BACKGROUND: P16 inactivation is frequently accompanied by telomerase reverse transcriptase ( TERT ) amplification in human cancer genomes. P16 inactivation by DNA methylation often occurs automatically during immortalization of normal cells by TERT . However, direct evidence remains to be obtained to support the causal effect of epigenetic changes, such as P16 methylation, on cancer development. This study aimed to provide experimental evidence that P16 methylation directly drives cancer development. METHODS: A zinc finger protein-based P16 -specific DNA methyltransferase (P16-Dnmt) vector containing a "Tet-On" switch was used to induce extensive methylation of P16 CpG islands in normal human fibroblast CCD-18Co cells. Battery assays were used to evaluate cell immortalization and transformation throughout their lifespan. Cell subcloning and DNA barcoding were used to track the diversity of cell evolution. RESULTS: Leaking P16-Dnmt expression (without doxycycline-induction) could specifically inactivate P16 expression by DNA methylation. P16 methylation only promoted proliferation and prolonged lifespan but did not induce immortalization of CCD-18Co cells. Notably, cell immortalization, loss of contact inhibition, and anchorage-independent growth were always prevalent in P16-Dnmt&TERT cells, indicating cell transformation. In contrast, almost all TERT cells died in the replicative crisis. Only a few TERT cells recovered from the crisis, in which spontaneous P16 inactivation by DNA methylation occurred. Furthermore, the subclone formation capacity of P16-Dnmt&TERT cells was two-fold that of TERT cells. DNA barcoding analysis showed that the diversity of the P16-Dnmt&TERT cell population was much greater than that of the TERT cell population. CONCLUSION P16 methylation drives TERT -mediated immortalization and transformation of normal human cells that may contribute to cancer development.
Humans ; Telomerase/genetics* ; DNA Methylation/physiology* ; Fibroblasts/cytology* ; Cyclin-Dependent Kinase Inhibitor p16/metabolism* ; Cell Line ; Cell Transformation, Neoplastic/genetics*

Copy number alteration (CNA) is a significant genetic change in pediatric B-cell acute lymphoblastic leukemia (B-ALL), with CDKN2A/B deletions, PAX5 deletions, and IKZF1 deletions being the most common. Recent studies have increasingly highlighted the potential prognostic significance of these gene deletions and multiple co-deletions in pediatric B-ALL. This paper reviews the main detection methods for CNA, as well as the prognostic characteristics and treatment approaches for common CNA in pediatric B-ALL.
Humans ; DNA Copy Number Variations ; Child ; PAX5 Transcription Factor/genetics* ; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics* ; Cyclin-Dependent Kinase Inhibitor p15/genetics* ; Ikaros Transcription Factor/genetics* ; Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics* ; Gene Deletion ; Cyclin-Dependent Kinase Inhibitor p16/genetics* ; Prognosis

OBJECTIVE: To investigate the relationship between CDKN2A copy number deletion and clinical features of patients with diffuse large B-cell lymphoma (DLBCL) and its prognostic value. METHODS: 155 newly diagnosed DLBCL patients with complete clinical data in the Department of Hematology of People's Hospital of Xinjiang Uygur Autonomous Region from March 2009 to March 2022 were included, formalin-fixed paraffin-embedded tumor tissues were obtained and DNA was extracted from them, and next-generation sequencing technology was applied to target sequencing including 475 lymphoma-related genes, the relationship between CDKN2A copy number deletion and clinical features, high-frequency mutated genes and overall survival (OS) of DLBCL patients were analyzed. RESULTS: CDKN2A copy number deletion was present in 12.9% (20/155) of 155 DLBCL patients, grouped according to the presence or absence of copy number deletion of CDKN2A, and a higher proportion of patients with IPI≥3 were found in the CDKN2A copy number deletion group compared to the group with no CDKN2A copy number deletion (80% vs 51.5%, P =0.015) and were more likely to have bulky disease (20% vs 5.2%, P =0.037). Survival analysis showed that the 5-year OS of patients in the CDKN2A copy number deletion group was significantly lower than that of the non-deletion group (51.3% vs 69.2%, P =0.047). Multivariate Cox analysis showed that IPI score≥3 (P =0.007), TP53 mutation (P =0.009), and CDKN2A copy number deletion (P =0.04) were independent risk factors affecting the OS of DLBCL patients. CONCLUSION CDKN2A copy number deletion is an independent risk factor for OS in DLBCL, and accurate identification of CDKN2A copy number deletion can predict the prognosis of DLBCL patients.
Humans ; Lymphoma, Large B-Cell, Diffuse/genetics* ; Prognosis ; Cyclin-Dependent Kinase Inhibitor p16/genetics* ; DNA Copy Number Variations ; Female ; Male ; Middle Aged ; Gene Deletion ; Adult ; Aged

Humans ; Squamous Cell Carcinoma of Head and Neck ; RNA, Messenger ; Immunohistochemistry ; Papillomavirus Infections/diagnosis* ; Oncogene Proteins, Viral/genetics* ; Head and Neck Neoplasms ; Cyclin-Dependent Kinase Inhibitor p16 ; Papillomaviridae ; Papillomavirus E7 Proteins/genetics* ; DNA, Viral

OBJECTIVE: To investigate paraffin section thickness in immunohistochemical detection of p16 expression in cervical tissue samples. METHODS: p16 immunohistochemical staining was performed in 150 cases of chronic cervicitis, 126 cases of low-grade squamous intraepithelial lesions(LSIL), 96 cases of high-grade squamous intraepithelial lesions (HSIL) and 78 cases of cervical cancer from January 2014 to March 2018 in Zhongshan Boai Hospital. The results of p16 protein expression in paraffin sections with thickness of 2, 3, 4, 5 and 6 μm were compared using Logistic regression analysis. RESULTS: With the increase of slice thickness, the staining of p16 protein in nucleus gradually increased. The thickness of cervical slices in chronic cervicitis and cervical cancer samples had no significant effect on the positive rate of p16 protein(=7.817 and 1.332, both >0.05), while the thickness of slices in LSIL and HSIL samples had significant effect on the positive rate of p16 protein (=17.688 and 10.182, <0.05 or <0.01). The stable and reliable results were obtained when the slices were between 3 and 5 μm thick. CONCLUSIONS Paraffin sections with thickness of 3.0-5.0 μm are recommended for immnohistochemical staining of p16 protein in cervical tissue samples.
Biomarkers, Tumor ; genetics ; metabolism ; Cervical Intraepithelial Neoplasia ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; metabolism ; Female ; Histocytological Preparation Techniques ; standards ; Humans ; Immunohistochemistry ; Paraffin ; Squamous Intraepithelial Lesions of the Cervix ; Uterine Cervical Neoplasms ; physiopathology

Gastric cancer is the result of multiple risk factors, including environmental factors, genetic factors and the interaction between them. The environmental factors mainly include dietary, Helicobacter pylori infection and family history of gastric cancer. Genetic factors mainly refer to the susceptible genes that cause epigenetic alterations in oncogenes, tumor suppress genes, cell cycle regulators, DNA repair genes and signaling molecules. This paper summarizes the susceptible genes of gastric cancer and explores the genetic basis of it.
Cyclin-Dependent Kinase Inhibitor p15 ; genetics ; Genes, Tumor Suppressor ; Genes, p16 ; Humans ; Oncogenes ; Stomach Neoplasms ; etiology ; genetics

We report three patients with normal karyotype (NK) ALL, who showed genetic aberrations as determined by high-resolution single nucleotide polymorphism array (SNP-A) analysis at both diagnosis and relapse. We evaluated the clinical relevance of the SNP-A assay for the detection of subtle changes in the size of affected genetic lesions at relapse as well as the prognostic value of the assay. In our patients, application of the SNP-A assay enabled sensitive detection of cryptic changes affecting clinically important genes in NK ALL. Therefore, this assay seems to be more advantageous compared to other conventional methods such as FISH assay, HemaVision (DNA Technology, Denmark), and conventional karyotyping for the detection of an "unstable genotype" at relapse, which may be associated with microscopic clonal evolution and poor prognosis. Further comprehensive studies are required to confirm the issues presented by our case patients in this report.
Adult ; Cyclin-Dependent Kinase Inhibitor p16/genetics ; Female ; Genotype ; Humans ; In Situ Hybridization, Fluorescence ; Karyotype ; Karyotyping ; Loss of Heterozygosity ; Male ; Middle Aged ; Oligonucleotide Array Sequence Analysis ; Polymorphism, Single Nucleotide ; Precursor Cell Lymphoblastic Leukemia-Lymphoma/*diagnosis/genetics ; Recurrence ; Retinoblastoma Protein/genetics

OBJECTIVETo investigate the expression level of COX-2, p16(INK4A) and p53 in patients with classic Hodgkin's lymphoma (cHL), and to evaluate their correlation with prognosis.

METHODSThe clinical data and samples of 52 cHL cases were collected. Immunohistochemical staining was performed to analyze the proteins level mentioned above and in situ hybridization of EBV encoded RNA (EBER) to clarify the tumor EBV infection state. Correlation between the protein expression and prognosis of patients was analyzed.

RESULTSOf 52 cases, the male and female ratio was 1.6∶1, the age was from 22 to 68 years old. All lesions located primarily in lymph nodes. All samples from 52 cases were stained with COX-2, p16(INK4A) and p53, and the positive expression of COX-2 was found in 28 cases (53.8%）, that of p16(INK4A) in 25 cases (48.1%)and p53 in 42 cases (80.8%）. All patients were divided into two groups according to differences in age (<40 years/ ≥ 40 years), gender (male/female), EBV infection (yes/no), B symptoms (yes/no), and the Ann Arbor staging (Ⅰ-Ⅱ/Ⅲ-Ⅳ), the correlation with COX-2, p16(INK4A) and p53 expression were analyzed, and only p53 expression was correlated with Ann Arbor staging (P=0.027). The statistical analysis of correlations between COX- 2, p16(INK4A) and p53 showed that the expression of COX-2 was strongly correlated with p53 (P=0.008), and p16 (INK4A) was not related to either COX-2 or p53 (P=0.246 and 0.958). Kaplan- Meier univariate OS analysis using SPSS17.0 software showed that only COX-2 expression was an adverse prognostic factor for patients'event free survival (EFS) (P=0.003). Meanwhile COX-2 expression was a unique independent prognostic factor analyzed by COX proportional hazards regression model (HR=0.091, 95% CI 0.017-0.505, P=0.006).

CONCLUSIONThe expression rate of COX-2, p16 (INK4A) and p53 in the cHL were relatively high; and they were not statistically correlated with tumor EBV infection status; the COX-2 positive group had poor prognosis, but only event free survival time becomes statistically significant shorter. COX proportional hazard regression model was used to analyze the COX-2 expression as a independent adverse prognostic factors for EFS.

Adult ; Aged ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; metabolism ; Cyclooxygenase 2 ; genetics ; metabolism ; Disease-Free Survival ; Epstein-Barr Virus Infections ; Female ; Hodgkin Disease ; diagnosis ; genetics ; metabolism ; Humans ; Immunohistochemistry ; Kaplan-Meier Estimate ; Male ; Middle Aged ; Prognosis ; Proportional Hazards Models ; Tumor Suppressor Protein p53 ; genetics ; metabolism ; Young Adult

OBJECTIVE: To investigate the effect of gene silencing of Bmi-1 on proliferation regulation of CD44+ nasopharyngeal carcinoma cancer stem-like cells (CSC-LCs). METHOD: The sequence-specific short hairpin RNA lentivirus targeting at human Bmi-1 gene (LV-Bmi-1shRNA) was constructed and was used to infect CD44+ nasopharyngeal carcinoma cells which were sorted by flow cytometry. A lentiviral which included a random sequence was also designed to serve as a negative control. We employed fluorescence microscope and flow cytometry to detect infection efficiency; real-time PCR was used to detect Bmi-1 and its downstream gene while each protein expression level was confirmed by western blotting protocol; CCK-8 proliferation assay was applied to measure proliferation capacity; tumor spheroid assay was used to evaluate the self-renewal capacity. Colony formation assay was used to measure cell colony formation capability; flow cytometry analyzed cell cycle distribution. RESULT: The constructed LV-Bmi-1shRNA successfully infected into the CD44+ nasopharyngeal carcinoma cells. The infection efficiency could reach above 95%; LV-Bmi-lshRNA effectively inhibited Bmi-1 mRNA and protein expression, while the downstream gene p16INK4a and p14ARF mRNA as well as protein expression level were upregulated (P < 0.05). Notablely, the proliferation, colony formation, self-renewal capabilities of the experimental group decreased significantly (P < 0.05). In addition, the cell cycle arrested at the G0-G1 phase. CONCLUSION Gene silencing of Bmi-1 inhibited the proliferation, colony formation and self-renewal capabilities of the CD44+ nasopharyngeal carcinoma CSC-LCs, inhibited the cell cycle processes, which may mediate through Bmi1-p16INK4a/p14ARF-p53 pathway. Our experimental results indicated that Bmi-1 gene may play an important role in the maintenance of the stem cell-like characteristics of CD44+ nasopharyngeal carcinoma cells. Bmi-1 gene may be a potential new target for the treatment of nasopharyng al carcinoma in the future.
Carcinoma ; Cell Cycle ; Cell Division ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p16 ; metabolism ; Gene Silencing ; Humans ; Hyaluronan Receptors ; metabolism ; Lentivirus ; Nasopharyngeal Carcinoma ; Nasopharyngeal Neoplasms ; genetics ; pathology ; Neoplastic Stem Cells ; cytology ; Polycomb Repressive Complex 1 ; genetics ; RNA, Messenger ; RNA, Small Interfering ; Tumor Suppressor Protein p14ARF ; metabolism ; Tumor Suppressor Protein p53 ; metabolism

OBJECTIVETo study the role of p16 gene mutation status as detected by fluorescence in-situ hybridization (FISH) and p16 protein expression as detected by immunohistochemistry in differential diagnosis of malignant mesothelioma and benign mesothelial hyperplasia.

METHODSp16 gene mutation status and protein expression were detected by FISH and immunohistochemistry respectively in 55 cases of pleural malignant mesothelioma and 30 cases of benign mesothelial hyperplasia.

RESULTSFISH study showed that the rate of p16 deletion in malignant mesothelioma (81.8%,45/55) was higher than that in benign mesothelial hyperplasia (3.3%,1/30). The difference was statistically significant (P<0.05). Immunohistochemical study showed that the rate of p16 protein expression in malignant mesothelioma (23.6%) was lower than that in benign mesothelial hyperplasia (76.7%). The difference was also statistically significant. The sensitivity and specificity of FISH in distinguishing between mesothelioma and reactive mesothelial hyperplasia were higher than those of immunohistochemistry.

CONCLUSIONSIn contrast to reactive mesothelial hyperplasia, p16 gene is deleted and p16 protein is not expressed in malignant mesothelioma. The sensitivity and specificity of FISH are higher than those of immunohistochemistry in the distinction.

Cyclin-Dependent Kinase Inhibitor p16 ; metabolism ; Diagnosis, Differential ; Epithelium ; pathology ; Genes, p16 ; Humans ; Hyperplasia ; diagnosis ; genetics ; Immunohistochemistry ; In Situ Hybridization, Fluorescence ; Mesothelioma ; diagnosis ; genetics ; metabolism ; Mutation ; Pleura ; pathology ; Pleural Neoplasms ; diagnosis ; genetics ; metabolism ; Sensitivity and Specificity