Abnormal methylation of the sex-determining region Y-box 17 (SOX17) promoter predicts poor prognosis in myelodysplastic syndrome.

BACKGROUND: The sex-determining region Y-box 17 (SOX17) is a member of the high mobility group (HMG) transcription factor family, which plays critical roles in the regulation of development and stem/precursor cell function. Recent evidence demonstrated that SOX17 acts as a tumor-suppressor gene, at least partly though repression of Wnt pathway activity. METHODS: Here we report that SOX17 methylation was detected in THP-1 and SKM-I cell lines and SOX17 mRNA levels was up-regulated by 5-aza-dC. To clarify the role of SOXI7 in MDS, methylation-specific PCR (MSP) was employed to examine the methylation status of SOX17 in 164 adult de novo MDS patients and 6 normal samples. RESULTS: We found that SOX17 methylation was presented in 58.5% (n = 96) of these patients and none of the normal samples. Methylation was correlated significantly with World Health Organization (WHO) subtypes and international prognostic scoring system (IPSS) risk group. Patients with advanced stages of WHO subtypes (69.6% vs. 44.4%, p = 0.001) and higher risk IPSS subgroups (69.8% vs. 48.8%, p = 0.010) exhibited a significantly higher frequency of SOX17 methylation. Though multivariate analysis indicated that SOX17 methylation status was not the independent factor that impacted overall survival (OS) (HR = 0.097), there were significant differences in marrow blast levels and the IPSS risk subgroups between patients with and without SOX17 methylation. CONCLUSIONS: These findings suggest that the hypermethylation of SOX17 promoter may be one of the early events in the development of MDS and predicts poor prognosis.

Methylation of Wnt antagonist genes: a useful prognostic marker for myelodysplastic syndrome.

Activation of the Wnt signaling pathway has been implicated in the pathogenesis of many tumors as well as in leukemia. However, its role in myelodysplastic syndrome (MDS) is unknown. In this study, we employed methylation-specific PCR to examine the methylation status of six Wnt antagonist genes in 144 MDS patients and in the MDS cell line SKM-1. We also used real-time PCR to examine the expression of Wnt antagonist genes and Wnt pathway genes in the SKM-1 cell line after treatment with 5-aza-2'-deoxycytidine. We found that methylation of the gene promoters of each of the six genes were observed in MDS patients at the following methylation frequencies: 41 % for sFRP1, 89.6 % for sFRP2, 43.1 % for sFRP4, 50.7 % for sFRP5, 44.4 % for DKK-1, and 69.4 % for DKK-3. In the SKM-1 cell line, the gene promoters sFRP1, sFRP2, sFRP5, DKK-1, and DKK-3 were methylated, while sFRP4 was not methylated. Treatment of the SKM-1 cell line with 5-aza-2'-deoxycytidine induced re-expression of methylated Wnt antagonists and inactivation of the Wnt pathway. Survival analysis showed that methylation status of sFRP1, sFRP4, and sFRP5 was associated with worse survival in MDS and sFRP5 methylation also predicted a high risk of leukemia evolution (P = 0.018). Our results indicate that epigenetic regulation of the Wnt pathway in MDS cell line, and the methylation status of Wnt antagonists predicts prognoses of MDS patients.

Methylation of the CpG island near SOX7 gene promoter is correlated with the poor prognosis of patients with myelodysplastic syndrome.

Myelodysplastic syndrome (MDS), characterized by the decreased production of blood cells, often progresses to acute myeloid leukemia (AML), a sign of poor prognosis of MDS. In AML, the Wnt/ß-catenin pathway is aberrantly activated, suggesting that the increased pathway activity may be correlated with the development and prognosis of MDS. SOX7 protein, encoded by the sex-determining region Y-box 7 (SOX7) gene, inhibits the activity of the Wnt/ß-catenin pathway. Because the DNA methylation can regulate the transcription of SOX7 gene, we used the methylation-specific PCR to investigate the methylation status of the CpG island in MDS patients to determine the potential correlation of the SOX7 methylation with the development and prognosis of MDS. We found that the CpG island of the SOX7 gene was methylated in 58.1% (97/167) of MDS patients, but not in any healthy control. Furthermore, the percentage of patients with the methylated CpG island of the SOX7 gene was significantly higher in patients at advanced stages of MDS than in the patients at early stages. The increased percentages of this SOX7 methylation were also correlated with age, marrow blast levels, and International Prognostic Scoring System (IPSS) risk. After prognostic analysis, we found that patients with the methylated CpG island of the SOX7 gene had shorter overall survival and cumulative survival than patients with unmethylated CpG island. Our findings suggest that the methylation of the CpG island of the SOX7 gene can be used as a predictive factor for the development and prognosis of MDS patients.

[Incidence of adult aplastic anemia in Shanghai, China].

OBJECTIVE: To survey the incidence of acquired adult aplastic anemia (AA) in Shanghai, China. Meanwhile, we compared it with the previous data from China in 1986 and other countries in order to explore the trends. METHODS: Newly diagnosed AA patients were registered in 6 districts (Jingan, Xuhui, Huangpu, Changning, Putuo, Yangpu) in Shanghai from 2004 to 2006. Then we calculated the crude and age-adjusted incidence of AA according to the population data from Shanghai Statistic Yearbook. RESULTS: There were 38 adult patients with acquired AA. The average crude incidence of AA was 0.33/100 000 from 2004 to 2006. The incidences per 100 000 persons per year were 0.40, 0.14 and 0.64 in 18 - 34, 35 - 59 and ≥ 60 years, respectively. The rate of severe AA was 0.17/100 000. CONCLUSION: The incidence of severe AA has no marked change, but the total rate is a little decreased compared with the data from China in 1986.

Therapy-related acute myeloid leukemia in a primary pulmonary leiomyosarcoma patient with skin metastasis.

Primary pulmonary leiomyosarcoma (LMS) is a very unusual tumor. Although LMS has well-known metastatic potential, cutaneous metastasis is a remarkably uncommon. Exposure to cytotoxic agents could lead to "therapy-related myeloid neoplasm" (t-MN). Starting from 2008, the World Health Organization (WHO) has adopted the term to cover the spectrum of malignant diseases previously known as therapy-related acute myeloid leukemia (t-AML), therapy-related myelodysplastic syndrome (t-MDS) and therapy-related myelodysplastic/myelo- proliferative neoplasm (t-MDS/MPN). We described the onset of t-MDS and progression to t-AML in one case diagnosed as primary pulmonary LMS with cutaneous metastasis. This patient achieved complete remission (CR) after three courses of IA regimen chemotherapy (idarubicin 5 mg/d, d 1-3; cytarabine 100 mg/d, d 1-5) and 1 course of HA chemotherapy regimen (homoharringtonine 3 mg/d, d 1-3; cytarabine 100 mg/d, d 1-7). This case presents the natural course of therapy-related neoplasm and provides therapeutic experience for t-AML.

[The value of flow cytometry for the differential diagnosis between refractory cytopenia with multiple dysplasia and aplastic anemia].

OBJECTIVE: To evaluate the value of flow cytometry (FCM) for the differential diagnosis between myelodysplasia (MDS) subtype refractory cytopenia with multiple dysplasia (RCMD) and aplastic anemia (AA). METHODS: The flow cytometric data of bone marrow samples from 168 cases of RCMD and 77 cases of AA were analyzed retrospectively in blind, and its results were compared with gold standard to evaluate its diagnosis values. RESULTS: The specificity of abnormal of single immunophenotype in the surface of granulocytes and myeloblasts was high (range 75.3%-100%), but the sensitivity was very low (range 5.4%-50%). In parallel tests, the sensitivity and specificity of the combination of CD34+ cells≥1%, myeloblasts≥3%, abnormal expression of CD117 in granulocytes and loss of CD13 in myeloblasts or increased intensity of CD33 in granulocytes were higher than other combinations. The sensitivity and specificity of above combination were more than 62% and 92%, respectively. In the scoring method, different score was given to 8 markers according to different diagnostic value, which were CD34+ cells≥1%, myeloblasts≥3%, abnormal expression of CD117 in granulocytes, loss of CD13 in myeloblasts, increased intensity of CD33 in granulocytes, loss of CD13 in granulocytes, loss of CD10 in granulocytes, and decreased SSC in granulocytes. The sensitivity and specificity were both high if we defined that the total score≥1.5 was RCMD and the score<1.5 was AA. CONCLUSIONS: The value of abnormal of single immunophenotype for differential diagnosis between RCMD and AA is low. Parallel tests can increase the diagnostic sensitivity obviously and not decrease the specificity. CD34+ cells≥1%, myeloblasts≥3% and abnormal expression of CD117 in granulocytes were the most important markers. The scoring method is precise to distinguish RCMD from AA.

Bone marrow blasts level predicts prognosis in patients with refractory cytopenia with multilineage dysplasia.

OBJECTIVES: Current prognostic models for myelodysplastic syndrome (MDS) do not consider the prognostic value of a bone marrow blast level that is <5%. Exploring the prognostic value of the International Prognostic Scoring System (IPSS) and a marrow blast level that is <5% may lead to better risk-adapted therapeutic strategies. METHODS: According to the World Health Organization classification, most of our patients (65.5%) fell into the new category 'refractory cytopenia with multilineage dysplasia' (RCMD). We evaluated the prognostic value of the IPSS in 435 adult patients with de novo MDS and in the 285 of them that had RCMD in a Chinese population. We also analyzed the prognostic value of bone marrow blast levels in patients with RCMD and in different IPSS risk groups. RESULTS: We found a significant difference in survival times between RCMD patients with a marrow blast level of 3.5% or higher vs. those with a blast level of <3.5%, with median survival times of 23.7 and 40.8 months, respectively. In addition, application of a marrow blast level cutoff of 3.5% in patients with RCMD could identify patients with a lower IPSS risk but with a potentially worse prognosis. Multivariate analysis showed marrow blast level (using 3.5% as the cutoff) to be an independent factor that impacted survival times of patients with RCMD. Furthermore, we also found that IPSS had strong prognostic value in Chinese RCMD population. CONCLUSION: In patients with RCMD, a higher percentage of marrow blasts was associated with a worse prognosis.

Retroperitoneoscopic distal pancreatectomy: a new surgical approach.

INTRODUCTION: The traditional laparoscopic surgery is difficult to deal with the deep lesions of the body and tail of the pancreas, which may damage the visceral organs of the abdominal cavity and cause abdominal adhesion and other related complications. AIM: This paper introduces the operation procedure of retroperitoneoscopy in pancreatic surgery, and evaluates its feasibility in clinical application. MATERIAL AND METHODS: Retrospective analysis was performed on patients with retroperitoneal pancreatectomy in our hospital. The anatomical features of the fascia, surgical plane composition and surgical pathway of the fascia of the retroperitoneoscopic pancreatectomy were observed during the operation, and the surgical safety and feasibility were analyzed. The following parameters were evaluated: operation time, blood loss, pancreatic fistula, postoperative gastro-intestinal recovery, hospital stay. RESULTS: All 3 patients had a smooth operation and no serious complications occurred. During retroperitoneal laparoscopic pancreatectomy, there is a vascularized plane between the posterior fascia of the pancreas and the prerenal fascia, which can avoid injury of the visceral organs and retroperitoneal vessels. The anterior renal fascia should be used as the posterior boundary of the safe separation plane. CONCLUSIONS: The surgical plane based on the anatomy of the fascia and interstitial dissection is the theoretical basis of modern surgery, which is safe, fast and effective. The inter-prerenal fascia plane is the correct and safe anatomical plane of posterior laparoscopic surgery.

Prognostic analysis of refractory anaemia in adult myelodysplastic syndromes.

BACKGROUND: Patients with myelodysplastic syndrome (MDS) display a very diverse pattern. In this study, we investigated prognostic factors and survival rate in adult patients with MDS refractory anaemia (MDS-RA) diagnosed according to French-American-British classification and evaluated the International Prognostic Scoring System (IPSS) for Chinese patients. METHODS: A multi-center study on diagnosis of MDS-RA was conducted to characterize the clinical features of Chinese MDS patients. The morphological criteria for the diagnosis of MDS-RA were first standardized. Clinical data of 307 MDS-RA patients collected from Shanghai, Suzhou and Beijing from 1995 to 2006 were analyzed using Kaplan-Meier curve, log rank and Cox regression model. RESULTS: The median age of 307 MDS-RA cases was 52 years. The frequency of 2 or 3 lineage cytopenias was 85.6%. Abnormal karyotype occurred in 35.7% of 235 patients. There were 165 cases (70.2%) in the good IPSS cytogenetic subgroup, 44 cases (18.7%) intermediate and 26 cases (11.1%) poor. IPSS showed 20 (8.5%) categorized as low risk, 195 cases (83.0%) as intermediate-I risk and 20 cases (8.5%) as intermediate-II risk. The 1-, 2-, 3-, 4- and 5-year survival rates were 90.8%, 85.7%, 82.9%, 74.9% and 71.2% respectively. Fifteen cases (4.9%) transformed to acute myeloid leukaemia (median time 15.9 months, range 3 - 102 months). Lower white blood cell count (< 1.5 x 10(9)/L), platelet count (< 30 x 10(9)/L) and cytogenetic abnormalities were independent prognostic factors by multivariate analysis, but age (= 65 years), IPSS cytogenetic subgroup and IPSS risk subgroup were not independent prognostic factors associated with survival time. CONCLUSIONS: Chinese patients were younger, and had lower incidence of cytogenetic abnormalities, more severe cytopenias but a more favourable prognosis than Western patients. The major prognostic factors were lower white blood cell count, lower platelet count and fewer abnormal karyotypes. The international prognostic scoring system risk group was not an independent prognostic factor for Chinese myelodysplastic syndrome patients with refractory anaemia patients.

[Assessment of the young rat model of visceral hypersensitivity by measuring electrical discharge of external oblique].

OBJECTIVE: To study the value of measuring electrical discharge of external oblique in assessment of young rat model of visceral hypersensitivity. METHODS: Eight-day-old neonatal Sprague-Dawley rats were randomly assigned to two groups: an experimental group and a control group (n=16 each). Rats in the experimental group were subjected to mechanical colorectal irritation daily for 7 consecutive days, while the rats in the control group did not received colorectal irritation treatment. On the 6th week of their lives, the spike amplitude of external oblique were measured to evaluate the bowel sensitivity. RESULTS: When the colorectal distention (CRD) pressure was 30 and 45 mmHg, the 95% confidence interval of the spike amplitude in the experimental group was significantly higher than that in the control group (P<0.01). When the CRD pressure were 60 and 75 mmHg, the 95% confidence interval of the spike amplitude in female rats was significantly higher than that in males (P<0.05). CONCLUSIONS: The electrical discharge of external oblique confirmed that chronic colorectal irritation in neonatal rats can result in a chronic visceral hypersensitivity in the juvenile stage, with gender differences. Electrophysiological assessment is a quantitative test, and can objectively reflect visceral sensibility of pain.

[A case-control study of risk factors for myelodysplastic syndromes].

OBJECTIVE: To determine the risk factors involved in myelodysplastic syndromes (MDS). METHODS: A 1:2 case-control study was conducted in 20 Shanghai' hospitals over a 3-year period, covering 266 "de novo" MDS cases corresponded to FAB criteria, and 532 age- and gender-matched controls from same hospitals with MDS cases. Subjects were all surveyed using the same standard questionnaire including histories of medications (Chloramphenicol, Sulfonamides, Meprobamate, Phenytoin, Colchicine, Cyclophosphamide, Propylthiouracil, Anti-TB medication, Tolbutamide, Primaquine and Chinese traditional herbs such as Bezoar, Angelica, Arsenic, Thunder cloud vine) at least 5 years prior to the onset of the disease, tumors, exposure to benzene, heavy metal, organic phosphates, pesticides, petrol/diesel, organic solvents, dye and hair dye products, radiation, house decorating, alcohol and smoking. RESULTS: Occupational exposure to benzene increased significantly the risk of MDS (OR: 8.52, 95% CI: 2.30 - 31.10). Living near high voltage power lines (100 m) increased significantly the risk of MDS (OR: 1.60, 95% CI: 1.10 - 2.32). House decorating (one year prior to the onset of the disease) increased significantly the risk of MDS (OR: 2.40, 95% CI: 1.38 - 4.14). Other investigated occupational poisons did not increase significantly the risk of MDS. Hair dye products, alcohol and smoking did not increase significantly the risk of MDS. CONCLUSION: Occupational exposure to benzene, living near high voltage power lines and house decorating are the risk factors of MDS.

[Genetic polymorphism of tumor necrosis factor-alpha in patients with chronic benzene poisoning].

OBJECTIVE: To study the relationship between genetic polymorphism of tumor necrosis factor-alpha (TNF-alpha, sites at -238 nt and -308 nt) and susceptibility to chronic benzene poisoning. METHOD: The polymorphism of TNF-alpha gene were detected by nested polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique from 50 benzene poisoning patients and 60 control individuals exposed to benzene. RESULTS: The frequency of the TNF-alpha-238G/G, A/G and A/A in controls (95%, 5% and 0%, respectively), were not significantly different from those in patients (92%, 6% and 2%, respectively, P > 0.05). The frequency of the TNF-alpha-308G/G, A/G and A/A in patients were 76%, 24% and 0% respectively, and that in control individuals were 93%, 7% and 0% respectively. Chi(2) test showed that frequency of TNF-alpha-308A/G in patients was significantly higher than that in controls (chi(2) = 6.22, P = 0.013). Logistic analysis showed that TNF-alpha-308A/G was the independent risk factor of benzene poisoning (OR = 5.3, P < 0.05). CONCLUSION: Individuals with TNF-alpha-308A/G gene polymorphism are susceptible to benzene poisoning.

[Establishment of Primary Adult MDS Nested Case-Control Study Cohort and Study of Risk Factors Associated with MDS Evolution to Leukemia].

OBJECTIVE: To establish a nested case-control study cohort in myelodysplastic syndrome (MDS) patients and investigate the clinical characteristics, WHO subtype and risk factors associated with MDS evolution to leukemia of this cohort. METHODS: All patients, ≥18 years of age, provided by 24 Shanghai hospitals with initial clinical findings consistent with a hematopoietic abnormality between June 2003 and April 2007, were the candidates for inclusion in this study. The blood and bone marrow samples of every patient should be provided at baseline. Diagnosis was made by incorporating morphologic, immunophenotypic, cytogenetic and molecular features according to WHO classification criteria. Cytogenetic analysis was performed using conventional G-banding karyotyping and fluorescence in situ hybridization (FISH) techniques. Cumulative risk of evolution was estimated by Kaplan-Meier method. Prognostic factors were evaluated by univariate Log-rank method and multivariate Cox proportional hazard models. RESULTS: A total of 435 patients were diagnosed as MDS. The median age of MDS onset was 58(18-90) years, with 248 male patients and 187 female patients (male: female 1.33: 1). The percentage of cases with refractory cytopenia with multilineage dysplasia (RCMD) was the highest (65.5%), while that of refraetory anemia (RA) (2.3%), refractory anenia with ring sideroblast (RARS) (1.1%) and 5q-syndrome (0.5%) was lower. Trisomy 8 (+8) was the most common chromosome abnormalities (71 cases, 12.7%). The mean follow-up time was 20.3 (4.2-57.1) months. Cases were patients with evolution by the end of follow-up, while controls were patients without evolution by that time. Case group included 41 patients and control group included 342 patients. Univariate analysis showed that the age, sex, WHO subtype, WBC count, absolute neutrophil count (ANC), IPSS cytogenetic subgroup, IPSS group and bone marrow blast percentage were significant risk factors for leukemia-free survival (LFS). Multivariate analysis of COX model showed that the age, sex, WHO subtype, IPSS cytogenetic subgroup and bone marrow blast were independent risk factors for LFS. CONCLUSION: A nested case-control study cohort of MDS patients is established. The clinical characteristics and WHO subtype of MDS patients in Chinese Shanghai are different from that in Western countries. The independent risk factors for MDS evolution are age, sex, WHO subtype, IPSS cytogenetic subgroup and bone marrow blast percentage.

Prognostic value of clinical characteristics and immunophenotypic biomarkers in 115 patients with primary central nervous system lymphoma.

BACKGROUND: Clinical outcome in patients with primary central nervous lymphoma (PCNSL) is variable and poorly predictable. This study investigated the association of clinical features and immune markers with prognosis of patients with PCNSL. METHODS: One hundred and fifteen newly diagnosed PCNSL patients at the study institution were considered eligible for this study. Clinical characteristics and biochemical assay data were collected. Immunohistochemical staining of Cyclin D3, Cyclin E, Foxp1, and LMO2 were performed. All cases were followed-up regularly. RESULTS: The common sites of involvement were frontal lobe (54.8%) and thalamus (16.5%). Diffuse large B-cell lymphoma composed of 96.5% of the cases. The median overall survival was 22 (4 - 41) months, and the 5-year survival rate was 22.8%. Age > 65 years, serum globulin > 40 g/L, large size of tumor, lymphocyte count ≥ 1 × 10(9)/L, and expression of Cyclin D3 and Cyclin E were associated with poor prognosis of PCNSL. Expressions of Foxp1, LMO2, and CD44 were not related to the survival. Expression of Cyclin E, large tumor size, and high serum globulin were independent prognostic factors for PCNSL. CONCLUSIONS: PCNSL prognosis is relatively poor. Age, high tumor burden, higher lymphocyte count, expression of Cyclin D3, and Cyclin E are inferior prognostic factors for PCNSL.

[Expression of FOXP1 and cyclinE in primary central nervous system lymphoma and its significance].

OBJECTIVE: To evaluate the value of FOXP1 and Cyclin E gene in primary central nervous system lymphoma(PCNSL) of immunocompetent patients on prognostic significance. METHODS: Clinical data of 71 patients with newly diagnosed PCNSL from 2002 to 2007 was analyzed retrospectively. Immunohistochemistry method (HRP-EnVision(TM)) was performed to observe the expression of FOXP1 and Cyclin E gene in tumor tissue samples. The survival was analyzed by Kaplan-Meier survival curve, survival factors analysis by the Log-rank test and COX proportional hazards regression model. RESULTS: FOXP1 positive was observed in 35 of 51 patients (68.63%) and Cyclin E staining was present in 29 of 50 cases (58.00%). FOXP1(+) patients had a shorter overall survival (OS) than FOXP1(-) ones. 2-year OS rate in FOXP1(+) and FOXP1(-) patients were 23.33% and 73.56%, respectively(P = 0.0015). Cyclin E(+) patients had a shorter overall survival(OS) than cyclinE(-) ones. 2-year OS rate in Cyclin E(+) and Cyclin E(-) patients were 17.56% and 69.76%, respectively (P = 0.0017). Multivariate analysis showed that Cyclin E expression was an independent prognostic factor for shorter OS (P = 0.048). FOXP1 expression might be an important prognostic factor for shorter OS (P = 0.065). CONCLUSION: Cyclin E expression is an independent prognostic factor and FOXP1 expression is a possible prognostic factor for poor clinical outcome in patients with PCNSL.

Multicentre hospital-based case-control study of diffuse large B-cell lymphoma in Shanghai, China.

BACKGROUND: Several potential risk factors have been identified for diffuse large B-cell lymphoma (DLBCL); however, epidemiological studies investigating the association between these risk factors and DLBCL have yielded inconsistent results. OBJECTIVES: To investigate potential medical, lifestyle, and environmental risk factors of DLBCL in Shanghai, China through a hospital-based case-control study. METHOD: One-hundred- and-forty-seven newly diagnosed DLBCL patients and 294 sex- and age-matched controls were recruited from 11 hospitals in Shanghai between 2003 and 2007. A standardized structured questionnaire was used to obtain patient data on demographics, medical history, family history, lifestyle, and environmental exposures. Conditional logistic regression models were used to estimate odds ratios (ORs), with 95% confidence intervals (CIs), for risk associated with each data category. RESULTS: History of tuberculosis (TB) infection and "living on a farm" were positively associated with DLBCL (TB: OR=3.05, 95% CI: 1.19-7.80; farm: OR=1.82, 95% CI: 1.21-2.73). In contrast, taking traditional Chinese medicine was negatively associated with DLBCL (OR=0.36, 95% CI: 0.14- 0.89). No significant correlation with DLBCL risk was found for any of the other potential risk factors (p>0.05), including but not limited to hair dyes, alcohol drinking, smoking, and home/workplace renovation within one year. CONCLUSIONS: Consistent with results from previous studies in other DLBCL case populations, traditional Chinese medicine appeared to have a direct or indirect protective effect against DLBCL. However, this study also identified a possible predisposition for DLBCL in TB sufferers and farmers.

[Establishment of human homoharringtonine-resistant SKM-1 cell line and its biological characteristics].

OBJECTIVE: To establish a homoharringtonine (HHT)-resistant SKM-1 cell line and explore its biologic characteristics and mechanisms for drug resistance. METHODS: The HHT-resistant SKM-1 cell line was established by repeatedly exposing the cells to comparatively large doses of HHT with a short-time duration, and gradually elevating the drug concentration to an endurable level. The morphology of the resistant and parental cell lines was observed through optical microscope. The MTT assay was used to determine the doubling time and the resistance index to draw growth curve. The immunophenotype, cell cycle distribution and DNR accumulation between SKM-1 and SKM-1/HHT were analyzed by flow cytometry, and the karyotypes by R-banding. Semi-quantitative real-time PCR was performed to evaluate the expression levels of mdr1, MRP and topo-IIa. RESULTS: The HHT-resistant cell line SKM-1/HHT was eventually established following 7-month drug induction. Both the resistant and the parental cell lines were similar with regard to morphology and immunophenotype. The karyotypes of the former was more complicated with differences located in chromosome 20, X, 4, 5, 9 and 11. The resistant cell line had more G(1) phase cells (64.04% vs 41.91%), less S phase cells (34.92% vs 53.53%), and less G(2) phase cells (1.04% vs 4.56%) compared with the parental cell line. The SKM-1/HHT cell line showed significant drug resistance to HHT, VCR, DNR and etoposide, the resistance indices of HHT, VCR, DNR and etoposide were 17.94, 8.75, 5.99 and 13.76 respectively. DNR accumulation was impaired in SKM-1/HHT cell line as less fluorescence of DNR (698 ± 36 vs 858 ± 54). The expression of mdr1 increased dramatically in the resistant cell line, its 2(-ΔCt) value was 20.1 higher than that of the parental cell line \[(3.42 ± 0.46)×10(-2) vs (0.17 ± 0.01)×10(-2), P < 0.05\], while MRP also increased in the resistant by 3.56 folds \[(4.77 ± 0.87)×10(-3) vs (1.34 ± 0.56)×10(-3), P < 0.05\]; However there was a slightly decrease of topo-IIa, the ratio of the resistant to the parental calculated by their 2(-ΔCt) values was 0.619:1 \[(1.91 ± 0.30)×10(-4) vs (3.08 ± 0.21)×10(-4), P < 0.05\]. CONCLUSION: A HHT-resistant cell line SKM-1/HHT was established. The prominent overexpression of mdr1 may be the main cause for multidrug resistance.

ID4 methylation predicts high risk of leukemic transformation in patients with myelodysplastic syndrome.

Epigenetic gene silencing due to promoter methylation is observed in human cancers like acute myeloid leukemia (AML). Little is known about aberrant methylation in myelodysplastic syndrome (MDS), a heterogeneous clonal stem cell disorder with a approximately 30% risk of transformation into secondary AML. Recent evidence demonstrated that ID4, a negative regulator of transcription, may act as a tumor-suppressor gene. To clarify the role of ID4 in MDS, we employed methylation-specific PCR (MSP) to examine the methylation status of ID4 in 144 adult de novo MDS patients. We found that ID4 methylation was present in 35.4% (n=51) of these MDS patients and methylaiton was correlated significantly with World Health Organization (WHO) subtypes and International Prognostic Scoring System (IPSS) risk groups. Patients with advanced stages of WHO subtypes (45.8% vs. 21.3%, P=0.002) and higher risk IPSS subgroups (45.7% vs. 26.0%, P=0.014) exhibited a significantly higher frequency of ID4 methylation. The median survival of patients with ID4 methylation was shorter than patients without ID4 methylation (12.2 months vs. 26.9 months, P=0.005). Multivariate analysis indicated that ID4 methylation status was the independent factor that impacted leukemia-free survival (LFS). Disease in patients with ID4 methylation progressed more rapidly than those without ID4 methylation (P=0.047, HR=2.11). Our results suggest that ID4 may be a therapeutic target in MDS.