P53 Gene Alterations Research Articles

The p53 gene mutation is of prognostic value in esophageal squamous cell carcinoma patients in unified stages of curability

Background: The p53 gene alteration is identified in approximately half of all human tumors, and is now thought to be a key gene for regulating the cell cycle through the induction of p21WAF1/CIP1 and inducing apoptosis through some genes such as BAX. In this study, we investigated the prognostic value of p53 mutation for postoperative esophageal squamous cell carcinoma patients.Methods: The subjects studied were 42 esophageal squamous cell carcinoma patients who underwent esophagectomy with complete curability in our department. The cases were limited to stage II and III. DNA was extracted from paraffin-embedded tissues. A p53 gene mutation was detected by polymerase chain reaction-single strand conformation polymorphism and subsequent direct sequencing method for exons 5 to 9. The 5-year survival rate was calculated and statistically compared between the p53 mutation(+) and (−) groups by the log rank test.Results: The p53 gene mutation was identified in 14 cases (33.3%). The 5-year survival rate of the p53 mutation(−) group (n = 28) was significantly higher than the (+) group (n = 14; 51.0% versus 35.7%, P <0.05 by the log rank test). Recurrence could be identified in 10 of 14 p53 mutation(+) cases (71.4%), whereas it was found in 12 of 28 (−) cases (42.8%).Conclusion: The current study indicated that p53 mutation of tumor tissues might be a prognostic factor for esophageal squamous cell carcinoma cases and one of the risk factors for its recurrence.

Correlations between p53-protein accumulation, serum antibodies and gene mutation in colorectal cancer.

Only half of colorectal-cancer patients elicit serum antibodies in response to intratumoral p53-gene mutations. Our study was designed to compare cellular events (p53-protein accumulation and gene mutations) with the presence of circulating anti-p53 antibodies (p53-Ab). Thirty-five colorectal-cancer patients were studied for their intratumoral p53-protein accumulation and circulating p53-Ab. Tumour DNA was analyzed for genomic mutations in a sub-set of 28 patients. In all, 18 tumours (51.4%) were positive by immunohistochemistry, and 17 tumour extracts were shown to contain "mutant" conformation p53 protein, 16 of them being were concordant by both methods. Of the 28 tumours tested by DGGE, 16 contained alterations in p53 exons 5 to 8 (57.1%). Of 12 tumours without detectable mutations, 10 were "mutant"-conformation-negative by immunohistochemistry and ELISA. Paradiploid tumors presented more frequently wild-type p53 genes and were significantly less frequently immunohistochemistry- or p53-Ab-positive than polyploid tumors. Circulating p53-Ab were detected in the serum of 11 patients (31%). In 9/11 cases, a gene mutation was found in the corresponding tumour. Three of four mutations in exon 8 and 3/3 mutations in exons 5-6 were associated with p53-Ab, in contrast with only 3/9 mutations in exon 7. We found good agreement in the detection of p53-gene alterations by different methods. However, our data suggest that all gene mutations may not be equivalent in term of immunogenicity.

The need for microdissectional tumor cell preparation during the molecular genetic analysis of prostate cancer.

For clinically localized prostate cancer, recent studies strongly indicate that the determination of p53 inactivation allows the identification of a highly aggressive subgroup of prostatic tumors associated with decreased recurrence-free and long-term survival following radical prostatectomy. However, several questions regarding the determination of p53 alterations in prostate cancer, such as the poor correlation between immunohistochemistry and molecular genetic analysis, remain to be clarified. On the DNA level, p53 gene alterations have been identified in only up to 64% of tumors exhibiting immunohistochemically detected overexpression of the p53 oncoprotein. This discrepancy can be explained either by the genetic microheterogeneity of prostate cancer or by stabilization of the wild-type protein due to posttranslational events. In the present study we tried to determine the concordance between an immunohistochemically detected p53 overexpression and the result of molecular genetic analysis. Therefore, tumor tissue obtained by microdissection from 40 prostate cancer specimens was subjected to DNA-sequence analysis. Microdissection was based either only on histopathologic criteria or on the result of the immunohistochemical staining reaction. In 8 of 14 (57%) tumors a positive immunohistochemical reaction could be confirmed by DNA sequencing, which revealed a missense point mutation at the p53 gene locus, mainly in the form of G-->A transversion in exon 5 of the p53 gene. Following the micropreparation of tumor cells exhibiting p53 oncoprotein overexpression, missense point mutation could be detected in an additional 4 cases. Following a microscopically guided tumor cell dissection according to the result of immunohistochemistry, DNA sequencing confirmed an immunohistochemically detected p53 overexpression in 86% of cases investigated. This result indicates that a microdissectional tumor cell preparation is recommended for molecular genetic analysis of histologically heterogeneous tissue specimens such as prostate cancer and should be performed according to and in addition to the result of immunohistochemistry when an immunohistochemical approach is available.

Detection of K-ras and p53 Gene Mutations in Pancreatic Juice for the Diagnosis of Intraductal Papillary Mucinous Tumors

The aim of this study was to investigate mutations of the K-ras oncogene and the p53 tumor suppressor gene in pancreatic juice and to evaluate our method for the diagnosis of intraductal papillary mucinous tumors (IPMT). Pancreatic juice was collected endoscopically from 12 patients with IPMT who underwent surgical resection (eight carcinomas and four adenomas) and eight cases without evident pancreatic diseases. DNA was extracted and both genes were examined by polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis and direct sequencing. In addition, surgically resected specimens were analyzed for both genes by the same methods, and p53 overexpression was investigated immunohistochemically. K-ras point mutations were detected in pancreatic juice from all 12 patients (100%) and p53 mutations were detected in five of 12 (42%). They were detected not only in carcinoma but also in adenoma and there was no difference between the mutations detected in pancreatic juice and surgical specimens. No mutations were found in any cases without pancreatic diseases. These findings suggest that alterations of K-ras and p53 gene are common events in the development of IPMT and that genetic analysis of them in pancreatic juice can be a useful tool for the clinical diagnosis of IPMT before surgery.

Discrimination of double primary lung cancer from intrapulmonary metastasis by p53 gene mutation.

When multiple synchronous lung tumours are identified, discrimination of multicentric lung cancers from intrapulmonary metastases by clinical findings is often difficult. We used genetic alterations in p53 gene as a discrimination marker of double primary lung cancers from single lung cancer with intrapulmonary metastasis. Twenty of 861 patients with primary lung cancer who underwent lung resection were selected as subjects because they showed synchronous double solid tumours of the same histological type in the unilateral lung without distant metastases. In addition, they had been diagnosed as lung carcinoma with intrapulmonary metastasis by clinical and histological findings. DNAs were extracted from paraffin-embedded tissue of paired tumours from these 20 patients. Exons 5–9 of the p53 gene were examined for genetic alterations in the tumours by polymerase chain reaction, single-strand conformation polymorphism analysis and subsequent DNA sequencing analysis. Three different patterns in the distribution of p53 mutations in double lung tumours were observed: [A] mutation in only one of the tumours (four cases), [B] different mutations in the tumours (two cases), and [C] same mutation in both tumours (one case). The cases of [A] or [B] patterns could be classified as double primary lung cancers, while the case of the [C] pattern was suggested to be lung cancer with intrapulmonary metastasis. These results suggested that the multicentric cancers were more frequent than the intrapulmonary metastatic cancers in double cancer cases. © 1999 Cancer Research Campaign

Mechanisms of relapse in acute leukaemia: involvement of p53 mutated subclones in disease progression in acute lymphoblastic leukaemia.

Mutations of the p53 tumour suppressor gene are infrequent at presentation of both acute myeloblastic leukaemia (AML) and acute lymphoblastic leukaemia (ALL), being found in between 5–10% of AML and 2–3% of ALL. Here we have studied the frequency of detection of p53 mutations at relapse of both AML and B-precursor ALL. In those patients with detectable mutations at relapse we investigated whether the mutation was detectable at presentation and was thus an early initiating event or whether it had arisen as a late event associated with relapse. Bone marrow samples from 55 adults and children with relapsed AML (n = 41) or ALL (n = 14) were analysed for p53 gene alterations by direct sequencing of exons 5–9. For samples where a p53 mutation was found at relapse, analysis of presentation samples was carried out by direct sequencing of the exon involved, or by allele-specific polymerase chain reaction (PCR) if the mutation could not be detected using direct sequencing. A p53 mutated gene was found at relapse in seven out of 55 cases. The frequency was higher in relapsed ALL (four out of 14 cases; 28.6%) compared to AML (three out of 41 cases; 7.3%). In five out of the seven cases presentation samples were available to study for the presence of the mutation. In two out of two AML patients the p53 mutation was detectable in the presentation sample by direct sequencing. In three ALL patients analysis of presentation material by direct sequencing showed a small mutant peak in one case, the other two being negative despite the sample analysed containing > 90% blast cells. However in both of these patients, the presence of p53 mutation was confirmed in the presentation sample using allele-specific PCR. In one of these patients the emergence of a subclone at relapse was confirmed by clonality analysis using IgH fingerprinting. Our results confirm that in ALL p53 mutations are present in a proportion of patients at relapse. Furthermore cells carrying the mutation are detectable at presentation in a minor clone suggesting that p53 mutations in ALL may be a mechanism contributing to disease relapse. © 1999 Cancer Research Campaign

P53 gene mutations and rearrangements in non-Hodgkin's lymphoma.

Alterations of the p53 gene have been associated with the progression of certain human malignancies. To establish further the correlation between p53 gene alterations and progression of non-Hodgkin's lymphomas (NHLs), the authors analyzed both mutations and rearrangements of the p53 gene in a cohort of 84 NHLs. Eighty-four NHLs were analyzed for p53 gene alterations. Point mutations of exons 5-9 were studied by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), and DNA rearrangements were studied by Southern blot analysis. RESULTS. Point mutations and DNA rearrangements of the p53 gene were detected in 6 (7.2%) and 3 (3.6%) patients, respectively. All p53 gene abnormalities were found exclusively in B-cell NHLs. Taken together, patients with p53 gene alterations had poorer survival than other patients (P = 0.024). However, of the three patients with p53 gene rearrangements, the two who appeared to have one normal allele showed a relatively better response to chemotherapy and had longer survival (27 and 47 months). In contrast, the remaining patient who had rearranged bands much stronger than the germline, and thus appeared to have both alleles rearranged, was refractory to chemotherapy and had poorer survival (6 months). Patients with NHLs of intermediate and high grades who carried point mutations or rearrangements of p53 genes had worse outcomes than other patients. Patients with one abnormal p53 allele and one residual normal allele had a more favorable prognosis than those with two abnormal alleles.

Detection of p16 gene alteration in cervical cancer using tissue microdissection and LOH study

The p16 gene was identified as cyclin-dependent kinase inhibitor (CDKI) and this may negatively regulate the cell cycle by acting as a tumor suppressor. Using tissue microdissection, the molecular changes at p16 and Rb genes were analysed in the spectrum of disease from dysplasia to invasive cancer of the uterine cervix. Six of 27 (22%) cases informative for D9S171 and IFNA of 9p21-22 marker ( p16INK4a) showed loss of one or both alleles in at least one of these loci. LOH of pRb was detected in 29% (5/17). Gene alterations at p16 and pRb loci were only detectable in some cases of HPV-16/18 DNA positive cervical cancer. Three cases demonstrated mutational changes of p16INK4a, and the alterations were determined to be G to T shift, suggesting transitional missense mutation. In summary, the inactivation of the p16/cdk-cyclin/Rb cascade may play an additional role during the malignant progression in HPV-16/18 positive cervical cancers.

P53 Gene Alterations in Prostate Cancer After Radiation Failure and Their Association with Clinical Outcome: A Molecular and Immunohistochemical Analysis

This study evaluates the prevalence of p53 gene mutations in prostate cancer in salvage prostatectomies after radiation failure using single strand conformational polymorphism (SSCP) and direct sequencing of the polymerase chain reaction (PCR) product. Findings were correlated with immunohistochemically (IHC) detectable p53 expression in residual prostate cancer. The usefulness of p53 as a marker of clinical outcome was evaluated. Thirty-three cases were available for molecular and immunohistochemical analysis. Immunohistochemical stains for p53 were performed with clone DO7. PCR-SSCP for mutations in the coding region of p53 DNA (exons 4-9) was performed on all immunopositive cases and 12 of 23 immunonegative cases. All samples with an SSCP shift were sequenced for the respective exon. Patients were evaluated for biochemical failure for 1-82 months (median 38 months) following surgery. Immunohistochemical p53 reactivity was noted in 10 of 33 (30%) patients. Among p53 immunopositive cases SSCP shifts were seen in 7 of 10 (70%) samples with 5 of the 7 (71%) showing p53 mutations. Univariate analysis revealed abnormal expression of p53 protein by immunohistochemistry to be a significant predictor of poorer outcome (p = 0.025, log rank), however this was not independent of pathologic stage, surgical margin status and Gleason score. The presence of p53 gene mutations by PCR-SSCP and direct sequencing did not predict for outcome. In our study 30% of prostate cancers at the time of salvage prostatectomy after radiation failure expressed immunohistochemically detectable p53. PCR-SSCP and sequencing shows that not all of these cases have detectable mutations in the most frequent mutation sites (exons 4-9). Clinical failure is more common in the group of prostate cancer patients with abnormal p53 immunoreactivity.

P53 Protein Overexpression and Gene Mutation in Mixed Mullerian Tumors of the Uterus

Alteration of the p53 tumor suppressor gene is associated with poor prognosis in many human cancer types. We examined the incidence and prognostic significance of p53 gene alterations in a series of uterine malignant mixed Müllerian tumors (MMT). Nuclear overexpression of p53 protein detected by immunohistochemistry (IHC) with the DO-7 antibody was observed in 12 of 24 (50%) tumors. Mutation of the p53 gene detected by single-strand conformation polymorphism (SSCP) of exons 5 to 8 was found in 11 of 24 (46%) tumors. The incidence of p53 alteration in uterine MMT was significantly higher than in several common epithelial tumor cell types previously investigated in our laboratory using identical techniques. However, unlike these tumors alteration of the p53 gene does not appear to be of prognostic importance in uterine MMT.

P53 gene mutations in osteosarcomas of low-grade malignancy

Alterations in tumor suppressor gene p53, localized on chromosome 17p13, are considered to play a significant role in the initiation and, to some extent, even in the progression of various malignant tumors. In this respect, investigations on conventional highly malignant osteosarcomas have shown a mutation rate of approximately 20%. However, currently, data on the mutation rate in the group of variant histology osteosarcomas of low-grade malignancy do not exist. Therefore, we investigated a panel of low malignant entities (five low malignant intramedullary osteosarcomas grade 1; one intramedullary osteosarcoma grade 2; eight parosteal osteosarcomas, including one local recurrence grades 1 and 2, and five periosteal osteosarcomas grade 2) with polymerase chain reaction/single-strand conformation polymorphism (PCR-SSCP) analysis focusing. on exons 4 to 8 of the p53 gene followed by direct sequencing. Point mutations were found in one low-grade osteoblastoma-like osteosarcoma and in two periosteal osteosarcomas grade 2 (one missense, one silent, and one nonsense mutation). This mutation rate of 15.7% (3 of 19) is comparable to that determined in highly malignant osteosarcomas. Moreover, the analysis of clinical data did not show any difference in the behavior of tumors with p53 mutations compared with those without. Therefore, we suggest that alterations in p53 gene are an early event in the tumorigenesis of malignant osteoblastic tumors without impact on progression of these tumors.

No evidence for overexpression of the p53 protein and mutations in exons 4–9 of the p53 gene in a large family with adenomatous polyposis

Objective: Familial adenomatous polyposis coli (FAP) is an autosomal dominant disease characterized by an early onset of numerous adenomatous polyps of the colon and a high risk of colon carcinoma. The role of the p53 gene in the multistage process of FAP is as yet poorly defined. In the present study, a large family with evidence of polyposis and colon cancer was screened for the mutations of the p53 gene and protein overexpression. Methods: We examined p53 protein expression from individuals with immunohistochemical techniques using monoclonal antibody PAb1801. Polymerase chain reaction products of exons 4–9 of the p53 were examined from individuals by single strand, conformational polymorphism analysis. Results: We could find no evidence of overexpression and mutations of the p53 in any lesion including adenomas and carcinomas. Conclusion: We found that p53 gene alterations do not contribute to the genesis of adenoma or carcinoma of FAP patients for this large family examined.

No evidence for overexpression of the p53 protein and mutations in exons 4–9 of the p53 gene in a large family with adenomatous polyposis

Objective: Familial adenomatous polyposis coli (FAP) is an autosomal dominant disease characterized by an early onset of numerous adenomatous polyps of the colon and a high risk of colon carcinoma. The role of the p53 gene in the multistage process of FAP is as yet poorly defined. In the present study, a large family with evidence of polyposis and colon cancer was screened for the mutations of the p53 gene and protein overexpression. Methods: We examined p53 protein expression from individuals with immunohistochemical techniques using monoclonal antibody PAb1801. Polymerase chain reaction products of exons 4–9 of the p53 were examined from individuals by single strand, conformational polymorphism analysis. Results: We could find no evidence of overexpression and mutations of the p53 in any lesion including adenomas and carcinomas. Conclusion: We found that p53 gene alterations do not contribute to the genesis of adenoma or carcinoma of FAP patients for this large family examined.

Deletion of the p16 gene and microsatellite instability in carcinoma arising in pleomorphic adenoma of the parotid gland.

Carcinoma arising in pleomorphic adenoma of the salivary gland is a rare tumor, and its molecular aspects are unknown. Recent studies have revealed that malignant transformation of various human cancers may involve two different genetic alterations: inactivation of the p16 gene, which is a putative tumor suppressor gene, and genetic instability represented by microsatellite instability (MSI). However, so far, molecular investigations including p16 gene alteration and MSI have not been performed on carcinoma arising in pleomorphic adenoma. Both inactivation of the p16 gene and MSI were studied using DNA extracted from paraffin-embedded sections of carcinoma arising in pleomorphic adenoma. Samples also were analyzed for cyclin D1 gene amplification, which is thought to have oncogenic effects similar to those with inactivation of the p16 gene. One case showed the homozygous deletion of the p16 gene in the carcinoma, although hypermethylation of the p16 gene and amplification of the cyclin D1 gene were not observed in any cases. In two of four cases, MSI was observed. One case in two showed MSI in both the pleomorphic adenoma and the carcinoma. Results of this study suggest that two different genetic alterations, the inactivation of the p16 gene and genetic instability, play roles in the malignant transformation of carcinoma in pleomorphic adenoma. The MSI observed in the adenoma suggests that genetic alterations occur in pleomorphic adenoma.

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.

Methylation of the p16INK4A gene in multiple myeloma.

The p16INK4A (p16) binds to both cyclin D-CDK4 and cyclin D-CDK6 and inhibits the progression of the cell cycle from G1 to S phase. Loss of expression of this protein can occur by several mechanisms including structural alterations. Recent studies have suggested that the loss of expression of p16 can occur by hypermethylation of the gene. The methylation status of the p16 gene in multiple myeloma was examined in three myeloma cell lines (U266, RPMI8226 and IM9) and 16 primary myeloma samples using methylation-specific polymerase chain reaction (MSP). The U266 and RPMI8226 cell lines contained a completely methylated p16 gene and the IM9 line had a partially methylated p16 gene. Identical results were obtained by another polymerase chain reaction (PCR)-based methylation assay system as well as Southern blotting after using a methylation-sensitive restriction enzyme. The U266 cell line expressed no p16, and the IM9 had weak expression as determined by reverse transcript (RT-)PCR. The U266 cells began to express, and IM9 increased the accumulation of, the p16 RNA after treatment with the demethylating agent 5'-aza-2-deoxycytidine (10(-6)-10(-5) M). This suggested that the levels of methylation of the p16 gene detected by the MSP technique correlated with the regulation of transcription of this gene. Examination of the primary myeloma samples showed that eight of 16 (50%) contained a methylated p16 gene. We have previously found that alterations of the p16 gene, such as deletions and point mutations, are rare in primary multiple myeloma; none of the 16 samples included in this study had p16 gene alterations. Our results suggest that methylation of the p16 gene may contribute to the development and/or progression of multiple myeloma.

Analysis of the p53 gene alterations in mouse embryo fibroblast cell line Balb 3T12 and its derivative 312

We have analysed the status of the p53 gene in the mouse embryo fibroblast cell line Balb 3T12 (TD50=106) and its transformed clonal derivative 312 (TD50=104) with an aim to determine whether there exists a correlation between increased tumorigenicity and clonal expansion of cells bearing a mutation in the p53 gene. While Southern hybridizations did not show any obvious changes in the p53 gene organization in 3T12 and 312 cells, sequencing the p53 cDNA revealed that 3T12 is mutated at the amino acid residue 233 (Tyr→ Asp) whereas 312 is mutated at the residue 132 (Cys→Trp). Exploiting the altered RFLP pattern due to mutations, we identified that 3T12 contains p53 alleles that are different from the already identified mutant p53. On the basis of these observations, we conclude that 3T12 and 312 have evolved independently.

P53 alteration, proliferating cell nuclear antigen, and nucleolar organizer regions in thymic epithelial tumors.

We examined p53 protein expression, p53 gene mutation, proliferating cell nuclear antigen (PCNA), and argyrophilic nuclear organizer regions (AgNOR), in 30 patients with surgically-treated thymic tumors (26 thymoma and 4 thymic carcinoma cases). p53 expression ratio with DO-1 was divided as p53 negative (0% positivity), low expressor (<10% positivity), high expressor (>10% positivity). The incidence of p53 low and high expressor in thymoma were 19% (5/26) and 8% (2/26), respectively. p53 immunopositivity in thymoma was significantly correlated with PCNA labeling index (LI). p53 expression ratio in invasive thymoma (33%) tended to be higher than that in non-invasive thymoma (18%). p53 expression was detected in one of the thymic carcinoma. There were no p53 gene mutations in 15 invasive thymoma, although one of four (25%) thymic carcinomas showed two point mutations. p53 gene alterations seem to be associated with malignant activity of tumor cells, and therefore detection of p53 gene mutations is useful as a diagnostic factor.

Genetic sub-types of human malignant astrocytoma correlate with survival.

In human malignant astrocytoma, age of the patient and histological grade of the tumor are important prognostic variables. Several genetic changes have been reported to occur in these tumors, which may be of additional and independent prognostic relevance. To determine their prognostic significance, we analyzed 75 high-grade tumors, 12 anaplastic astrocytomas and 63 glioblastomas multiforme, for the presence of genetic changes that occur frequently in high-grade astrocytoma, i.e., loss of heterozygosity (LOH) for chromosome 10, p53-gene alteration (mutation and/or LOH), and EGFR-gene amplification. We defined 4 groups of patients who showed a specific combination of genetic changes in the tumor: group 1, p53-gene alteration without complete LOH 10; group 2, complete LOH 10 only; group 3, p53-gene alteration + complete LOH 10; group 4, complete LOH 10 + EGFR-gene amplification. In univariate analysis, the log-rank test revealed significant differences in survival between patients of group 1 (median survival of 13 months) and group 3 (median survival of 5.2 months, p = 0.0058) and between patients of group 1 and group 4 (median survival of 4 months, p = 0.0033). In multivariate analysis, age and genetic sub-type proved to be important prognostic variables, whereas histological grading was less important. The age-corrected survival time for group-4 patients is significantly shorter than that for group-1 patients (relative risk = 3.79, p = 0.0075). Our data indicate that genetic sub-type is an important prognostic variable in human high-grade astrocytoma.

P16INK4a Gene Inactivation by Deletions, Mutations, and Hypermethylation Is Associated With Transformed and Aggressive Variants of Non-Hodgkin’s Lymphomas

The molecular mechanisms underlying the pathogenesis of aggressive lymphomas and the histological transformation of indolent variants are not well known. To determine the role of p16(INK4a) gene alterations in the pathogenesis of non-Hodgkin's lymphomas (NHLs) and the histological progression of indolent variants, we have analyzed the expression, deletions, and mutations of this gene in a series of 112 NHLs. Hypermethylation of the gene was also examined in a subset of tumors with lack of protein expression but without mutations or deletions of the gene. p16(INK4a) gene alterations were detected in 3 out of 64 (5%) indolent lymphomas but in 16 out of 48 (33%) primary or transformed aggressive variants. In the low-grade tumors, p16(INK4a) alterations were detected in 1 (4%) chronic lymphocytic leukemia (hemizygous missense mutation), 1 (6%) follicular lymphoma (homozygous deletion), and 1 (5%) typical mantle cell lymphoma (homozygous deletion). The two later cases followed an aggressive clinical evolution. In the aggressive tumors, p16(INK4a) gene alterations were observed in 2 (29%) Richter's syndromes (2 homozygous deletions), 3 (33%) transformed follicular lymphomas (1 homozygous deletion and 2 nonsense mutations), 3 (43%) blastoid mantle cell lymphomas (2 homozygous and 1 hemizygous deletions), 5 (28%) de novo large-cell lymphomas (1 homozygous deletion and 4 hypermethylations), 2 lymphoblastic lymphomas (2 homozygous deletions), and 1 of 2 anaplastic large cell lymphomas (hypermethylation). Protein expression was lost in all tumors with p16(INK4a) alterations except in the typical chronic lymphocytic leukemia (CLL) with hemizygous point mutation. Sequential samples of the indolent and transformed phase of three cases showed the presence of p16(INK4a) deletions in the Richter's syndrome but not in the CLL component of two cases, whereas in a follicular lymphoma the deletion was present in both the follicular tumor and in the diffuse large-cell lymphoma. In conclusion, these findings indicate that p16(INK4a) gene alterations are a relatively infrequent phenomenon in NHLs. However, deletions, mutations, and hypermethylation of the gene with loss of protein expression are associated with aggressive tumors and they may also participate in the histological progression of indolent lymphomas.