest that the p53 gene mutations may be involved in the formation not only of carcinomas but also of adenomas which occur in familial polyposis coli patients.     

p53 mutation and protein accumulation during multistage human esophageal carcinogenesis.

Preinvasive lesions of squamous cell carcinoma are well defined morphologically and provide a model for multistage carcinogenesis. Since alterations in the p53 tumor suppressor gene occur frequently in invasive esophageal squamous cell carcinoma, we examined a set of preinvasive lesions to investigate the timing of p53 mutation. Surgically resected tissues from nine patients with esophageal squamous cell carcinoma contained precursor lesions which had not yet invaded normal tissues. Immunohistochemistry showed high levels of p53 protein in both preinvasive lesions and invasive carcinomas in six cases; sequence analysis of all invasive tumors identified p53 missense mutations in two cases. Preinvasive lesions from both tumors with mutations plus one wild-type tumor were microdissected and sequenced. In one patient there were different mutations in the invasive carcinoma (codon 282, CGGarg > TGGtrp) and a preinvasive lesion (codon 272, GTGval > T/GTGleu/val). In a second case, an invasive carcinoma had a mutation in codon 175 (CGCarg > CAChis), and adjacent preinvasive lesions contained a wild-type sequence. A carcinoma and preinvasive lesion from the third case contained high levels of protein and a wild-type DNA sequence. Therefore, p53 mutation may precede invasion in esophageal carcinogenesis, and multifocal esophageal neoplasms may arise from independent clones of transformed cells. The timing of p53 protein accumulation is favorable for an intermediate biomarker in multistage esophageal carcinogenesis.