Promoter Methylations of p16 and p14 Genes in Early and Advanced Gastric Cancer. Correlations of the Modes of Their Occurrence with Histologic Type

The INK4a/ARF locus encodes two cell cycle-regulatory proteins, p16^INK4a and p14^ARF. These share an exon using different reading frames, and act through Rb and p53 pathways. Recently, it has been found that silencing of p16^INK4a and p14^ARF expressions by aberrant methylation of the CpG islands in the promoter regions is an alternative mechanism that inactivates possible tumor suppressor functions in various tumors. To clarify the features of gastric cancers with promoter methylation of p16^INK4a and p14^ARF, we investigated the methylation status in gastric cancer cell lines and primary gastric cancers using methylation-specific PCR (MSP), and correlated the methylation status with microsatellite instability (MSI), DNA ploidy pattern, p53 immunohistochemistry, and various clinicopathologic factors, paying attention to the correlations with the histologic types. Of 10 cell lines studied, silencing of the expression of p16^INK4a and p14^ARF due to promoter methylation was detected by MSP and RT-PCR in six (60%) and two (20%) cell lines, respectively. p14^ARF silencing was detected only in cell lines derived from gastric cancer of the diffuse type, while p16^INK4a silencing was found in cell lines derived from both diffuse and intestinal types. In 59 primary gastric cancers, promoter methylation of p16^INK4a and p14^ARF was found in 10 (17%) and 14 (24%) of the tumors independently, there being an association with DNA diploidy, but not with p53 immunohistochemistry. p16^INK4a methylation was found irrespective of tumor stages and histology. Whereas p14^ARF methylation was found more frequently in intestinal type cancers in an early stage and in diffuse type cancers in an advanced stage, MSI tended to be related especially to p14^ARF methylation in cancers of the intestinal type. Thus, the significance of p14^ARF methylation differed between intestinal and diffuse types, while such a difference was not observed in p16^INK4a methylation.     

TP53 R72P and MDM2 SNP309 polymorphisms in modification of childhood acute lymphoblastic leukemia susceptibility

Genomic and immunologic surveillance mechanisms are crucial in protection from cancer. The tumor suppressor protein p53, encoded by TP53, is a major regulator of genome surveillance. Among the natural sequence variants of TP53, rs1042522 (R72P) modifies the risk for solid tumors. To investigate its relevance in childhood acute lymphoblastic leukemia (ALL) susceptibility, we genotyped 114 cases and 414 newborn controls from Wales (UK) for polymorphisms in TP53 (R72P), its negative regulator MDM2 (single-nucleotide polymorphism SNP309, rs2279744), and selected HLA complex genes whose products interact with TP53. TP53 R72P showed a risk association with gene dosage effect (P=0.002) resulting in a strong association of homozygous genotype (OR=2.9, 95% CI=1.5–5.6) and no sex effect. SNP309 did not show any association with primary susceptibility to childhood ALL, even after stratification by sex. However, females with SNP309 minor allele had earlier onset of childhood ALL (median age at diagnosis was 36 months in females, but 60 months in males; P=0.002). The HLA complex genes did not show any statistically significant interaction with R72P. We have therefore identified TP53 R72P as a possible risk modifier for childhood ALL and the association of MDM2 with age at onset with sex effect suggests prenatal hormonal programming of childhood ALL susceptibility.     

Analysis of the p16INK4, p14ARF, p15, TP53, and MDM2 genes and their prognostic implications in osteosarcoma and Ewing sarcoma

We examined alterations of the p16INK4, p14ARF, p15, TP53, and MDM2 genes in 30 osteosarcomas and 24 Ewing sarcomas. Among 21 osteosarcomas and 24 Ewing sarcomas, p16INK4, p14ARF, and p15 abnormalities were found in 4 (19%), 2 (9%), and 3 (14%) osteosarcomas, respectively, and in 4 (17%), 3 (13%), and 4 (17%) Ewing sarcomas, respectively. The alterations of p16INK4, p14ARF, and p15 included homozygous deletions spanning all 3 genes, methylation of p16INK4 or p15, and a nonsense mutation of p16INK4, which simultaneously caused a missense mutation of p14ARF. Alterations of TP53 were found in 15 (50%) of 30 osteosarcomas and 1 (3%) of 24 Ewing sarcomas. None of the sarcomas showed MDM2 amplification. While TP53 abnormalities were far more frequent in osteosarcoma than in Ewing sarcoma, alterations of p16INK4, p14ARF, and p15 were present at similar frequencies in the two types of sarcoma. The event-free survival (EFS) was worse in Ewing sarcoma patients with p16INK4 and p14ARF mutation/deletion than in those without the mutation/deletion (P = 0.019), and EFS was worse in osteosarcoma patients with TP53 alterations than in those without TP53 alterations (P = 0.048). The different incidence of TP53 abnormalities in the 2 types of sarcoma may reflect differences of the molecular processes through which the 2 types of tumor develop.     

Homozygous deletion of CDKN2A (p16, p14) and CDKN2B (p15) genes is a poor prognostic factor in adult but not in childhood B-lineage acute lymphoblastic leukemia: a comparative deletion and hypermethylation study

The biological behavior of childhood B-lineage acute lymphoblastic leukemia (B-ALL) is different from that of adults. We performed a comprehensive analysis of the deletion and the methylation profile of CDKN2A (hereafter identified separately as p16 and p14, for the different proteins encoded) and CDKN2B (hereafter p15) in 91 newly diagnosed B-ALL patients (61 children, 30 adults). The prognostic significance of the profiles of these genes and the association between alterations in these genes and known cytogenetic prognostic factors (BCR/ABL; ETV6/RUNX1, formerly TEL/AML1; MLL rearrangement; and ploidy changes of chromosomes) were also assessed. The prevalence of homozygous deletion, hemizygous deletion, and no deletion of the 9p21 region was 11.5%, 16.4%, and 72.1%, respectively, in children and 30.0%, 20.0%, and 50.0%, respectively, in adults; the higher incidence of homozygous deletion in adults was significant (P=0.029). Homozygous deletion was associated with poor overall survival in adults (P=0.019), but not in children. The incidence of promoter methylation of p16, p14, and p15 was 34.4%, 14.8%, and 34.4%, respectively, in children and 26.7%, 10.0%, and 40.0%, respectively, in adults, with no significant difference between the two groups. No significant association was observed between deletion and methylation or with known cytogenetic prognostic factors. The difference in incidence, distribution, and prognostic effect of homozygous deletion in children and adults may explain the prognostic disparity.     

Mutant Brca2/p53 mice exhibit altered radiation responses in the developing mammary gland

Appropriate balance between proliferation and apoptosis is critical for mammary gland development and is often altered during tumorigenesis. Carcinogens like radiation induce DNA damage and activate protective responses such as cell cycle arrest and apoptosis. We used mice carrying Brca2(-/-) and/or p53(-/-) mutations to evaluate the individual and combined effects of these genes on cell proliferation and apoptosis in the developing mammary gland. Mice were exposed to 5Gy of radiation or chamber exposure (controls) followed by injection with BrdU. Mammary glands were collected 6 h post-radiation exposure and evaluated for proliferation (BrdU) and apoptosis (TUNEL) in terminal end buds (TEB) and ducts. Under control conditions, the Brca2 mutation reduced proliferation and apoptosis in TEB but not ducts, whereas the p53 mutation reduced apoptosis in TEB and ducts but did not influence proliferation. Despite these alterations in proliferation and/or apoptosis, neither mutation, either individually or combined, significantly altered the overall balance between the two as measured by the proliferation to apoptosis ratio (growth index). Following irradiation, the Brca2 mutation had no significant effect on proliferation or apoptosis, whereas the p53 mutation resulted in reduced apoptosis in TEB and ducts but did not significantly influence proliferation. Neither mutation by itself altered the growth index in the TEB after irradiation although combined Brca2/p53 mutation caused significantly increased proliferation, reduced apoptosis, and an elevated growth index in TEB and ducts. These results reveal both independent and collaborative growth regulatory roles for Brca2 and p53 under normal and adverse environmental conditions. Additionally, we demonstrate the importance of gene-environment interactions by showing that Brca2- and p53-deficient mice can compensate for their genetic deficiencies under control conditions but not after exposure to radiation. We also demonstrate distinct spatial differences in the cellular functions of Brca2 and p53 and show that combined mutation of both genes is more detrimental than loss of either gene alone.     

P53, p15INK4B, p16INK4A and p57KIP2 mutations during the progression of chronic myeloid leukemia

The occurrence of acute transformation during the treatment of chronic myeloid leukemia (CML) is still a poorly understood mechanism. In this disease p53, p16INK4A, p15INK4B, p57KIP2 mutations and p15INK4B/p16INK4A homo/hemizygous deletions were analyzed in the initial diagnosis phase and during the treatment phase of twelve CML cases, in order to establish whether there was a consistent molecular genetic alteration in its progression. During the treatment period, four of twelve cases had blastic crisis. All the mutations observed in p53, p16INK4A and p15INK4B cumulated in three out of four CML cases who had blastic crises. In one case, p53 codon 282 mutation (CGG-->TGG; arg-->trp) were observed in initial diagnosis. Seven months later, G-->C transition in the 3' side of p15 cDNA (778. nucleotide) was observed in the accelerated phase with the same p53 codon 282 mutation. Thirteen months later, this patient died as a result of blastic crisis. The patient in blastic crises in the initial diagnosis phase had a mis-sense point mutation in p16 codon 69 (ACT-->AGT; thr-->ser) and a polymorphism in codon 68 (GCC-->GCG). Six months later, this patient also died. In one case, p53 codon 237 mutation (ATG-->ATA; met-->ile) were observed in the initial diagnosis phase. Then months later, the patient died as a result of blastic crises. No p15INK4B/p16INK4A homo/hemizygous deletion and p57KIP2 gene mutation which was described in the same pathway were observed in CML progression. These results indicate that p15INK4B and p16INK4A gene alterations may have an affect on the progression of CML-like p53 mutation. A correlation was found with the progression of CML and p53, p15INK4B and p16INK4A somatic mutations. Finding p15INK4B and p16INK4A gene alteration as well as p53 mutations may be a prognostic marker in patients with CML.     

Prognostic significance of p16INK4a/p53 in Tunisian patients with breast carcinoma

Infiltrating ductal carcinoma (IDC) of the breast is a result of genetic alterations that affect the regulation of the cell cycle check-point and apoptosis. The aim of the present study was analysis using immunohistochemical localization of mouse double minute-2 (mdm2), p16INK4a, p53, bax and bcl-2 markers in Tunisian patients with breast IDC and to determine if there was correlation with the major clinico-pathological parameters and with survival of patients. We showed that the expression of p53, p16INK4a, mdm2, bcl-2, and bax was observed in 46.3%, 20.7%, 38%, 50% and 11.9% of cases, respectively. Statistical analysis revealed that positive expression of mdm2 was associated with larger tumors (P = 0.013), whereas bax positivity was more prevalent in younger patients and in tumors of smaller size (P = 0.008 and P = 0.012 respectively). Furthermore, the expression of p16INK4a correlated with advanced grade (P < 0.0001), triple negative tumors (ER-/PR-/HER2-, P = 0.001) and mdm2 expression (P = 0.017). The absence of nuclear p53 accumulation was predictive of good prognosis as well as when it was associated with negative expression of p16INK4a. Our findings suggest that among the biomarkers tested, p16INK4a might have a useful clinical and prognostic significance in infiltrating ductal carcinoma of the breast.     

Expression of p14ARF, p15INK4b, p16INK4a, and DCR2 increases during prostate cancer progression.

Prostate carcinoma is a hormonally driven age-related neoplasm. Cellular senescence is an age-related process where cells remain metabolically active but in a growth-arrested state at the G1 phase. p14(ARF), p15(INK4b), and p16(INK4a), which are known to regulate G1 cell cycle arrest, and the tumor necrosis factor receptor superfamily member decoy receptor 2 (DCR2), have been recently identified as senescence markers. The purpose of this study was to characterize and compare the expression of p14(ARF), p15(INK4b), p16(INK4a), and DCR2 in tissue microarrays containing cases of normal prostate, nodular hyperplasia, prostate intraepithelial neoplasia (PIN), and malignant prostate cancer tissue. We performed immunohistochemical staining for p14(ARF), p15(INK4b), p16(INK4a), and DCR2 in tissue microarray blocks containing 41 cores of normal prostate, 65 cores of nodular hyperplasia, 21 cores of PIN, 69 cores of low-grade prostate carcinoma, and 42 cores of high-grade prostate carcinoma, derived from 80 cases of prostatectomy with adenocarcinomas. We detected positive staining of p16(INK4a) in 19% of the PIN, 25% of the low-grade carcinoma, and 43% of the high-grade carcinoma specimens but none in the normal prostate and nodular hyperplasia specimens. Expression of p14(ARF) revealed very high levels of expression in normal tissues (83%), nodular hyperplasia (88%), PIN (89%), and cancer cells (100%). P15(INK4b) and DCR2 were found positive in 81 and 33% normal, 46 and 10% nodular hyperplasia, 74 and 36% PIN tissues, 87 and 89% low-grade carcinomas, and 100 and 93% high-grade carcinomas. There is an increased protein expression of senescence-associated molecular markers, indicating that cellular senescence might play a role in prostate carcinoma. Because p16(INK4a)-positive cells were detected only in premalignant lesions and carcinomas but not in normal or benign tissues, p16(INK4a) may aid in the diagnosis of PIN and prostate cancer in difficult cases.     

Investigation of germline PTEN,p53, p16INK4A/p14ARF,and CDK4 alterations in familial glioma

Epidemiological studies suggest that some familial aggregations of glioma may be due to inherited predisposition. Many genes involved in familial cancers are frequently altered in the corresponding sporadic forms. We have investigated several genes known to be altered in sporadic gliomas for their potential contribution to familial glioma. Fifteen glioma patients with a family history of brain tumors were identified through the Mayo Clinic Department of Neurology (nine diffuse astrocytomas, two oligodendrogliomas, two mixed oligoastrocytomas, one pilocytic astrocytoma, and one pineal glioma). Eleven of the propositi had one or more first degree relative with a glioma. Lymphocyte DNA was derived from each of the patients and analyzed by polymerase chain reaction (PCR) and direct sequencing of the PTEN, p53, p16^INK4A/p14^ARF, and CDK4 genes. In addition, fluorescence in situ hybridization (FISH) was performed on EBV‐transformed lymphocytes from each affected individual to detect germline copy number of the p16^INK4A/p14^ARF tumor suppressor region. A p53 germline point mutation was identified in one family with some findings of Li‐Fraumeni syndrome, and a hemizygous germline deletion of the p16^INK4A/p14^ARF tumor suppressor region was demonstrated by FISH in a family with history of both astrocytoma and melanoma. Thus, whereas germ‐line mutations of PTEN, p53, p16^INK4A/p14^ARF, and CDK4 are not common events in familial glioma, outside of familial cancer syndromes, point mutations of p53 and hemizygous deletions and other rearrangements of the p16^INK4A/p14^ARF tumor suppressor region may account for a subset of familial glioma cases. Collectively, these data lend genetic support to the heritable nature of some cases of glioma. Am. J. Med. Genet. 92:136–141, 2000. © 2000 Wiley‐Liss, Inc.     

Analysis of p53 expression in osteosarcoma of the jaw: Correlation with clinicopathologic and DNA ploidy findings

The objective of this study was to determine whether p53 expression correlated with clinicopathological and DNA ploidy in osteosarcoma of the jaw, a particular subtype of osteosarcoma associated with an older age at presentation, longer median survival, rare metastasis, and death due to uncontrolled local recurrence. Seventeen cases of osteosarcoma of the jaw were stained for p53 using CM1 antibody (Novocastra). Eight cases (47.5%) showed nuclear staining in more than 10% of the neoplastic nuclei. Ploidy analysis showed 13 aneuploid (eight p53+) and three diploid (none p53+) tumors. No significant relationship was found between p53 positivity and clinicomorphological features, DNA ploidy or survival. p53-positive cases were, however, more commonly associated with aneuploidy and chondroblastic differentiation. These results indicate that p53 immunostaining does not seem to provide prognostic information in osteosarcoma of the jaw.     

Prognostic value of p16INK4a and p14ARF gene hypermethylation in human colon cancer

The INK4a/ARF locus (9p21) encodes two unique and unrelated cell cycle regulators, p16INK4a and p14ARF. This study was performed to evaluate the methylation status of p16INK4a and p14ARF genes, as well as its association with p16 and p53 expression, microsatellite instability (MI) status, and various clinicopathologic parameters in sporadic colorectal cancer. Sixty-five cases of primary colorectal adenocarcinoma with a series of clinicopathological parameters were obtained. We performed methylation-specific PCR of p16INK4a and p14ARF genes in colorectal cancer paraffin blocks with its paired normal samples, as well as immunohistochemical stainings for p16 and p53, and MI analysis. Aberrant methylations of p16INK4a and p14ARF gene were present in 21 (32.3%) and 33 (50.8%) out of 65 cases, respectively. p16INK4a aberrant methylation was correlated with p16 negativity (P=0.021) and p53 overexpression (P=0.007). p16INK4a aberrant methylation was more frequently present in poorly differentiated adenocarcinomas (P=0.002). Aberrant methylation of p14ARF gene occurred more frequently in patients under 50 years of age and in left-sided colon cancers, and was not statistically significant. Compared with the group with simultaneous absence of methylation in both promoters, the group showing concomitant alterations in both p16INK4a and p14ARF genes (n=10) more frequently presented lymph node metastasis (P=0.020) and higher tumor grade (P=0.014). There was no correlation between p16INK4a and p14ARF gene hypermethylation or MI status. This study suggests that simultaneous hypermethylation of both p16INK4a and p14ARF genes is greater prognostic value in sporadic human colorectal cancer.     

Search for the sex-determining switch in monotremes: Mapping WT1 , SF1 , LHX1 , LHX2 , FGF9 , WNT4 , RSPO1 and GATA4 in platypus

The duck-billed platypus has five pairs of sex chromosomes, but there is no information about the primary sex-determining switch in this species. As there is no apparent SRY orthologue in platypus, another gene must acquire the function of a key regulator of the gonadal male or female fate. SOX9 was ruled out from being this key regulator as it maps to an autosome in platypus. To check whether other genes in mammalian gonadogenesis could be the primary switch in monotremes, we have mapped a number of candidates in platypus. We report here the autosomal location of WT1, SF1, LHX1, LHX9, FGF9, WNT4 and RSPO1 in platypus, thus excluding these from being key regulators of sex determination in this species. We found that GATA4 maps to sex chromosomes Y₁ and X₂; however, it lies in the pairing region shown by chromosome painting to be homologous, so is unlikely to be either male-specific or differentially dosed in male and female.     

Investigation of germline PTEN, p53, p16(INK4A)/p14(ARF), and CDK4 alterations in familial glioma

Epidemiological studies suggest that some familial aggregations of glioma may be due to inherited predisposition. Many genes involved in familial cancers are frequently altered in the corresponding sporadic forms. We have investigated several genes known to be altered in sporadic gliomas for their potential contribution to familial glioma. Fifteen glioma patients with a family history of brain tumors were identified through the Mayo Clinic Department of Neurology (nine diffuse astrocytomas, two oligodendrogliomas, two mixed oligoastrocytomas, one pilocytic astrocytoma, and one pineal glioma). Eleven of the propositi had one or more first degree relative with a glioma. Lymphocyte DNA was derived from each of the patients and analyzed by polymerase chain reaction (PCR) and direct sequencing of the PTEN, p53, p16(INK4A)/p14(ARF), and CDK4 genes. In addition, fluorescence in situ hybridization (FISH) was performed on EBV-transformed lymphocytes from each affected individual to detect germline copy number of the p16(INK4A)/p14(ARF) tumor suppressor region. A p53 germline point mutation was identified in one family with some findings of Li-Fraumeni syndrome, and a hemizygous germline deletion of the p16(INK4A)/p14(ARF) tumor suppressor region was demonstrated by FISH in a family with history of both astrocytoma and melanoma. Thus, whereas germ-line mutations of PTEN, p53, p16(INK4A)/p14(ARF), and CDK4 are not common events in familial glioma, outside of familial cancer syndromes, point mutations of p53 and hemizygous deletions and other rearrangements of the p16(INK4A)/p14(ARF) tumor suppressor region may account for a subset of familial glioma cases. Collectively, these data lend genetic support to the heritable nature of some cases of glioma.     

No p16INK4A/CDKN2/MTS1 mutations independent of p53 status in soft tissue sarcomas

The p16^INK4A/CDKN2/MTS1 gene encodes a specific inhibitor of cyclin-dependent kinases (CDKs) 4 and 6. This study investigates p16^INK4A gene status and expression in mesenchymal tumours, in particular soft tissue sarcomas (STSs). Employing non-radioactive polymerase chain reaction–single strand conformational polymorphism (PCR–SSCP) sequencing, no p16^INK4A mutation was found in 86 samples taken from 74 mesodermal tumours with known p53 gene status. This suggests that p16^INK4A gene alterations, in contrast to p53, are not involved in the progression of STS. This finding is supported by the reports of a low frequency of deletions and intragenic mutations in STS. Furthermore, by immunohistochemistry (IHC), an inverse correlation was established between p16^INK4A and RB positivity for 62 per cent of the frozen tumour samples investigated. However, alterations in other components of the pRb/p16^INK4A/CDK4/cyclin D1/E2F pathway have been proven crucial for tumourigenesis in human sarcomas. © 1998 John Wiley & Sons, Ltd.     

Evaluation of p16INK4a, minichromosome maintenance protein 2, DNA topoisomerase IIalpha, ProEX C, and p16INK4a/ProEX C in cervical squamous intraepithelial lesions

p16INK4a has been shown to be overexpressed in nearly all high-grade squamous intraepithelial lesions (HSILs). Other cell-cycle regulators, such as minichromosome maintenance protein 2 (MCM2), DNA topoisomerase IIalpha (TOP IIA), and ProE(X) C (a cocktail of MCM2 and TOP IIA), have also demonstrated some value in identifying squamous intraepithelial lesions. Data on direct comparison of those cell regulatory proteins in the detection of squamous intraepithelial lesions, with a focus on low-grade squamous intraepithelial lesions (LSILs), are limited. We immunohistochemically evaluated the diagnostic value of p16, MCM2, TOP IIA, ProE(X) C, and a cocktail of p16 and ProE(X) C in 62 cervical biopsy specimens, including 14 cases of benign squamous mucosa (group 1), 34 cases of LSILs (group 2), and 14 cases of HSILs (group 3). The staining intensity and distribution were recorded. The results demonstrated that positive staining for p16 and the p16/ProE(X) C was observed in 100% of cases in group 3, whereas 79%, 86%, and 79% of cases were positive for CM2, TOP IIA, and ProE(X) C, respectively. ProE(X) C and the p16/ProE(X) C showed positive staining in 94% and 100% of cases in group 2, respectively. In contrast, immunoreactivity for p16, MCM2, and TOP IIA was detected in only 76% of cases in group 2. Importantly, all 8 p16-negative cases in group 2 were positive for p16/ProE(X) C (P = .003). Our data indicate that (1) p16 is a more sensitive and specific marker for identifying HSILs; (2) ProE(X) C is a better marker for the detection of LSILs; and (3) p16/ProE(X) C provides the highest diagnostic value for the detection of both HSILs and LSILs.     

Hypermethylation of the CDKN2/p16INK4A promotor in thyroid carcinogenesis

Functional inactivation of the p16INK4A gene has been reported to be involved in the development of a variety of human malignancies. In thyroid carcinomas, mutations of the p16INK4A gene or homozygous deletions of the gene locus 9p21 are rare. This study investigated whether p16INK4A promotor methylation is an alternative mechanism for p16INK4A gene inactivation during thyroid carcinogenesis. A methylation-specific polymerase chain reaction protocol was applied. A total of 77 thyroid tumor specimens, including 18 follicular adenomas, 18 follicular carcinomas, 16 papillary carcinomas, 12 poorly differentiated carcinomas, and 13 undifferentiated carcinomas were analyzed longitudinally. In addition, 15 tumor-free thyroid tissues were investigated. The p16INK4A promotor status was compared with p16INK4A protein expression and patient-specific data. p16INK4A promotor hypermethylation was detected in 13% of non-tumorous tissue; in 33% of follicular adenomas; in 44% of papillary carcinomas; in 50% of follicular carcinomas; in 75% of poorly differentiated carcinomas; and in 85% of undifferentiated carcinomas. With the exception of two cases, the p16INK4A protein was lost as a result of promotor hypermethylation. Comparing the methylation status with tumor stage, no correlation was found. However, lymph node and distant metastasis status showed a statistically significant prevalence for the p16INK4A promotor methylation (p = 0.035). There was no association between p16INK4A promotor methylation and age and sex. These results suggest that hypermethylation of the p16INK4A promotor region is a frequent and an early event during thyroid carcinogenesis and is associated with tumor progression and dedifferentiation.     

Mutations of the CDKN2/p16INK4 gene in Australian melanoma kindreds

The cyclin dependent kinase inhibitor 2 (CDKN2) gene on chromosome9p21 is potentially involved in the genesis of many cancersand is currently under intense investigation as a possible melanomasusceptibility locus. We have analyzed 18 Australian melanomakindreds for mutations within the coding and neighboring splicejunction portions of the CDKN2 gene. In seven kindreds (includingour six largest), CDKN2 mutations were found to segregate withthe putative melanoma chromosome previously assigned by 9p haplotypeanalysis. These changes included the duplication of a 24 bprepeat, a deleted C residue resulting in the introduction ofa premature stop codon, and four single basepair changes causingamino acid substitutions. Mutations segregated to 46 of 51 affectedindividuals in these seven klndreds, with three apparent sporadiccases In one famiiy and one in each of another two families.Penetrance was variable (55–100%) among the differentmutations. These data provide additional strong support thatthe CDKN2 gene is the chromosome 9p21 familial melanoma locus.     

Epstein-Barr virus infection correlates with the expression of COX-2, p16INK4A and p53 in classic Hodgkin lymphoma

Classic Hodgkin lymphoma (cHL), a germinal-center related B cell neoplasm in almost all cases, is characterized by scarcity of the neoplastic Hodgkin/Reed-Sternberg (H/RS) cells. Epstein-Barr virus (EBV) has been shown to affect cell cycle and regulation of apoptosis. In total, 95 cases of cHL were studied. Five-micrometer sections were prepared and stained with hematoxylin and eosin and immunohistochemical streptavidin-biotin methods for EBV-LMP-1, COX-2, p53, p16, ki-67 and cleaved caspase-3. In-situ hybridization for EBV encoded RNA was used to confirm the detection of EBV in H/RS. There were 49 nodular sclerosis, 32 mixed cellularity, 8 lymphocyte-rich, and 6 lymphocyte-depleted subtypes in this series of cases. EBV, COX-2, p16^INK4A and p53 were detected in 55% (52/95), 64% (61/95), 62% (59/95), and 65% (62/95) of the cases respectively. EBV was detected in 62% (38/61), 70% (41/59), and 69% (43/62) of COX2, p16 and p53 positive cases respectively. On the other hand, EBV-non-infected cases of cHL are associated with 59% (20/34), 69% (25/36), and 73% (24/33) of COX2, p16 and p53 negative cases respectively. In conclusion, EBV infection is associated with the expression of COX-2, p16^INK4A and p53. EBV might be the dominant factor in determining the expression of these three proteins.