p14ARF, p15INK4b and p16INK4a methylation status in chronic myelogenous leukemia

The INK4 family of proteins p15^INK4b, p14^ARF and p16^INK4a function as cell cycle inhibitors where they are involved in the inhibition of G1 phase progression. Methylation of the p15^INK4b promoter never seems to occur in solid tumors but is a major gene silencing mechanism in hematological malignancies. p14^ARF and p16^INK4a promoter methylation often occurs in solid tumors but also in leukemias and lymphomas. In chronic myelogenous leukemia (CML), only a few reports have been published regarding INK4 methylation and the results of the literature are discordant. Thus clearly, more works on large series have to be performed independently.     

Risk assessment in chronic myelomonocytic leukemia (CMML)

The clinical course of chronic myelomonocytic leukemia (CMML) is extremely variable, and disease progression can occur at any time from diagnosis. Median survival is about 20 months. About 20% of patients develop acute myeloid leukaemia (AML). Multivariate analyses performed by several groups showed that elevated medullary blast count, low haemoglobin, elevated serum lactate dehydrogenase (LDH), and perhaps an increased lymphocyte count, are the most important independent prognostic parameters, whereas karyotype analysis was not consistently shown to yield additional prognostic information. Applying different scoring systems to 288 CMML patients included in the Düsseldorf MDS Registry, we found that the International Prognostic Scoring System (IPSS) was not useful for defining risk groups in CMML, while the Spanish Score, the modified Bournemouth Score, the Düsseldorf Score, and probably the MDAP Score, identified patient groups differing significantly in survival. These scores should therefore be employed for clinical decision making and for risk stratification in the context of clinical trials.     

P15INK4b gene methylation and myelodysplastic syndromes

Myelodysplastic syndromes (MDS) are clonal disorders, which frequently undergo leukemic transformation. It was recently shown that the promoter of the p15INK4b but not the p16INK4a gene is frequently and selectively hypermethylated in MDS. The p15INK4b gene is a cyclin dependent kinase inhibitor gene, which is actively transcribed after TGFbeta exposure. Methylation of the p15INK4b gene is significantly correlated with blastic bone marrow involvement, and sequential analyses have shown that methylation increases with disease evolution toward AML. These data strongly suggest that p15INK4b gene methylation is a mechanism allowing leukemic cells to escape to inhibitory signals from the bone marrow environment, however the exact role of p15INK4b gene methylation in disruption of the signal mediated by TGFbeta remains to be investigated.     

Expression of the cell cycle regulators p14(ARF) and p16(INK4a) in chronic myeloid leukemia

Expression of p14(ARF) and p16(INK4a) tumor suppressor genes was investigated in 109 patients with chronic myeloid leukemia (CML). The p14(ARF) and p16(INK4a) mRNA levels were significantly low in patients in chronic phase (CP) at presentation and high in patients treated with interferon-alpha (IFN-alpha), especially in non-responders. A moderate overexpression of p14(ARF) with a normal expression of p16(INK4a) was observed in imatinib-resistant patients. Although protein expression did not consistently match mRNA levels, a role for the two cell cycle regulators in the IFN-alpha signaling pathway is suggested as well as a relation with the resistance to IFN-alpha or imatinib therapy.     

Aberrant methylation of p16INK4a and deletion of p15INK4b are frequent events in human esophageal cancer in Linxian, China

p16^INK4a and p15^INK4b genes, which encode two functionally relatedCDK inhibitors, recently emerged as candidate tumor suppressorgenes since they were both localized to 9p21, which frequentlyundergoes hemizygous and homozygous deletion in a variety oftumor types. To determine the mode of inactivation of thesetwo genes in human esophageal squamous cell carcinoma (ESCC),we performed multiple molecular analyses in 60 ESCC specimensfrom Linxian, China using DNA methylation assay, LOH analysis,deletion screening and SSCP-sequencing. We observed that p16^INK4ainactivation was predominantly associated with aberrant methylationin the CpG island of its promoter region, whereas p15^INK4b frequentlyhad homozygous deletions. Compared with aberrant methylation,which occurred in 17 of 34 cases, homozygous deletion of p16^INK4aand LOH at its nearby D9S942 microsatellite marker were observedat a much lower frequency (17%). Intragenic mutation in p16^INK4agene was rare. In contrast, homozygous deletion in p15^INK4band LOH at the nearby D9S171 marker were observed at frequenciesof 35 and 47%, respectively, and the two events were significantlyassociated with each other. On the other hand, aberrant methylationof p15^INK4b was relatively infrequent (6/34) and occurred concomitantlywith p16^INK4a methylation. Among the 60 cases, only four containeda continuous homozygous deletion spanning both p15^INK4b andp16^INK4a. Six cases were exclusively deleted at p16^INK4a and17 exclusively deleted at p15^INK4b. LOH at D9S942 and D9S171was also found to be mutually exclusive. Our results suggestthat the alteration mode at 9p21 was not uniform, and the twogenes were inactivated by distinct mechanisms. Altogether, 68%of the samples harbor at least one type of alteration in p16^INK4agene and 50% of the samples were altered in p15^INK4b gene, indicatingthat they are the frequent inactivating targets during ESCCdevelopment.     

Aberrant promoter methylation of p15 INK4b and p16 INK4a genes may contribute to the pathogenesis of multiple myeloma: a meta-analysis

We carried out the current meta-analysis aiming to comprehensively assess the potential role of p15 ^INK4b and p16 ^INK4a aberrant promoter methylation in the pathogenesis of multiple myeloma (MM). The MEDLINE (1966?~?2013), Cochrane Library (Issue 12, 2013), EMBASE (1980?~?2013), CINAHL (1982?~?2013), Web of Science (1945?~?2013), and Chinese Biomedical (CBM) (1982?~?2013) databases were searched without language restrictions. Meta-analyses were conducted using Stata software (Version 12.0, Stata Corporation, College Station, TX, USA). Odds ratios (ORs) and their 95 % confidence intervals (95 %CIs) were calculated. Thirteen clinical case–control studies, which enrolled a total of 465 MM patients and 180 healthy subjects, were included in the meta-analysis. The results of our meta-analysis demonstrated that the frequencies of p15 ^INK4b and p16 ^INK4a promoter methylation in cancer samples were significantly higher than in normal samples (p15 ^INK4b : OR?=?6.26, 95 %CI?=?3.87?~?10.12, P?p16 ^INK4a : OR?=?2.26, 95 %CI?=?1.22?~?4.20, P?p15 ^INK4b was significantly related with the risk of MM among both Caucasians and Asians (all P?p16 ^INK4a promoter methylation and the pathogenesis of MM among Asians (OR?=?5.17, 95 %CI?=?3.45?~?7.74, P?P?>?0.05). The current meta-analysis confirms and reinforces existing findings that p15 ^INK4b and p16 ^INK4a promoter methylation may be closely implicated in the pathogenesis of MM.     

Hypermethylation of p16(INK4a) and p15(INK4b) genes in non-small cell lung cancer.

Hypermethylation of CpG island is a common mechanism by which tumor suppressor genes are inactivated. The tumor suppressor genes p16(INK4a) and p15(INK4b) are important components of the cell cycles. We have studied the feasibility of detecting tumor-associated aberrant p16(INK4a) and p15(INK4b) methylation in non-small cell lung cancer (NSCLC) using methylation-specific PCR. We found a high frequency of hypermethylation of the p16(INK4a) gene in 17 of 45 cases of NSCLC. In this study, there was no difference between the clinicopathological features or overall survival of patients with and without p16(INK4a) methylation. On the other hand, p15(INK4b) promoter hypermethylation is rare (5/45) in lung cancer and occurs in association with p16(INK4a) methylation. The overall survival of patients with p15(INK4b) methylation was markedly shortened in this series. We also analyzed cells in bronchial washings, and p16(INK4a) methylation was detected in 4 of 17 cases of NSCLC. Moreover, 1 of 10 plasma samples from patients with NSCLC was positive for p16(INK4a) methylation. Our results suggest a possible prognostic role of p15(INK4b) methylation in NSCLC, and that the detection of aberrant p16(INK4a) methylation in both bronchial washings and plasma may be useful for cancer diagnosis.     

Efficacy of decitabine in the treatment of patients with chronic myelomonocytic leukemia (CMML)

Chronic myelomonocytic leukemia (CMML) characterized by cytopenias, bone marrow and peripheral blood cell dysplasia is notoriously hard to treat. Recent reclassification of CMML as a myelodysplastic/myeloproliferative (MDS/MPS) disease rather than a myelodysplastic syndrome (MDS) by the World Health Organisation (WHO) has led to a review of CMML patients treated with decitabine. Overall response rates (ORR) (complete response [CR]+partial response [PR]) in the subset of patients with CMML in one pivotal phase 3 trial (D-0007) and two phase 2 trials (PCH 95-11, PCH 97-19) decitabine were reviewed. For consistency across trials, all decitabine-treated patients were evaluated using the phase 2 response criteria (CR was defined by normocellular bone marrow with <5% blasts and normal Hgb, WBC, and platelet counts, and PR required 50% decrease in blast count, increases in Hgb by >1.5 mmol/L, WBC count by >1000, and platelet count by >50,000). A total of 31 patients diagnosed with CMML are included in this review. Similar demographics and disease characteristics were observed in all three studies, with an average age of 70.2 years and 71% of patients male. Baseline WBC of >20,000 were observed in 8/28 (29%) patients and baseline bone marrow blasts >5% in 11/28 (39%) patients. All clinical responses were centrally reviewed. The ORR was 25% (14% CR+11% PR). Hematologic improvement was observed in 11% of patients and stable disease in 39% of patients. The decitabine adverse event profile seen in CMML patients was similar to observations in other hematologic patient populations, with myelosuppression and related infectious complications. These data demonstrate encouraging activity for decitabine in CMML, and suggest that studies in other myeloproliferative diseases may be warranted.     

p15(INK4b) in bladder carcinomas: decreased expression in superficial tumours.

The p15 gene which encodes a cyclin-dependent kinase inhibitor, is located in the 9p21 chromosomal region that is frequently deleted in human bladder transitional cell carcinomas (TCCs). The aim of the present paper is to study the potential involvement of the p15 gene in the evolution of TCCs. p15 mRNA expression was investigated by semi-quantitative RT-PCR in a series of 75 TCCs, 13 bladder cell lines and 6 normal bladder urothelia by semi-quantitative RT-PCR. p15 was expressed in the normal urothelium but p15 mRNA levels were significantly decreased in 66% of the superficial (Ta-T1) TCCs (P = 0.0015). In contrast, in muscle-invasive (T2-T4) TCCs, p15 expression differed widely between samples. p16 mRNA levels were also studied and there was no correlation between p15 and p16 mRNA levels, thus indicating that the two genes were regulated independently. Lower p15 expression in superficial tumours did not reflect a switch from quiescence to proliferative activity as normal proliferative urothelial controls did not present decreased p15 mRNA levels relative to quiescent normal urothelia. We further investigated the mechanisms underlying p15 down regulation. Homozygous deletions of the p15 gene, also involving the contiguous p16 gene, were observed in 42% of the TCCs with decreased p15 expression. No hypermethylation at multiple methylation-sensitive restriction sites in the 5;-CpG island of p15 was encountered in the remaining tumours. Our data suggest that decreased expression of p15 may be an important step in early neoplastic transformation of the urothelium and that a mechanism other than homozygous deletions or hypermethylation, may be involved in p15 down regulation.     

Pericardial effusion in chronic myelomonocytic leukemia (CMML): a case report and review of the literature

Pericardial effusion is a rare but potentially life-threatening complication of chronic myelomonocytic leukemia. A case of a 70-year-old female with CMML is reported, who developed a severe pericardial effusion after an initially stable course of disease. She underwent pericardiocentesis and subsequent intracardial instillation of mitoxantrone with poor response. Subxiphoid pericardiotomy was successfully performed. Leukocytosis did not respond to hydroxyurea. Because of decreasing performance status of the patient systemic chemotherapy was not applied and the patient died within 2 weeks. A review of the literature shows that there is no effective treatment for pericardial effusion in CMML, and some common features may be noted.     

Prognostic factors and survival in chronic myelomonocytic leukaemia (CMML)

Ninety-seven cases of chronic myelomonocytic leukaemia (CMML) were examined retrospectively for survival and possible prognostic factors including age, total white cell count, peripheral blood and bone marrow monocyte counts, % double esterase (DE) positive cells in bone marrow and serum lysozyme. Age, absolute monocyte counts and serum lysozyme proved to be significant independent prognostic indicators but Cox model analyses showed serum lysozyme to be the most important factor whether taken as a continuous or discrete (two groups) variable. Twelve cases of second malignancy were found, including 2 cases of multiple myeloma, but this was not significantly greater than expected when compared with an age and sex matched group.     

The prognostic significance of p16INK4a/p14ARF and p15INK4b deletions in adult acute lymphoblastic leukemia.

Cytogenetic/molecular abnormalities significantly influence the prognosis of patients with acute leukemia. Recently, two genes, p16INK4a and p15INK4b, encoding two cyclin-dependent kinase inhibitor proteins of the INK4 family of Mr 15,000 and 16,000, respectively, have been localized to 9p21. Remarkably, the p16INK4a locus has been found to encode a second protein, p14ARF, known as p19ARF in mice, with a distinct reading frame. Like p16INK4a, p14ARF is involved in cell cycle regulation, blocking cells at the G1 restriction point through the activity of MDM-2 and p53. We studied bone marrow samples of 42 newly diagnosed and untreated patients with acute lymphoblastic leukemia for the incidence of deletions of p16INK4a/p14ARF and p15INK4b using Southern blot analysis and determined the clinical outcome with regard to complete remission (CR) duration, event-free survival, and overall survival. We found deletions of p16INK4a/p14ARF in 17 of 42 patients (40%), with homozygous deletions in 11 of 42 patients (26%) and hemizygous deletions in 6 of 42 patients (14%). The gene for p15INK4b was codeleted in most, but not all, cases and was never deleted without deletion of p16INK4a/ p14ARF. No correlation was observed between molecular studies and karyotype abnormalities as determined by conventional cytogenetics. Furthermore, no difference was found in the CR rate, CR duration, event-free survival, and overall survival in patients with homozygous gene deletions compared to patients with no deletions or loss of only one allele.