Jin-Hou Wu

Yeast carriage on hands of hospital personnel working in intensive care units

The frequency and distribution of yeast carriage on the hands of hospital personnel working in intensive care unit (ICUs), was investigated. Hand carriage of yeast and Candida species was 46 and 29%, respectively. Rhodotorula sp. and Candida parapsilosis were most frequently recovered. There was no significant difference in frequency or distribution of yeasts and Candida sp. recovered among the three ICUs. Seventeen C. parapsilosis isolates and three Candida albicans isolates were genotyped by electrophoretic karyotyping using contour-clamped homogenous electric-field gel electrophoresis. Eleven separate types of C. parapsilosis and two types of C. albicans were identified. There was no common genotype among these isolates, even within the same unit. We conclude that yeast carriage on the hands of personnel working in ICU is common, but these yeasts are usually not acquired from a common source in the ICU.

Heterogeneous Patterns of FLT3 Asp835 Mutations in Relapsed de Novo Acute Myeloid Leukemia: A Comparative Analysis of 120 Paired Diagnostic and Relapse Bone Marrow Samples

Purpose: We analyzed Asp835 mutations of FLT3 on paired marrow samples at diagnosis and relapse from 120 adult patients with de novo acute myeloid leukemia (AML) to determine the role of FLT3 Asp835 mutation in the relapse of AML. Experimental Design: Asp835 mutation was analyzed by DNA PCR amplification of exon 20 of FLT3 gene followed by EcoRV digestion. All of the mutations were confirmed by sequence analysis. Mutant to wild-type allelic ratio was determined by Genescan analysis. The Expand Long Template PCR System was used to determine the allelic location of internal tandem duplication of FLT3 (FLT3/ITD) and Asp835 mutations. Results: Thirteen patients had Asp835 mutations at diagnosis, of them 8 lost the mutations at relapse, and the remaining 5 patients carrying Asp835 mutations at diagnosis relapsed with the identical mutation types. Another 6 patients acquired Asp835 mutations at relapse. Five samples harbored both FLT3/ITD and Asp835 mutations that were found on different alleles by cloning analysis in the 3 patients studied. There were no differences in WBC count, French-American-British subtype, percentage of marrow blasts, or circulating blasts between patients with and without Asp835 mutations, whereas the difference in the prevalence of Asp835 mutations among cytogenetic/molecular subgroups was statistically significant (P = 0.025). Conclusions: The present study showed that patients with AML had heterogeneous patterns of FLT3 Asp835 mutations, either acquisition or loss of the mutations at relapse. Asp835 mutant clone may develop as a secondary event in a subset of patients with AML.PURPOSE We analyzed Asp(835) mutations of FLT3 on paired marrow samples at diagnosis and relapse from 120 adult patients with de novo acute myeloid leukemia (AML) to determine the role of FLT3 Asp(835) mutation in the relapse of AML. EXPERIMENTAL DESIGN Asp(835) mutation was analyzed by DNA PCR amplification of exon 20 of FLT3 gene followed by EcoRV digestion. All of the mutations were confirmed by sequence analysis. Mutant to wild-type allelic ratio was determined by Genescan analysis. The Expand Long Template PCR System was used to determine the allelic location of internal tandem duplication of FLT3 (FLT3/ITD) and Asp(835) mutations. RESULTS Thirteen patients had Asp(835) mutations at diagnosis, of them 8 lost the mutations at relapse, and the remaining 5 patients carrying Asp(835) mutations at diagnosis relapsed with the identical mutation types. Another 6 patients acquired Asp(835) mutations at relapse. Five samples harbored both FLT3/ITD and Asp(835) mutations that were found on different alleles by cloning analysis in the 3 patients studied. There were no differences in WBC count, French-American-British subtype, percentage of marrow blasts, or circulating blasts between patients with and without Asp(835) mutations, whereas the difference in the prevalence of Asp(835) mutations among cytogenetic/molecular subgroups was statistically significant (P = 0.025). CONCLUSIONS The present study showed that patients with AML had heterogeneous patterns of FLT3 Asp(835) mutations, either acquisition or loss of the mutations at relapse. Asp(835) mutant clone may develop as a secondary event in a subset of patients with AML.

Clonal leukemic evolution in myelodysplastic syndromes with TET2 and IDH1/2 mutations

Somatic mutations of TET2, IDH1, and IDH2 have been described in myelodysplastic syndrome. The impact of these mutations on outcome of myelodysplastic syndrome and their progression to secondary acute myeloid leukemia remains unclear. Mutation status of TET2, IDH1 and IDH2 was investigated in a cohort of 46 paired myelodysplastic syndrome/acute myeloid leukemia samples and 122 non-paired cases with de novo myelodysplastic syndrome, to clarify their roles in the evolution of myelodysplastic syndrome to acute myeloid leukemia. Among the 168 de novo myelodysplastic syndrome patients, the frequency of TET2, IDH1, and IDH2 mutations was 18.5%, 4.2% and 6.0%, respectively. TET2/IDH mutations had no impact on survivals, while TET2 mutations were significantly associated with rapid progression to acute myeloid leukemia. Seventeen of the 46 paired myelodysplastic syndrome/secondary acute myeloid leukemia samples harbored TET2/IDH mutations; none acquired these mutations in acute myeloid leukemia phase. Progression to acute myeloid leukemia was accompanied by evolution of a novel clone or expansion of a minor pre-existing subclone of one or more distinct mutations in 12 of the 17 cases with TET2/IDH mutations. A minor subclone in 3 cases with biallelic TET2 inactivation subsequently expanded, indicating biallelic TET2 mutations play a role in acute myeloid leukemia progression. Twelve patients acquired other genetic lesions, and/or showed increased relative mutant allelic burden of FLT3-ITD, N/K-RAS, CEBPA or RUNX1 during acute myeloid leukemia progression. Our findings provide a novel insight into the role of TET2/IDH mutation in the pathogenesis of myelodysplastic syndrome and subsequent progression to acute myeloid leukemia.

Heterogeneous Patterns of CEBPα Mutation Status in the Progression of Myelodysplastic Syndrome and Chronic Myelomonocytic Leukemia to Acute Myelogenous Leukemia

Purpose: We aimed to assess the role of CEBPα mutations in the progression of myelodysplastic syndrome (MDS) to acute myelogenous leukemia (AML) and their cooperating mutations. Experimental Design: Mutational analysis of CEBPα with direct sequencing for each PCR product was done on matched bone marrow samples obtained from 50 adult patients with MDS at diagnosis and at AML transformation. Cloning analysis was used to determine the allelic distribution. Results: CEBPα mutations were identified in four patients at diagnosis of MDS, including one with refractory anemia with excess blasts and three with chronic myelomonocytic leukemia. At AML transformation, three patients retained the identical mutant clones as their initial diagnosis, three acquired the mutations, and one lost CEBPα mutation when she gained FLT3/ITD mutation. Together, seven patients had CEBPα mutations throughout the disease course; four patients had NH2-terminal mutations resulting in a frameshift and truncation of the protein, three of them had two different mutations either on the same alleles or on different alleles, two had missense mutations, and one had a deletion in the basic region leucine zipper domain. Except for one with coexistence of N-ras mutation, no sample harbored cooperating mutations with FLT3 or N-ras genes. CEBPα mutations had no influence on the time to AML progression or overall survival. Conclusions: Our results show that CEBPα mutations play a role in a subset of patients with MDS, especially in chronic myelomonocytic leukemia. The mutation status was heterogeneous, exhibiting identical clone, clonal change, or clonal evolution during the progression to AML.

Clonality analysis using X-chromosome inactivation patterns by HUMARA-PCR assay in female controls and patients with idiopathic thrombocytosis in Taiwan

OBJECTIVE Analysis of X-chromosome inactivation patterns (XCIPs) is a useful tool in the diagnosis of clonal disorders. The human androgen receptor (HUMARA) locus is especially useful for clonality study. The present study was conducted 1) to determine the heterozygosity rate for HUMARA locus in Taiwanese women, 2) to determine the frequency of excessive skewing in different cell types, and 3) to determine the utility of XCIPs in the differential diagnosis of thrombocytosis. PATIENTS AND METHODS XCIPs by HUMARA-PCR assay were performed on purified granulocytes and T cells from 73 female patients presenting with idiopathic persistent thrombocytosis (IT), 10 patients with reactive thrombocytosis (RT), and 46 bone marrow samples from female controls. XCIPs of buccal mucosa cells were also compared with those of T cells in 57 patients with IT. The percentage of clonal granulocytes was calculated after correcting for the degree of Lyonization in T cells. RESULTS The heterozygosity rate for the HUMARA gene was 89.1% in Taiwanese females. The median age of informative IT patients and controls was 59 (18-92) and 58 (19-89), respectively. Excessive skewing (allele ratio <0.33) was more frequent in granulocytes than in T cells in both controls (12/43 vs 9/43, p = 0.080) and IT patients (56/64 vs 25/64, p < 0.001). XCIPs were the same for both buccal mucosa and T cells in 43 patients but were different in 14 patients. Of the 43 informative controls, 31 had a polyclonal pattern; an ambiguous pattern was found in nine; and the remaining three, aged 71, 73, and 80, respectively, had a clonal pattern. A clonal pattern was found in 42 IT patients, a polyclonal pattern in 12, and an ambiguous pattern in 10 of the 64 IT patients. The frequency of clonal, polyclonal, and ambiguous patterns in the 40 IT patients with age < or = 65 was 55.0%, 30.0%, and 15.0%, respectively. None of the IT patients aged >65 had a polyclonal disease. IT patients aged >65 had a significantly higher frequency of clonal pattern (p = 0.030) and a significantly lower frequency of polyclonal pattern (p = 0.002) than those with age <65. Of the eight heterozygous patients with RT, one aged 80 exhibited a clonal pattern, and the remaining seven had a polyclonal pattern. CONCLUSIONS The present study on Taiwanese females showed a heterozygosity rate of 89.1% for the HUMARA gene. Our results confirmed that IT is a heterogeneous disorder in terms of clonality. Twenty-three percent of IT patients exhibited a greater than 20% difference in allele expression for buccal mucosa and T cells. Presence of a clonal XCIP in young patients with IT can serve as a positive marker for the diagnosis of clonal thrombocytosis, and elderly patients with polyclonal XCIPs are unlikely to have essential thrombocythemia.

Gene Mutation Patterns in Patients with Minimally Differentiated Acute Myeloid Leukemia

Minimally differentiated acute myeloid leukemia (AML-M0) is a rare subtype of AML with poor prognosis. Although genetic alterations are increasingly reported in AML, the gene mutations have not been comprehensively studied in AML-M0. We aimed to examine a wide spectrum of gene mutations in patients with AML-M0 to determine their clinical relevance. Twenty gene mutations including class I, class II, class III of epigenetic regulators (IDH1, IDH2, TET2, DNMT3A, MLL-PTD, ASXL1, and EZH2), and class IV (tumor suppressor genes) were analyzed in 67 patients with AML-M0. Mutational analysis was performed with polymerase chain reaction–based assays followed by direct sequencing. The most frequent gene mutations from our data were FLT3-ITD/FLT3-TKD (28.4%), followed by mutations in IDH1/IDH2 (28.8%), RUNX1 (23.9%), N-RAS/K-RAS (12.3%), TET2 (8.2%), DNMT3A (8.1%), MLL-PTD (7.8%), and ASXL1 (6.3%). Seventy-nine percent (53/67) of patients had at least one gene mutation. Class I genes (49.3%) were the most common mutated genes, which were mutually exclusive. Class III genes of epigenetic regulators were also frequent (43.9%). In multivariate analysis, old age [hazard ratio (HR) 1.029, 95% confidence interval (CI) 1.013-1.044, P = .001) was the independent adverse factor for overall survival, and RUNX1 mutation (HR 2.326, 95% CI 0.978-5.533, P = .056) had a trend toward inferior survival. In conclusion, our study showed a high frequency of FLT3, RUNX1, and IDH mutations in AML-M0, suggesting that these mutations played a role in the pathogenesis and served as potential therapeutic targets in this rare and unfavorable subtype of AML.

High frequency of additional gene mutations in acute myeloid leukemia with MLL partial tandem duplication: DNMT3A mutation is associated with poor prognosis.

The mutational profiles of acute myeloid leukemia (AML) with partial tandem duplication of mixed-lineage leukemia gene (MLL-PTD) have not been comprehensively studied. We studied 19 gene mutations for 98 patients with MLL-PTD AML to determine the mutation frequency and clinical correlations. MLL-PTD was screened by reverse-transcriptase PCR and confirmed by real-time quantitative PCR. The mutational analyses were performed with PCR-based assays followed by direct sequencing. Gene mutations of signaling pathways occurred in 63.3% of patients, with FLT3-ITD (44.9%) and FLT3-TKD (13.3%) being the most frequent. 66% of patients had gene mutations involving epigenetic regulation, and DNMT3A (32.7%), IDH2 (18.4%), TET2 (18.4%), and IDH1 (10.2%) mutations were most common. Genes of transcription pathways and tumor suppressors accounted for 23.5% and 10.2% of patients. RUNX1 mutation occurred in 23.5% of patients, while none had NPM1 or double CEBPA mutation. 90.8% of MLL-PTD AML patients had at least one additional gene mutation. Of 55 MLL-PTD AML patients who received standard chemotherapy, age older than 50 years and DNMT3A mutation were associated with inferior outcome. In conclusion, gene mutations involving DNA methylation and activated signaling pathway were common co-existed gene mutations. DNMT3A mutation was a poor prognostic factor in MLL-PTD AML.

Primary colonic lymphoma: an analysis of 74 cases with localized large-cell lymphoma

Background:  Surgical resection is considered a crucial treatment in patients with primary colonic lymphoma, but combining surgery with chemotherapy has provided additional therapeutic benefits in some studies. To further explore the optimal therapeutic approach in different clinical scenarios, we reviewed cases with localized large‐cell lymphoma and analyzed the factors related to the outcomes.

Acute promyelocytic leukemia-associated thrombosis.

Patients with acute promyelocytic leukemia (APL) are prone to both bleeding and thrombosis. The bleeding complications are well known. In contrast, APL-associated thrombosis is relatively underappreciated. We aimed to explore the issue of APL-associated thrombosis events. In the past 20 years, 127 cases with APL were found in our hospital database. We collected their coagulation laboratory profiles, including leukemia burdens, white blood cell and platelet counts, prothrombin time, activated partial thromboplastin time, fibrinogen levels, and disseminated intravascular coagulation scores. Data were compared between patients with or without thrombosis. Clinical outcomes and potential risk factors were obtained for analysis. Ten cases with APL-associated thrombosis were found. The incidence of thrombosis was 7.9% in our cohort. Five patients had cerebral infarction, 5 had catheter-related thrombosis and 1 had acute myocardial infarction. No laboratory data were associated with clinical thrombosis. Three patients died during the induction phase but thrombosis was not the direct cause of death for any of them. We conclude that patients with APL are susceptible to thrombosis in addition to bleeding. Laboratory coagulation parameters did not predict thrombosis in our series. Ischemic stroke and catheter-related thrombosis were the most common events in our Taiwanese cohort. Such a thrombosis pattern is unique and worth further investigation.

Classical Hairy Cell Leukemia and Its Variant: A 17-Year Retrospective Survey in Taiwan Chinese

Background: Classical hairy cell leukemia (HCL-C) and its variant (HCL-V) are rare chronic B-cell lymphoproliferative disorders. Only a few reports in Chinese patients are available. Methods: We retrospectively reviewed 16 patients with HCL-C and HCL-V in Taiwan over a 17-year period. Results: Eight were HCL-C and 8 were HCL-V. All HCL accounted for 0.7% of all adult leukemias. Compared to HCL-V, HCL-C was characterized by profound leukopenia, monocytopenia, thrombocytopenia and fewer circulating hairy cells. One HCL-C and 2 HCL-V patients had second malignancies. Seven HCL-C patients achieved hematological remission after splenectomy (n = 1) or 2-chlorodeoxyadenosine (n = 6). Of the 8 HCL-V patients, 6 received splenic irradiation. Only one achieved complete remission and another had partial remission; relapse or disease progression was noted 13.4 or 25.7 months later, respectively. Two of three HCL-V patients who underwent splenectomy had stable disease. All patients with HCL-C were alive while 3 with HCL-V expired. Compared to HCL-C, HCL-V had a significantly shorter leukemia-free survival. Conclusion: A relatively higher proportion of HCL-V in all HCL comparing to Westerners is observed. Second malignancies are common. With an inferior outcome and dismal response to most treatment, enrollment in a clinical trial should be considered for HCL-V.