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.     

Association of EGFR Gene Amplification and CDKN2 (p16/MTS1) Gene Deletion in Glioblastoma Multiforme

Glioblastoma multiforme (GBM) can be divided into genetic subsets: approximately one-third of GBM, primarily in older adults, have EGFR amplification; another one-third, primarily in younger adults, have TP53 mutation. The majority of GBM also have homozygous deletions of the CDKN2 (p16/MTS1) gene, resulting in cell cycle deregulation and elevated proliferation indices. We evaluated the relationship between CDKN2 deletions and the GBM subsets as defined by EGFR amplification or TP53 mutation in 70 GBM. Twenty-eight cases (40%) had EGFR amplification, 21 (30%) had TP53 mutation, and 21 (30%) had neither change. CDKN2 deletions were present in 36 (51%) GBM. Of the 28 GBM with EGFR amplification, 20 (71%) had CDKN2 deletion (p = 0.0078). The remaining 16 cases with CDKN2 loss were divided between GBM with TP53 mutations (6 cases) and GBM with neither EGFR amplification nor TP53 mutation (10 cases). Thus, CDKN2 deletions occur twice as commonly in GBM with EGFR amplification (71%) than in GBM with TP53 mutation (29%). CDKN2 deletions occurred in GBM from patients somewhat older than those patients with GBM lacking CDKN2 deletion (mean age 53 vs. 48 years). Specifically among GBM with EGFR amplification, those with CDKN2 deletions also occurred in patients slightly older than those few GBM without CDKN2 deletions (mean age 55 vs. 51 years). The presence of CDKN2 deletions in most GBM with EGFR amplification and in generally older patients may provide one explanation for the potentially more aggressive nature of such tumors.     

MTS1/CDKN2 gene mutations are rare in primary human astrocytomas with allelic loss of chromosome 9p

Human astrocytomas frequently have allelic losses of chromosome9p, suggesting the presence of a 9p astrocytoma tumor suppressorgene. The MTS1 (or CDKN2) gene on chromosome 9p encodes a cell-cycleregulator and is deleted in approximately 80% of astrocytomacell lines. To determine whether MTS1 is the tumor suppressorgene involved in human astrocytoma formation in vivo, we haveanalyzed chromosome 9p allelic loss and the MTS1 gene in 30primary astrocytomas. Deletion mapping demonstrated 15 caseswith allelic loss of chromosome 9p, with all losses either flankingor involving the MTS1 gene. Direct analysis of the MTS1 gene,however, revealed only a single missense mutation in a high-gradetumor that had lost the second allele. The low frequency ofMTS1 mutations in primary astrocytomas with allelic 9p losssuggests that MTS1 may be more important for in vitro than invivo astrocytoma growth, and that another 9p tumor suppressorgene may be involved in astrocytoma formation in vivo. Analysisof the MTS1 gene also demonstrated two intragenic polymorphisms,one in exon 2 and one in the 3' untranslated region, that canbe used to assay allelic loss directly at MTS1.     

HER-2/neu expression in glioblastoma multiforme.

BACKGROUND: The HER-2/neu oncogene encodes for a transmembrane glycoprotein with intracellular tyrosine kinase activity. The HER-2/neu receptor belongs to the family of epidermal growth factor receptors that are crucial in the activation of subcellular signal transduction pathways controlling epithelial cell growth and differentiation. Overexpression of HER-2/neu is observed in 20% to 40% of breast cancers and other solid tumors. Although information is limited, one study suggested that 15% of glioblastoma multiforme (GBM) express HER-2/neu by immunohistochemistry (IHC); gene amplification by fluorescence in situ hybridization (FISH) was not investigated. Studies in this area are potentially significant owing to the role of recombinant monoclonal anti-HER-2/neu antibody traztuzumab (Herceptin) in the treatment of tumors. DESIGN: A retrospective clinicopathologic review of 49 patients with GBM with HER-2/neu IHC staining and HER-2/neu gene amplification by FISH was performed. RESULTS: The study included 44 patients (17 women, 27 men; age range 20 to 79 y, mean 57.9 y). Initial surgery involved tumor debulking or subtotal resection in 34 patients. Thirty-six patients received adjuvant radiation therapy and 19 patients received adjuvant chemotherapy. At follow-up (range 1.0 to 49.5 mo, mean 10.5 mo), 40 patients died with tumor and 4 patients were lost to follow-up. All tumors were negative for HER-2/neu protein by IHC and for HER-2/neu gene amplification by FISH. CONCLUSIONS: No GBM demonstrates HER-2/neu protein by IHC or amplification of the HER-2/neu gene by FISH. The HER-2/neu oncogene does not seem to play a role in the pathogenesis of GBM.     

p16/CDKN2 alterations and pRb expression in oesophageal squamous carcinoma.

BACKGROUND: Upregulation of the cell cycle associated genes, p16/CDKN2 and the retinoblastoma susceptibility gene (Rb), is commonly seen during the proliferation of normal cells. An inverse relation between the expression of p16/CDKN2 and Rb has been noted in many tumours, but has not yet been determined in oesophageal squamous carcinoma. AIMS: To investigate p16/CDKN2 genetic alterations and both the p16/CDKN2 and the Rb protein (pRb) immunophenotypes in oesophageal squamous carcinoma. METHODS: Twenty primary oesophageal squamous carcinomas were examined for mutations in p16/CDKN2 by the polymerase chain reaction, single stranded conformational polymorphism, and DNA sequencing. Synthesis of p16/CDKN2 and pRb proteins was determined by immunohistochemistry in 19 specimens of formalin fixed, paraffin wax embedded tissues. RESULTS: Mutations of p16/CDKN2 were not detected in exons 1 and 2. In only one case, G to C and C to T base changes were detected in a non-coding region of exon 3. Expression of p16/CDKN2 and Rb was observed in both normal and neoplastic areas of tissue sections, indicating neither consistent homozygous deletion nor consistent hypermethylation of the genes in tumours. Fourteen tumours showed an inverse expression of p16/CDKN2 and Rb. An increased percentage of cells that immunostained positively for p16/CDKN2 but not for pRb was observed in eight tumours, five of which had no detectable pRb, suggesting defective Rb expression in these oesophageal squamous carcinomas. CONCLUSIONS: These results indicate that p16/CDKN2 mutations occur infrequently in oesophageal squamous carcinoma. The alteration of the Rb gene is suggested as an important step in the development of these tumours.     

肺癌CDKN2/p16基因纯合缺失的研究

目的　研究 Ｃ Ｄ Ｋ Ｎ２／ｐ１６ 基因的缺失与肺癌发生、发展的关系。方法　采用多重聚合酶链反应技术,对８９ 例肺癌进行了 Ｃ Ｄ Ｋ Ｎ２／ｐ１６ 基因第１ 、２ 外显子纯合缺失的分析研究。结果　标本取材方法的改良提高了聚合酶链反应技术对基因缺失的检出率,８９ 例肺癌中检出第１ 外显子缺失率１９ ．１ ％ （１７／８９） ,第２ 外显子缺失率２２ ．５ ％ （２０／８９） ,有１４ 例第１ 、２ 外显子共同缺失,第１ 或（ 和） 第２ 外显子总缺失率为２５８ ％ （２３／８９） 。 Ｃ Ｄ Ｋ Ｎ２／ｐ１６ 基因的缺失集中发生于非小细胞肺癌, 并与转移和分期有关。结论 Ｃ Ｄ Ｋ Ｎ２／ｐ１６ 基因的缺失是非小细胞肺癌的遗传易感因素,并在其恶性进展中起一定作用。     

Parental preferences for CDKN2A/p16 testing of minors

PurposeGenetic testing of minors is controversial, as ethical considerations depend on multiple aspects of the particular disease and familial context. For melanoma, there is a well-established and avoidable environmental influence and a documented benefit of early detection.MethodsWe surveyed 61 CDKN2A/p16 mutation-tested adults from two kindreds about their attitudes toward genetic testing of minors immediately posttesting and 2 years later.ResultsOverall, 86.9% expressed support of melanoma genetic testing of minors, with the importance of risk awareness (77.4%) and the likelihood of improved prevention and screening behaviors (69.8%) as the most frequently cited potential benefits. Among mutation carriers, 82.6% wanted genetic testing for their own children. These preferences remained stable over a 2-year period. Most respondents (62.3%) favored complete involvement of their children in genetic counseling and test reporting; 19.7% suggested that children be tested but not informed of the results. Concerns about inducing psychological distress or compromising children's decision autonomy were infrequently cited. Testing preferences did not vary by respondent age, gender, or melanoma history.ConclusionRespondents strongly supported melanoma genetic testing of minors, with most citing improved health behavior as a likely outcome. We discuss options for melanoma genetic counseling and testing of minors.     

喉鳞状细胞癌MTS1／p16基因纯合子缺失的检测

为探讨MTS1／p16基因在喉癌中纯合子缺失情况及其与喉癌生物学行为的关系,本研究用比较PCR技术对59例喉鳞状细胞癌（LSCC）MTS1／p16基因纯合子缺失进行检测。剔除3例不可比样本,9／56 LSCC有MTS1／p16基因纯合子缺失,缺失率为16．07％。MTS1／p16基因纯合子缺失与LSCC病理分级无关;LSCC晚期（Ⅲ-Ⅳ）期的MTS1／p16基因纯合子缺失率（36．84％）明显高于早期（Ⅰ-Ⅱ期）肿瘤（5．4％）,P＜0．01,显示MTS1／p16基因纯合子缺失与LSCC的自然病程有关。认为MTS1／p16基因纯合子缺失是LSCC发生发展过程中的分子机制之一,可能与LSCC的恶性发展有关。     

Malignant transformation of neurofibromas in neurofibromatosis 1 is associated with CDKN2A/p16 inactivation

Patients with neurofibromatosis 1 (NF1) are predisposed to develop multiple neurofibromas (NFs) and are at risk for transformation of NFs to malignant peripheral nerve sheath tumors (MPNSTs). Little is known, however, about the biological events involved in the malignant transformation of NFs. We examined the CDKN2A/p16 gene and p16 protein in NFs and MPNSTs from patients with NF1. On immunohistochemical analysis, all NFs expressed p16 protein. The MPNSTs, however, were essentially immunonegative for p16, with striking transitions in cases that contained both benign and malignant elements. None of the benign tumors had CDKN2A/p16 deletions, whereas three of six MPNSTs appeared to have homozygous CDKN2A/p16 deletions. Methylation analysis and mutation analysis of CDKN2A/p16 in MPNSTs did not reveal any abnormalities. These results show that malignant transformation of NF is associated with loss of p16 expression, which is often secondary to homozygous deletion of the CDKN2A/p16 gene. The findings suggest that CDKN2A/p16 inactivation occurs during the malignant transformation of NFs in NF1 patients and raises the possibility that p16 immunohistochemistry may provide ancillary information in the distinction of NF from MPNST.     

Deletion and methylation of the tumour suppressor gene p16/CDKN2 in primary head and neck squamous cell carcinoma.

AIMS: To study the homozygous deletion and methylation status of the 5' CpG island of the p16 and p15 genes (9p21) in a set of primary advanced head and neck squamous cell carcinomas (SCC) and to test whether inactivation of these genes by these mechanisms contributes to head and neck SCC development. METHODS: DNA was extracted from fresh tumours. Homozygous deletion was determined by the polymerase chain reaction (PCR) followed by hybridisation with the corresponding probe, radioactively labelled by the random priming method. Methylation status of the CpG island of the 5' region of these genes was assessed by digestion with the appropriate restriction enzymes followed by PCR and subsequent hybridisation with the corresponding probe. The presence of point mutations was determined by PCR-SSCP (single strand conformation polymorphism). RESULTS: The p16 and p15 genes were homozygously deleted in 20% and 10% of the tumours, respectively. No point mutations were found at p16 and p15. The 5' CpG island at the p16 gene was methylated in 20% of the cases. CONCLUSIONS: The tumour suppressor gene p16 is inactivated through homozygous deletion or methylation in a significant proportion of cases of head and neck SCC.     

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.     

肺癌组织CDKN2/p16基因点突变的分析

目的 研究我国肺癌组织中CDKN2／p16基因点突变的发生情况。方法 采用聚合酶链反应－单链构象多态性和序列分析的方法，对89例肺癌组织中CDKN2／p16基因第2外显子的点突变进行了研究。结果 在未发生第2外显子缺失的69例肺癌组织中，发现CDKN2／p16基因第2外显子变异或可疑变异者16例；对该16例进行序列分析，共发现有9例存在不同类型的基因点突变。结论 点突变是CDKN2／p16基因失活的一种形式，但不是主要形式。由点突变引起的CDKN2／p16基因失活在肺癌的发生发展中起一定的作用。     

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.     

CDKN 2/p16基因甲基化失活与肺癌关系的研究

目的 研究ＣＤＫＮ２／ｐ１６基因５’端调控序列区ＣｐＧ岛甲基化的状态与肺癌发展的关系。方法 采用甲基化敏感性核酸酶切基因组ＤＮＡ的方法,对８９例肺癌的ＣＤＳＮ２／ｐ１６基因进行了Ｓｏｕｔｈｅｒｎ杂交分析。结果 ８９例肺癌发现该基因甲基化２１例,甲基化频率为２３．６％（２１／８９）,其中１７例发生于４２例Ｐ１６蛋白 阴性表达的患者。结论 ＣＤＫＮ２ｐ／１６基因５’端ＣｐＧ岛甲基化可能是该基因失活的重     

CDKN2A/p16 inactivation is related to pituitary adenoma type and size

p16 (CDKN2A, MTS1, INK4A) status at genomic and protein levels was analysed and correlated with clinico-pathological features in 72 pituitary adenomas. Methylation of CpG islands of promoter/exon 1 sequences was found in most gonadotroph, lactotroph, plurihormonal, and null cell adenomas (36 of 44, 82%), but it was rare in somatotroph (1 of 13 cases, 8%) and corticotroph adenomas (1 of 15 cases, 7%). Homozygous CDKN2A deletion was restricted to rare somatotroph (15%) and corticotroph adenomas (13%). Immunohistochemical p16 protein expression was observed in the normal adenohypophysis, whereas it was absent in 60 of 72 (83%) tumours and reduced in another ten (14%) tumours. Staining for p16 was only seen in 5 of 15 (33%) corticotroph, 3 of 13 (23%) somatotroph, 3 of 5 (60%) plurihormonal, and 1 of 19 (5%) null cell adenomas. p16 immunonegativity without CDKN2A methylation or deletion occurred in 22 tumours, including most somatotroph and corticotroph adenomas (15 of 28, 54%). Both CDKN2A alterations and p16 negativity were related to larger tumour size. Patients with p16-negative tumours were older than patients with p16-positive tumours. These data suggest that p16 down-regulation is common in all adenoma types. The mechanisms of p16 down-regulation probably involve CDKN2A methylation in most types, but remain to be determined in somatotroph and corticotroph adenomas. These findings also suggest that p16 down-regulation is usually not an initial event, but is acquired during adenoma progression.