Effective Management of Polycythemia Vera With Ropeginterferon Alfa-2b Treatment.

Background: Polycythemia vera (PV) is a myeloproliferative neoplasm. Ropeginterferon alfa-2b is a new-generation polyethylene glycol-conjugated proline-interferon. It is approved for the treatment of PV at a starting dose of 100 µg (50 µg for patients receiving hydroxyurea (HU)) and dose titrations up to 500 µg by 50 µg increments. The study was aimed at assessing its efficacy and safety at a higher starting dose and simpler intra-patient dose escalation. Methods: Forty-nine patients with PV having HU intolerance from major hospitals in China were treated biweekly with an initial dose of 250 µg, followed by 350 µg and 500 µg thereafter if tolerated. Complete hematological response (CHR) was assessed every 12 weeks based on the European LeukemiaNet criteria. The primary endpoint was the CHR rate at week 24. The secondary endpoints included CHR rates at weeks 12, 36 and 52, changes of JAK2V617F allelic burden, time to first CHR, and safety assessments. Results: The CHR rates were 61.2%, 69.4% and 71.4% at weeks 24, 36, and 52, respectively. Mean allele burden of the driver mutation JAK2V617F declined from 58.5% at baseline to 30.1% at 52 weeks. Both CHR and JAK2V617F allele burden reduction showed consistent increases over the 52 weeks of the treatment. Twenty-nine patients (63.0%) achieved partial molecular response (PMR) and two achieved complete molecular response (CMR). The time to CHR was rapid and median time was 5.6 months according to central lab results. The CHRs were durable and median CHR duration time was not reached at week 52. Mean spleen index reduced from 55.6 cm2 at baseline to 50.2 cm2 at week 52. Adverse events (AEs) were mostly mild or moderate. Most common AEs were reversible alanine aminotransferase and aspartate aminotransferase increases, which were not associated with significant elevations in bilirubin levels or jaundice. There were no grade 4 or 5 AEs. Grade 3 AEs were reversible and manageable. Only one AE led to discontinuation. No incidence of thromboembolic events was observed. Conclusion: The 250-350-500 µg dosing regimen was well tolerated and effectively induced CHR and MR and managed spleen size increase. Our findings demonstrate that ropeginterferon alfa-2b at this dosing regimen can provide an effective management of PV and support using this dosing regimen as a treatment option.

[Analysis of Early-Death and Factors Affecting Prognosis of Patients with Acute Promyelocytic Leukemia].

OBJECTIVE: To investigate the factors affecting the early-death, overall survival (OS) and relapse-free survival (RFS) of acute promyelocytic leukemia (APL) patients. METHODS: The clinical and laboratorial charachteristics of 176 APL patients in our center were analyzed retrospectively during January 2002 to Mar 2016. The risk factors of early death and factors affecting OS and RFS of patients were analyzed. RESULTS: Among total of 176 patients, early death occured in 10 patients. Multivariate analysis showed both age ≥60 years and fibrinogen<1.5 g/L (HR=6.4, 95%CI 1.4-28.2) (P=0.015), (HR=12.2, 95%CI 1.5-102.8) (P=0.021), respectively were the independent risk factors for the early death during the induction therapy. Among 154 patients with full follow-up data (median follow-up time was 101(2-262) months), the estimated 5-year OS and RFS rate were (98± 1)% and (77± 4)%, respectively. Cox regression analysis showed relapse during treatment as well as initial WBC count≥30× 109/L were independent prognostic indicators for OS. Accompanied psoriasis indicated higher relapse rate of APL(HR=4.8, 95%CI 1.8-12.5)(P=0.002), while the low-risk APL indicated lower relapse rate (HR=0.4, 95%CI 0.2-0.99)(P=0.048). CONCLUSION: Importance should be attached to the early-death events in elder and low-fibrinogen APL patients. As for patients with psoriasis or non low-risk group, emphasizing the intensified dynamic supervision during the treatment helps to detect the early-relapse events. For relapsed patients and patients with ≥30× 109/L WBC count, seeking more optimized therapy strategy seems allow this cohorts to get better prognosis.

[Role of Aberrant Splicing in Pathogenesis of Myelodysplastic Syndromes-Review].

The spectrum of genetic abnormalities in myelodysplastic syndromes(MDS) has been revealed by high-throughput sequencing. However, the functional role of these mutations in occurrence and development of MDS was not delineated. The mutations in splicing factors have been identified as the commonest gene mutations in MDS. Recently, it was reported that the mutated or dysregulated splicing factors, including SF3B1, SRSF2 and U2AF1, attribute to aberrant mRNA splicing, which leads to hematopoietic dysfunction and drives MDS. These findings will be of great help in searching for candidate therapeutic targets in mis-splicing pathway in MDS. In this review the role of aberrant splicing in pathogenesis of MDS is summarized briefly.

Long-term outcomes of imatinib in patients with FIP1L1/ PDGFRA associated chronic eosinophilic leukemia: experience of a single center in China.

BACKGROUND: The FIP1L1/PDGFRA (F/P) fusion gene is the most common clonal genetic abnormality of chronic eosinophilic leukemia (CEL). Tyrosine kinase inhibitors (TKI), such as imatinib, have been demonstrated to be effective therapies for F/P mutated disease. The aim of this study was to analyze the treatment response and long term prognosis in patients with F/P mutated CEL. METHODS: The clinical features and treatment responses of 33 consecutive patients with F/P mutated CEL between August 2006 and October 2014 were analyzed. The 33 cases received imatinib therapy at an initial dose of 100 mg/day (30 patients) or 200 mg/day (3 patients); the maintenance dose depended on the response condition and patient willingness. Through the follow up, the molecular responses were regularly monitored. RESULTS: With a median follow up of 64 months, 94% of the 33 patients with F/P mutated CEL achieved a complete hematologic remission (CHR), and 97% achieved a complete molecular remission (CMR) after a median of 3 (1.5-12) months. Twenty-four cases received maintenance therapy, with a median CMR duration of 43 (5-88) months. Imatinib therapy was discontinued in 8 cases, including 4 cases who experienced relapse, and 4 patients who maintained CHR or CMR after discontinuing therapy with a median time of 47 (2-74) months. One case exhibited primary resistance with a PDGFRA T674I mutation. CONCLUSIONS: F/P mutated CEL has an excellent long-term prognosis following imatinib therapy. A 100 mg daily dose of imatinib is sufficient to induce remission, and a single 100 mg weekly dose maintains a durable remission. A subgroup of patients may maintain a durable remission after discontinuing therapy with a CMR.

[Relationship between polymorphisms of tumor necrosis factor alpha gene and primary myelodysplastic syndromes].

OBJECTIVE: To investigate the association of single nucleus polymorphisms(SNP)of tumor necrosis factor alpha (TNF-α) gene (-308 G>A and -238 G>A genotypes) with susceptibility to primary myelodysplastic syndromes (MDS). METHODS: Two SNPs (TNF-α-308 G>A,TNF-α-238 G>A) of TNF-α gene were detected by Taqman probes in 341 MDS patients and 365 unrelated-healthy controls. RESULTS: Compared to healthy controls, the frequency of TNF-α-308 AA+AG genotype and A allele increased (18% vs 10%, P=0.015, 9% vs 5%, P=0.021, respectively) in refractory cytopenia with multilineage dysplasia (RCMD) patients. There was no correlation of TNF-α-308 G>A genotype and allele frequency between MDS and controls. No difference in the genotype and allele frequency of TNF-α-238 G>A were found between controls and MDS or the subtypes of MDS (P>0.05). We did not find any linkage between plasma level of TNF-α and TNF-α-308 G>A or TNF-α-238 G>A genotype. Statistic differences were observed between platelet count[58(1-611)×109/L vs 90(7-352)×109/L]and bone marrow blasts in MDS patients carrying TNF-α-308 G>A GG and AA+AG genotype (P=0.024, 0.019, respectively). CONCLUSION: TNF-α-308 G>A polymorphism was correlated with susceptibility to MDS-RCMD.

[The effect of transmission electron microscopy on diagnosis of acute myeloid leukemia].

OBJECTIVE: To analyze coincidence rate of acute myeloid leukemia (AML) sub-typing between transmission electron microscopy (TEM) and clinical discharge diagnosis. METHODS: Reviewing sub-typing results of TEM, light microscopy, flow cytometric analyzing, molecular biological detection and karyotype in 793 AML cases, comparing their coincidence rates with discharge diagnosis to reveal advantages of AML sub-typing by TEM. RESULTS: General coincidence rates of TEM, light microscopy, flow cytometric analyzing, molecular biological detection and karyotype on AML sub-typing were 63%, 59%, 52%, 47%, 26% and 23% respectively, and clinical coincidence rates of TEM on M1, M2a, M4 and M5, M6, M7, t (8; 21) and t (15; 17) were 39%, 34%, 17%, 74%, 50%, 73%, 87% and 89% respectively. CONCLUSION: TEM has a higher coincidence rate in general AML sub-typing, especially strong screenings on t (15; 17), t (8; 21), M7, M5 and M6, but lower coincidence rates on M1, M2a and M4 sub-typing than other methods.

[The relevance between quantitative and type of chromosomal abnormality and leukemia transformation in myelodysplastic syndrome].

OBJECTIVE: To investigate leukemia transformation rate in myelodysplastic syndrome (MDS) and the relationship with quantitative and type of chromosomal abnormality. METHODS: This study retrospectively analyzed and rediagnosed 138 MDS patients with complete data, investigated the rate and time of leukemia transformation, and analyzed characteristics of chromosome karyotype of de novo patients. RESULTS: 29 (21.01%) of 138 patients transformed into leukemia, the rate and the median time of leukemia transformation were 21.01% and 8 (3-24) months, respectively, among which, the rate of leukemia transformation in normal karyotype, abnormal karyotype analysis of ≤5 mitotic cells, and >5 mitotic cells in split phase groups were 6.2%, 23.8% and 38.5%, respectively, and median time of which were 17(13-22), 13(5-23), and 7(3-10) months, respectively. Increased trend of leukemia conversion rate along with increased quantity of chromosomal abnormality was observed (χ²=14.185, P＜0.01). Leukemia transformation time negatively correlated with quantity grade of abnormal karyotype (r=-0.631, P<0.01), The leukemia transformation rates in monosomy 7/del 7q, trisomy 8, trisomy 11, complex karyotype and normal karyotype groups were 65.0%, 50.0%, 30.8% and 28.6%, being significantly different (χ²=21.555, P<0.01). Leukemia transformation rate of complex karyotype and monosomy 7/del 7 q was slightly higher than of trisomy 8 and trisomy 11, but both of them were significantly higher than of normal karyotype (χ²=8.054, P=0.005). There were no leukemia transformation cases in del 5q, del 20q, monosomy Y, and trisomy 21 group. CONCLUSION: With or without abnormal chromosome karyotype, quantity and types of abnormal karyotype had important clinical value to predict leukemia transformation in patients with MDS.

Distinct clinical and experimental characteristics in the patients younger than 60 years old with myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) mainly occur in elderly individuals in Western countries. However, MDS is commonly found in young individuals (<60 years) in Asia. The reason for the high incidence in younger individuals is still unclear, and the differences in disease features between young and elderly patients with MDS have been not well recognized. To explore these issues, in this study, we analyzed the clinical and experimental characteristics of MDS in the patients younger and older than 60 years old and characterized the potential age-associated differences. The results showed that over half of the patients with MDS (61.9%) were younger than 60 years old upon the first diagnosis. The younger patients were more likely to be female, who have lower risk and less advanced MDS. The occurrence of trisomy 8 and bone marrow failure were more frequent in the younger patients than the older ones. The marrow CD34+ cells in the younger patients showed lower proliferation and higher apoptosis in comparison with that in the older ones. Obvious amplification of T cells and low CFU formation could be found in the younger patients. CFU formation was significantly increased in the younger patients after the removal of activated T cells. In addition, the younger patients had a lower frequency of p15(INK4B) methylation, longer survival expectancy and less AML transformation. In summary, the younger patients with MDS in China may show more benign disease features than the older ones. Enhanced immunological response may be involved in the pathogenesis of MDS in the patients younger than 60 years.

[Characteristics of cytogenetics and molecular biology in patients with eosinophilia].

The aim of study is to explore the characteristics of cytogenetics and molecular biology in patients with eosinophilia. Bone marrow samples from 79 cases of eosinophilia (AEoC ≥ 1.5×10(9)/L) were detected for PDGFRA/B and FGFR1 gene rearrangement by fluorescence in situ hybridization and reverse transcription polymerase chain reaction (RT-PCR). Forty-four samples were detected for T cell receptor (TCR) clonal rearrangement by PCR. The results showed that among 76 cases the FIP1L1/PDGFRA (F/P) fusion gene was detected in 19 cases, the CHIC2 deletion was detected in 19 cases, the PDGFRA rearrangement was detected in 4 cases, and no FIP1L1 rearrangement was detected. According to the 2008 WHO classification, diagnosis were revised as myeloid neoplasms with PDGFRA/B rearrangement in 20 (42%) of 48 patients and 5 (83%) of 6 patients with hypereosinophilia syndrome (HES) or chronic eosinophilic leukemia (CEL), respectively. The diagnosis in (17%) of 6 patients with CEL was revised as chronic eosinophilic leukemia, not otherwise as specified (CEL-NOS). Clonal cytogenetic abnormalities were detected in 1 case of CEL-NOS and 3 cases with PDGFRB rearrangement. Karyotypic abnormalities involved in chromosome 4q12 were not detected in all of the 21 cases with PDGFRA rearrangement. The clonal TCR gene rearrangement were detected in 33% (5/15), 40% (6/15), and 36% (5/14) cases with PDGFRA/B rearrangement, HES, or secondary eosinophilia, respectively. There was no statistical difference in incidence rate among 3 subgroups. It is concluded that PDGFRA/B rearrangement can be detected in many cases of HES or CEL. Interphase FISH and PCR testing can enhance the diagnostic rate of myeloid neoplasms with PDGFRA/B rearrangement.

[Analysis of in vitro characteristics of colony-forming cells in myelodysplastic syndrome and comparison with that in non-severe aplastic anemia].

OBJECTIVE: To investigate in vitro characteristics of colony-forming cells (CFC) in patients with myelodysplastic syndrome (MDS) and to compare that in patients with non-severe aplastic anemia (NSAA). METHODS: Data of in vitro CFC and correlation with other related laboratory tests in 155 newly diagnosed MDS patients were analyzed retrospectively, and to compare with data of in vitro CFC in 122 newly diagnosed NSAA patients. RESULTS: Median number of burst-forming units-erythroid (BFU-E) was 9 (0 - 157)/10(5) bone marrow mononuclear cells (BMMNC), colony forming unit-erythroid (CFU-E) 30 (0 - 425)/10(5)BMMNC and colony forming unit-granulocytes/macrophages (CFU-GM) 14 (0 - 125)/10(5)BMMNC in patients with MDS, being significantly lower than those in healthy control; number of BFU-E and/or CFU-E was lower than the lower limit of normal control in 66 cases (42.6%), CFU-GM lower in 3 cases (1.9%) and BFU-E and/or CFU-E with CFU-GM lower in 70 cases (45.2%). Cluster/CFU-GM ratio was significantly lower in low blast group (MDS < 5% blast in bone marrow smear) than that in high blast group (MDS ≥ 5% blast) (0.65 vs 1.0, P = 0.049). In all MDS patients, cluster had positive correlation with each type of CFC (r = 0.415, 0.338, 0.642 for BFU-E, CFU-E, CFU-GM, respectively, P = 0.000), but had negative correlation with neutrophil alkaline phosphatase (N-ALP) positive rate and scores (r(rate) = -0.315, P = 0.001 and r(scores) = -0.257, P = 0.006). The median number of each type of CFC was significantly higher in MDS group than that in NSAA group (BFU-E 9 vs 5/10(5)BMMNC, P = 0.017; CFU-E 30 vs 19.5/10(5)BMMNC, P = 0.023; CFU-GM 14 vs 10/10(5)BMMNC, P = 0.003, respectively). Positive correlation between BFU-E and CFU-E were revealed in both MDS and NSAA group (r(MDS) = 0.712, P = 0.000 and r(NSAA) = 0.757, P = 0.000), with a lower correlation coefficient in MDS (P < 0.05). CONCLUSIONS: Early onset MDS present markedly decreased hematopoietic progenitor cells (HPC), and particularly in erythroid progenitors extensively and severely. The number of BFU-E, CFU-E and CFU-GM can reflect HPC number in vivo but not stand for normal hematopoietic clones, the number of clusters represent pathologic HPC clones but not exactly leukemic blasts.

[Clinical features and survival analysis in primary myelodysplastic syndromes patients with immunological abnormalities].

OBJECTIVE: To analyze the clinical features and survival time in primary myelodysplastic syndromes (MDS) patients accompanied with immunological abnormalities. METHODS: The clinical information, laboratory findings and survival time in 194 untreated primary MDS patients with complete immunological laboratory tests or a past history of autoimmune disease were analyzed retrospectively. RESULTS: There were 37/194 cases (19.07%) with autoimmune abnormalities, including 16/194 (8.25%) with autoimmune disease and 21/194 asymptomatic cases (10.82%) with serologic immunological abnormalities only. There was significant differences in the distribution of age < 60 years old, female, CD4(+)T-cell/CD8(+)T-cell ration < 1 and trisomy 8 (P < 0.05) between the cases with autoimmune disease and without autoimmune abnormalities. The former had a higher 2-year OS, but there was no significance (P = 0.065). There was no significant differences in the distribution of age, MDS-subtype, IPSS risk groups, haemoglobin, absolute neutrophil count, platelets count, the severity of anemia and neutropenia, high level of serologic TNF, chromosomal abnormalities, cytogenetic risk groups and bone marrow cellularity (P > 0.05). CONCLUSION: MDS patients with autoimmune disease are mainly female and younger than 60 years old, with high proportion of trisomy 8 and better prognosis.

[A preliminary study of prognostic value of thrombocytopenia in patients with primary myelodysplastic syndromes].

OBJECTIVE: To investigate the prognostic value of thrombocytopenia in patients with primary myelodysplastic syndromes (MDS). METHODS: Four hundred and nineteen primary MDS patients were retrospectively analyzed. Kaplan-Meier method, Log-rank test and COX regression model were used to evaluate factors that influence the prognosis. RESULTS: Two hundred and fifty-six cases (61.1%) had thrombocytopenia (PLT < 100×10(9)/L), one hundred and three cases (24.6%) had severe thrombocytopenia (PLT < 30×10(9)/L). Overall survival (OS) tended to shorten along with the decreasing of platelet count. Univariate analysis indicated that PL < 30×10(9)/L, MCV ≤ 95 fl, LDH ≥ 300 U/L, lymphocyte-like micromegakaryocyte, nucleated RBC PAS positive, IPSS cytogenetic intermediate- and poor-risk were all related with poor prognosis. Moreover, the prognosis of patients with RCMD, RAEB-Ior RAEB-IIwas poorer than that of the other subgroups. Among these parameters, PLT < 30×10(9)/L, MCV ≤ 95 fl, IPSS cytogenetic intermediate- and poor-risk group and RCMD, RAEB-I and RAEB-II had independent prognostic significance in multivariate analysis. Modified WPSS prognostic model was proposed by adopting PLT, MCV, chromosomal karyotype and WHO classification. The OS of patients with low risk, intermediate-1 risk, intermediate-2 risk and high risk were 59, 28, 14 and 4 months, respectively, and there was a statistically significant difference between the groups (P < 0.05). CONCLUSION: Severe thrombocytopenia indicated unfavorable prognosis, in combination with MCV, chromosomal karyotype and WHO classification, a modified WPSS prognostic model was proposed and worked well for prognostic indication in patients with MDS.

[Clinical efficacies and safety of HAG regimen for patients with high-risk myelodysplastic syndromes: a multicentre study].

OBJECTIVE: To evaluate the efficacies and toxicity of HAG (HHT + Ara-C + G-CSF) regimen in patients with high-risk myelodysplastic syndromes (MDS). METHODS: A total of 97 patients with high-risk MDS received HAG regimen as the induction therapy. RESULTS: The complete remission (CR) rate of all the patients was 52.3% (45/86). The overall response (OR) rate was 66.3% (57/86). The early mortality rate was 9.3% (9/97). There was no significant difference in CR rate and OR rate between the patients aged ≥ 60 and those < 60. The OR rate was 29/34, 9/12 and 6/13 in patients with favorable karyotype, intermediate karyotype and unfavorable karyotype respectively. The OR rate was higher in patients with favorable karyotype than those with unfavorable karyotype (P = 0.038). The major adverse effect was infection. CONCLUSION: HAG regimen provides higher CR rate and OR rate for patients with high-risk MDS.

[Determination of hematopoietic clonality by detection of multiple X-linked gene exonic polymorphic loci using transcription-based clonality assays].

OBJECTIVE: To explore the frequencies of heterozygosity in X-linked G6PD, P55, BTK, and FHL-1 gene exonic polymorphic loci among Chinese females and the value of determination of hematopoietic clonality by detection of these X-chromosome exonic polymorphisms based on X-chromosome inactivation patterns (XCIP)-transcription-based clonality assays (TCA). METHODS: Genomic DNA was extracted from peripheral blood of 446 Chinese healthy females. Allele-specific PCR (ASPCR) or PCR-restriction enzyme digestion method was applied for detecting G6PD, P55, BTK and FHL-1 polymorphisms. Those heterozygotic loci were used as markers to examine the hematopoietic clonality of bone marrow mononuclear cells by TCA from essential thrombocythemia (ET) patients with JAK2V617F mutation and myelodysplastic syndrome (MDS) patients with abnormal karyotype. RESULTS: Among the total 446 genomic DNA samples, the frequencies of heterozygosity in G6PD, P55, BTK and FHL-1 loci were 12.8%, 29.4%, 52.0% and 46.4%, respectively. About 81.4% of females were heterozygous at one or more loci. All 10 ET patients with JAK2V617F mutation and 2 MDS patients with abnormal karyotype, which were heterozygotic in either locus, had monoclonal/oligoclonal hematopoiesis. CONCLUSION: Clonality detection based on X chromosome inactivation patterns-transcription based clonality assays is applicable to about 80% of Chinese females.

[Analysis of clinical features and prognosis of primary myelodysplastic syndromes with myelofibrosis patients].

OBJECTIVE: To analyze the clinical features and prognosis of the primary myelodysplastic syndrome with myelofibrosis (MDS-MF) patients and to improve the cognition of MDS-MF. METHODS: Four hundred and sixty-six primary MDS patients with bone marrow (BM) biopsy were divided into two groups according to whether BM associated with fibrosis, the clinical features and prognosis of the two groups were analyzed retrospectively. RESULTS: 167 (35.8%) MDS cases revealed myelofibrosis, of which MF-1 123 cases (26.4%), MF-2 40 cases (8.6%), MF-3 4 cases (0.9%). The proportion of hepatosplenomegaly in MDS-MF group was significantly higher than in MDS without MF group, the difference had statistical significance (P = 0.031). The proliferation of BM biopsy in MDS-MF group was significantly more active than in MDS without MF group. The number of blasts, megakaryocytes and abnormal megakaryocytes in MDS-MF group were significantly higher than in MDS without MF group, the differences had statistical significance (P < 0.05). Among the 345 patients who had available results of cytogenetic analysis, 121 cases were MDS-MF patients, the proportion of middle and high-risk prognostic group according to IPSS karyotype prognosis groups in MDS-MF group were significantly higher than in MDS without MF group, the differences had statistical significance (P = 0.047). The median survival was 17 (1 - 60) months in MDS-MF group, and was 32 (1 - 62) months in MDS without MF group. The difference had statistical significance (P = 0.001). Myelofibrosis had independent prognostic significance by multi-variable analysis (P = 0.019). CONCLUSION: The myelofibrosis in MDS is main the proliferation of reticular fiber. The proliferation of reticular fiber is closely related with the number of blast cells, the proliferation and developmental abnormalities of megakaryocytes and the karyotype. The prognosis of MDS-MF patients is poor.

[Study on the role of fluorescence in situ hybridization in cytogenetic evaluation of myelodysplastic syndrome].

OBJECTIVE: To exploit the role of bone marrow (BM) and peripheral blood (PB) fluorescence in situ hybridization (FISH) in cytogenetic evaluation of myelodysplastic syndrome (MDS). METHODS: The metaphase cytogenetics and BM interphase FISH were prospectively compared in 112 cases of de novo MDS. At the same time, comparison of BM and PB FISH was conducted in 56 cases. RESULTS: The differences between metaphase cytogenetics and BM FISH were observed in 22 (54%) of 41 cases with clonal karyotypic abnormalities, most of differences were caused by the limitation of FISH probe panel which could not target all of the regions with aberrations. Only 6 (27%) of 22 differences were involved in our probe regions, the FISH results did not change their cytogenetic risk categories. BM FISH testing was abnormal in 15 (21%) of 71 cases with normal karyotypes, FISH testing was abnormal in 14/51 (27%) and 1/20 (5%) cases with fewer than 20 normal metaphases or more than 20 normal metaphases. Comparison of FISH results of PB and BM samples showed abnormal PB FISH results in 21 (72%) of 29 cases with abnormal BM FISH results, and in 1 (4%) of 27 cases with normal BM FISH results. CONCLUSION: BM FISH should be used to MDS cases with fewer than 20 normal metaphases. Although PB FISH testing is limited by a relatively high false negative rate, it is a reasonable choice to cases with failure of BM aspiration.

[JAK2 exon 12 mutations in patients with Philadelphia (Ph) chromosome-negative myeloproliferative neoplasms].

OBJECTIVE: To investigate JAK2 exon 12 mutations in patients with Philadelphia (Ph) chromosome-negative myeloproliferative neoplasms (MPN) and the clinical characteristics of patients with JAK2 exon 12 mutants. METHODS: Allele-specific PCR (AS-PCR) was applied to identify JAK2 V617F mutation. Genomic DNA corresponding to exon 12 of JAK2 gene and epigenetic regulator gene (TET2, ASXL1, EZH2) were amplified by polymerase chain reaction (PCR). Identification of mutants was by direct sequencing and classification of mutation types by sequencing followed by plasmid cloning. SNP genotyping of two 46/1 tag SNPs, rs12340895 and rs10974944, was analyzed using commercially available Taqman assays on the 7500HT real-time PCR instrument according to standard protocols. RESULTS: No JAK2 exon 12 mutation was detected in patients with ET, PMF or JAK2 V617F positive PV. Among 13 JAK2 V617F negative PV patients, JAK2 exon 12 mutation was detected as N542-E543del in 2(15.4%) patients who presented with a phenotype of predominant erythrocytosis and erythroid colonic grown from their bone marrow samples in the absence of exogenous EPO, reduced serum erythropoietin (EPO) level, and no mutations in TET2, ASXL1 or EZH2 genes. One of the affected patients was heterozygous for 46/1 but the second was negative for this haplotype. CONCLUSION: There was no need to detect JAK2 exon 12 mutation in ET, PMF or MPN-U patients without JAK2 V67F mutation. Ph negative MPN patients with JAK2 exon 12 mutations had somewhat unique clinical and laboratory features.

[An update on epigenetic regulator gene mutations and pathogenesis of myelodysplastic syndromes].

The myelodysplastic syndrome (MDS) is a group of heterogeneous clonal disorders. So far, the etiology and pathogenesis of MDS is poorly understood. Recently, more and more epigenetic regulator gene such as TET2, ASXL1, EZH2, DNMT3A and UTX mutations were detected in patients with MDS: TET2 may convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (hmC). TET2 is the most frequently mutated gene in MDS known so far and it may act as tumor-suppressor gene. ASXL1 belongs to the enhancer of trithorax and Polycomb (ETP) gene group. MDS phenotypes may be caused not only by loss-of-function of ASXL1 but also by gain-of-function mutations, overexpression of this gene and so on. EZH2 is a kind of histone methyltransferase. EZH2 is frequently over-expressed in a wide variety of cancerous tissue types, which reveals it has oncogenic activity. While, defined mutations resulted in dysfunction of histone methyltransferase activity, suggesting that EZH2 acts as a tumor suppressor for myeloid malignancies. DNMT3A belongs to the DNA methyltransferases (DNMT) gene family. It may be correlated with abnormal methylation status in patients with MDS. UTX coding protein is a histone demethylase, and UTX can affect cell proliferation as well as cell fate decision. Inactivating UTX mutations are found in multiple cancer types recently. These gene mutations may play key roles in the pathogenesis of MDS, which are summarized in this review.