Variation of nicotinic subtype α7 and muscarinic subtype M3 acetylcholine receptor expression in three main types of leukemia.

Cholinergic receptors, such as α7-nicotinic acetylcholine receptor (α7-nAChR) and M3-muscarinic acetylcholine receptor (M3-mAChR), have been demonstrated to serve a significant role in the proliferation, differentiation and apoptosis of leukemic cells. However, the expression of these receptors in samples from patients with leukemia remains unclear. The present study aimed to determine the expression of M3-mAChR and α7-nAChR in the bone marrow or peripheral blood of 51 patients with leukemia, including acute myeloid leukemia (AML; n=33), acute lymphoblastic leukemia (ALL; n=13), and chronic myeloid leukemia (CML; n=5). Peripheral blood mononuclear cells (PBMCs) were also isolated from healthy subjects (n=5) for comparison. Western blot analysis was performed to determine the protein expression profiles, and a pattern of decreased α7-nAChR levels in patients with leukemia was observed. Among the leukemia types, the lowest expression of α7-nAChR and M3-mAChR were identified in patients with T-cell ALL/lymphoma (T-ALL). CML exhibited the highest level of M3-mAChR, which was significantly different from APL and AML-M4, yet not from healthy subjects (P<0.05). Therefore, different expression profiles of α7-nACR and M3-mAChR were detected amongst the leukemia types. Collectively, the present study supports the potential role of cholinergic signaling in mediating leukemogenesis. However, further studies in larger cohorts are required to validate these findings.

Utilization of flow cytometry for diagnosis of hematologic malignancies in Thailand: increasing trends and diagnostic yields in 7,982 samples.

BACKGROUND: Diagnosis of hematologic malignancies requires a multidisciplinary approach. Flow cytometry (FCM) has become an essential tool for immunophenotypic studies of malignant hematopoietic cells. OBJECTIVE: To evaluate the utilization trend of FCM and its diagnostic yields for hematologic malignancy at a major teaching hospital in Thailand. MATERIAL AND METHOD: FCM results of bone marrow (BM) and peripheral blood (PB) specimens during 2000-2013 were analyzed and compared to clinical diagnosis. RESULTS: Overall, 7,982 specimens were submitted for diagnostic FCM including 6,561 BM and 1,421 PB. The number of specimens analyzedwas 121, 142, 164, 299, 491, 431, 690, 611, 719, 744, 725, 863, 955 and 1,027, respectively, from 2000 to 2013. The most common clinical diagnoses requested for FCM were acute leukemia (5,911 cases, 74%) followed by lymphoma (1,419 cases, 17.8%), and chronic lymphocytic leukemia (CLL) (634 cases, 7.94%). The highest diagnostic yield of FCM was found in acute leukemia cases (69.71%) followed by CLL (35.33%). Only 15.43% of clinically suspected lymphoma cases were positive by FCM. Overutilization of PB (35.6% of cases) instead of BM for lymphoma staging significantly contributed to low diagnostic yields of lymphoma by FCM as circulating tumor cells may not be present in such cases. CONCLUSION: FCM has an increasing role in the diagnosis of hematologic malignancies in Thai patients over the past 14 years with the highest diagnostic yield in acute leukemia. Appropriate specimen types and study indications are required in order to reduce futility of costly diagnostic tests and improve diagnostic yields.

A comparative study of cytomorphology and flow cytometry to detect malignant cells in the body cavity fluid.

BACKGROUND: Many types of cancer metastasize to the epithelial linings of the body cavity causing malignant fluid to accumulate in such spaces. Cytomorphological evaluation is considered essential in the diagnosis of malignant body fluid. Nevertheless, the accuracy of cytomorphological results is subjective and can vary depending on the cytopathologists' experience. OBJECTIVE: To determine if DNA content analysis using propidium iodide (PI), anti-CD45 (leukocyte common antigen) and anti-AE1/AE3 (pan-cytokeratin) as analyzed by flow cytometry could be used to detect and differentiate malignant cells in the body fluid when compared to cytomorphological evaluation. MATERIAL AND METHOD: A cross-sectional, laboratory-based, observational study on 90 specimens was conducted. Flow cytometric analysis was done. Sensitivity, specificity, positive predictive value, negative predictive value and accuracy were reported. RESULTS: The DNA index (DI) cut-off value, as determined by the receiver operating characteristic curve of 1.215 or more, had 51.7% sensitivity and 89.1% specificity to detect malignant cells. When DI was combined with AE1/AE3 positivity, the sensitivity increased to 62.1% with 80.3% specificity. When such techniques were used in adjunct to cytospin preparation, the sensitivity and specificity increased to 89.7% and 65.66%, respectively. Twelve specimens (13.3%) had positive results by flow cytometry but negative cytomorphological results by pathologists, 4 of which were later confirmed as cancer from pleural biopsy. Eleven specimens (12.2%) had false negativity, 6 of which were unspecified metastatic carcinoma. Four specimens with negative flow cytometric results were cerebrospinal fluid (CSF) specimens with a low cell count. Subgroup analysis in the cases of non-CSF fluid showed 72% sensitivity and 72.1% specificity. CONCLUSION: Immunophenotypic analysis using DI and AE1/AE3 in conjunction with cytospin preparation had a moderately high sensitivity to detect malignant cells in the body fluid (-90%). Non-CSF specimens yielded better results than CSF Further modifications are ongoing in order to increase the detection capabilities of our screening panel.

Detection of lymphoid neoplasms in bone marrow by flow cytometric analysis and comparison to bone marrow aspiration and biopsy.

BACKGROUND: Lymphoid neoplasms are a heterogeneous group of hematologic malignancies. Bone marrow (BM) can be involved in a certain proportion of lymphoid neoplasms, necessitating accurate and rapid diagnosis of such involvement for early therapeutic decision. OBJECTIVE: To evaluate the diagnostic utility of flow cytometry (FC) for assessment of BM involvement by lymphoid neoplasms. BM biopsy (BMBx) was used as the gold standard and BM aspiration (BMA) was used as a comparison. MATERIAL AND METHOD: Two hundred and eighty-three samples with a clinical suspicion for lymphoid neoplasms were received in FC laboratory and analysed using various lymphoid markers. The FC results were then compared to BMA and BMBx. RESULTS: Of 283 cases, 94 had lymphoid neoplasms by BMBx (33%). Among the positive BMBx cases, concordant agreement of all three investigations was found in 45 cases (48%). FC was positive in 52/94 cases (55%) while BMA was positive in 62/ 94 cases (66%). Among the negative BMBx cases, FC was positive in 8/189 cases (4%) and BMA was positive in 56/189 cases (30%). FC and BMA were both negative in 25/94 cases (27%). The specificity of FC and BMA was 96% and 60% while the sensitivity was 55% and 65%, respectively. Subtype agreements were better between FC and BMBx than BMA and BMBx, particularly in small lymphoid neoplasms. CONCLUSION: BMA tended to overdiagnose lymphoid neoplasms and could not accurately differentiate subtypes of B-cell and T-cell neoplasms. FC correlated more with BMBx and had less false positivity than BMA. Further utilzation of broader FC markers may help to improve the diagnostic capability and sensitivity of FC.

Molecular alterations of isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) metabolic genes and additional genetic mutations in newly diagnosed acute myeloid leukemia patients.

BACKGROUND: Isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) metabolic genes encode cytosolic and mitochondrial enzymes that catalyze the conversion of isocitrate to α-ketoglutarate. Acquired somatic mutations of IDH1 and IDH2 have recently been reported in some types of brain tumors and a small proportion of acute myeloid leukemia (AML) cases. METHODS: Two-hundred and thirty newly diagnosed AML patients were analyzed for the presence of IDH1 and IDH2 heterozygous mutations by polymerase chain reaction-denaturing high performance liquid chromatography (PCR-DHPLC) followed by direct sequencing. Clinical and biological characteristics were analyzed and correlated to the IDH mutational status. Coexisting mutations such as FLT3, PML-RARA, RAS, AML1, and NPM1 mutations were additionally explored. RESULTS: The prevalence of IDH1 and IDH2 mutations was 8.7% (20/230) and 10.4% (24/230), respectively. Six missense mutations were identified among IDH1-mutated cases; p.R132H (n = 8), p.R132C (n = 6), p.R132S (n = 2), p.R132G (n = 2), p.R132L (n = 1), and p.I99M (n = 1). Two missense mutations were found in IDH2-mutated cases; p.R140Q (n = 20) and p.R172K (n = 4). No patients had dual IDH1 and IDH2 mutations. About 18% of AML with normal cytogenetics and 31% of acute promyelocytic leukemia had IDH mutations. Half of the IDH-mutated cohort had normal karyotype and the major FAB subtype was AML-M2. Interestingly, IDH1- and IDH2-mutated cases predominantly had NPM1 mutations (60-74%) as compared to the wild type (P < 0.001). Very few IDH-mutated cases had FLT3 and/or RAS abnormalities and none of them had AML1 mutations. Older age and higher median platelet counts were significantly associated with IDH2 mutations although the clinical impact of either IDH1 or IDH2 mutations on patients' overall survival could not be observed. CONCLUSION: Overall, 19% of newly diagnosed AML patients had alterations of IDH genes. No patients concurrently carried both IDH1 and IDH2 mutations suggesting that these mutations were mutually exclusive. NPM1 mutation appears as a major coexisting genetic mutation in IDH-mutated patients. Our present data failed to support the prognostic relevance of IDH mutations although alterations of these metabolic genes potentially have an important role in leukemia development.

Utilization of a scoring system for diagnosis of chronic lymphocytic leukemia in Thai patients.

BACKGROUND: Chronic lymphocytic leukemia (CLL) is a rare B-lymphoid malignancy in Southeast Asia. We evaluated whether a scoring system based on the expression of CDS, CD23, FMC7, CD79b and surface immunoglobulin (Slg) could be utilized to distinguish CLL from other types of lymphoid neoplasms in the Thais. MATERIAL AND METHOD: One-hundred and forty-five samples with a clinical suspicion for CLL were analysed by flow cytometry. A score of one was assigned if the following marker was identified: CD5+, CD23+, FMC7-, CD79b- and SIg(-/weak). A cut-off score of > or =3 was required for the definitive diagnosis of B-CLL. RESULTS: Only 50 cases (34.5%) were confirmed as B-CLL (scores > or =3). Cases with scores > or =3 had significantly higher leukocyte counts and marrow/blood lymphocytes than cases with scores < or =2. Dual CD5/CD23 expression was found in 87.5% of CLL cases. In 81 cases with scores < or =2, a variety of non-CLL disorders predominated, such as marginal zone lymphoma, splenic lymphoma with villous lymphocytes, mantle cell lymphoma, and prolymphocytic leukemia. CONCLUSION: A score of 3 and dual CD5/CD23 expression are essential for the diagnosis of CLL while a score of 2 mostly indicative of non-CLL. The majority of clinical cases of CLL turned out to be non-CLL by flow cytometry. Increased utilization of this scoring system should increase the accuracy of diagnosis of this rare type of leukemia in the Thai population.

WT1 mutations and polymorphisms in Southeast Asian acute myeloid leukemia.

Genomic alterations of the Wilms' Tumor 1 (WT1) gene have been reported to occur in patients with acute myeloid leukemia (AML). No data presently exists regarding the frequency of WT1 mutations in the Southeast Asian AML population. This study focused on WT1 exons 7-10 mutations and their correlation with other molecular markers and patients' characteristics. The zinc finger domain of WT1 gene covering exons 7-10 was directly sequenced. Six types of mutations were identified among 49 cases (12.24%); 4 localized on exon 7 and 2 on exon 9. Two novel mutations were identified including the insertion within codon 313 and codon 314. Patients harboring WT1 mutations seemed to have a younger age (29.5 vs 45.4 years), a higher white blood cell count (120.3 vs 19.8×10(9)/L), and a lower platelet count (54.2 vs 104.3×10(9)/L) as compared to those without the mutations although statistical differences could not be demonstrated. All exon 7 mutations were frameshift mutations and had NRAS mutation while exon 9 mutations were base substitutions and had FLT3-ITD mutation. Interestingly, the major allele for rs16754 single nucleotide polymorphism was G (25-homozygous and 6-heterozygous) which was in contrast to A in the Western reports. The frequency of WT1 mutation in the Southeast Asian AML was thus comparable to the figures reported from the West although the designated major allele for rs16754 polymorphism was different.

Positive and negative predictive values of HLA-DR and CD34 in the diagnosis of acute promyelocytic leukemia and other types of acute myeloid leukemia with recurrent chromosomal translocations.

The predictive value of HLA-DR and CD34 in the diagnosis of four distinct genetic entities of acute myeloid leukemia (AML) is presently not established. We evaluated the positive and negative predictive values (PPV and NPV, respectively), sensitivity, specificity, and correlation coefficients of HLA-DR and CD34 in AML patients with t(15;17), t(8;21), inv(16), and abn(11q23). In AML with t(15;17) (n = 64), HLA-DR was expressed in 4.68% and CD34 was expressed in 15.62% and none of the cases expressed both HLA-DR and CD34. In AML with t(8;21) (n = 99), HLA-DR, CD34 or both antigens were expressed in the majority of cases (90.90%, 80.80%, and 79.79%, respectively). AML patients with inv(16) (n = 18) and abn(11q23) (n = 31) also highly expressed HLA-DR and CD34. Eight cases of t(8;21) and 1 case of abn(11q23) did not express either antigen. The highest correlation between CD34 and HLA-DR expression values was observed in cases with t(8;21) (r = 0.72) with the lowest correlation in inv(16) (r = 0.035). The PPV and NPV of HLA-DR-negativity plus CD34-negativity to predict t(15;17) was 85% and 100%, respectively, with 100% sensitivity and 92.74% specificity. The PPV and NPV of other myeloid markers such as CD117, MPO and CD11c to diagnose t(15;17) were much lower than those of HLA-DR and CD34. It was concluded that the absence of double negativity of HLA-DR and CD34 strongly predicts against t(15;17). Rare HLA-DR-positive/CD34-negative cases exist in patients with t(15;17) and 8% of t(8;21) cases expressed neither antigen. Further studies should determine whether HLA-DR-positive t(15;17) and HLA-DR-negative/CD34-negative t(8;21) represent a special entity associated with significant prognostic relevance.

AML1 mutation and its coexistence with different transcription factor gene families in de novo acute myeloid leukemia (AML): redundancy or synergism.

AML1 mutations were identified in 6.3% of AML patients with chromosomal translocations involving CBF, PML-RARalpha, HOX, or ETS transcription factor (TF) gene families. Rare chromosomal abnormalities, t(16;21) and t(7;11), were also found. This study represents the first series to demonstrate the coexistence of known and novel AML1 mutations with different TF gene mutations. Although the occurrence of two TF gene mutations may appear unnecessary, the possible synergistic mechanism between different TF gene families cannot be excluded and needs to be further explored.

C-kit receptor tyrosine kinase (CD117) expression and its positive predictive value for the diagnosis of Thai adult acute myeloid leukemia.

We examined the expression of c-kit receptor tyrosine kinase in 195 Thai adult patients with acute leukemia and determined its specificity and predictive values for the diagnosis of adult acute myeloid leukemia (AML). CD117 was used to detect c-kit expression on CD45 and side-scatter-gated blast cells by flow cytometry. Of 163 AML cases, 67% expressed CD117. None of acute lymphoid leukemia (ALL) had CD117 expression, except one case of T-ALL. The majority of AML patients carrying t(8;21), inv(16), and t(15;17) had high CD117 expression. High proportion of AML cases without c-kit expressed monocytic markers. Significant associations between CD117 and CD34 (P<0.001), CD13 (P=0.006), CD7 (P=0.034), and CD19 (P<0.001) were found in AML cases. The calculated specificity of CD117 for the diagnosis of AML was 0.97, which was higher than CD13 (0.78) and CD33 (0.75) but comparable to MPO (0.97). The positive predictive value (PPV) of CD117 for AML was 0.99, with the negative predictive value of 0.35. In conclusion, the majority of Thai adult AML cases expressed c-kit. C-kit is infrequently expressed in ALL and appeared to be specific for AML with high PPV. Future targeting therapy using c-kit as a therapeutic target should benefit the majority of Thai AML patients who had high c-kit expression.

Immunophenotypes and outcome of Philadelphia chromosome-positive and -negative Thai adult acute lymphoblastic leukemia.

Little evidence exists in Asian countries regarding the incidence, immunologic characteristics, and clinical outcomes of adult patients with Philadelphia chromosome-positive (Ph+) and -negative (Ph-) acute lymphoblastic leukemia (ALL). In this study, we prospectively studied 324 Thai adult acute leukemia patients, 79 (24%) of whom were identified as having ALL. Immunophenotyping was performed by 5-parameter flow cytometry, and karyotyping was conducted by standard banding methods. The Ph chromosome was detected in 18.5% of cases. The mean age of Ph+ ALL patients was 29 years (50% male), and that of Ph- ALL patients was 33 years (62% female). The Ph+ ALL patients had significantly higher white blood cell (WBC) counts (mean, 93 x 10(9)/L), with 67% having WBC counts higher than 50 x 10(9)/L. In contrast, most Ph- ALL patients had WBC counts lower than 50 x 10(9)/L (mean, 36 x 10(9)/L; P < .05). CD10 and CD34 were more highly expressed in the Ph+ ALL patients (mean expression, 83% and 87%, respectively) than in the Ph- ALL patients (45% and 57%; P < .005). The aberrant expression of myeloid antigens, including CD33 and CD13, was also significantly observed in the Ph+ ALL patients. The median survival time of Ph+ ALL patients was 8 months, compared with 22 months for the Ph- ALL patients. In conclusion, immunophenotyping results showed that Ph+ ALL in Thai adults arises from B-cells at an earlier stage of development. Extreme leukocytosis, a younger age, male sex, high expression levels of CD10 and CD34, aberrant myeloid antigens, and poorer rates of survival appeared to be associated with the Ph chromosome in Thai adult ALL cases. The incidence of the Ph chromosome among Thai adult ALL patients was not different from that found in Western countries.

Collection and processing of umbilical cord blood for cryopreservation.

Umbilical cord blood (UCB) is being increasingly used as an alternative source of hematopoietic stem cells for allogeneic bone marrow transplantation. UCB transplantation has been successfully used to treat a variety of genetic, hematological, and oncological disorders in children and adults. The objectives of this study was to establish a closed-system technique for UCB collection and buffy coat separation by Optipress I device. Thirty-four UCB were collected by triple-bag system from pregnant mothers whose fetuses were not affected by thalassemic diseases after prenatal diagnosis. The mean volumn of UCB collection were 120 +/- 5 ml (range 65-180 ml). Total WBC, CD34+ cells, the progenitor cell erythroid burst-forming unit (BFU-E) and granulocyte-macrophage colony-forming unit (CFU-GM) in the UCB units were (9.36 +/- 0.84) x 10(8), (3.61 +/- 0.52) x 10(6), (9.12 +/- 1.60) x 10(5), and (5.32 +/- 1.23) x 10(5), respectively. Good correlation between the nucleated cell and net cord blood volume could be demonstrated (p < 0.0001). The correlation between CD34+ cells and the following parameters: nucleated cell, BFU-E or CFU-GM were also demonstrated (p = 0.001, 0.0105 or 0.0001, respectively). Buffy coat was subsequently separated from 18 UCB units by Optipress I device. 70 +/- 3 ml of buffy coat were collected and cryoprocessing was done by automatic controlled-rate freezer. Good recovery of total WBC, CD34+ cells, progenitor cells BFU-E and CFU-GM after buffy coat separation were observed 89 per cent, 95 per cent, 109 per cent, and 102 per cent respectively. There was no aerobic bacterial or fungal contamination in the separated blood products. By using this technique, the UCB units were easily collected, rapidly separated within one hour, and high recovery of the hematopoietic progenitor cells could be obtained.

Immunophenotypic profile of adult acute myeloid leukemia (AML): analysis of 267 cases in Thailand.

Little data exists in Thailand and other Southeast Asian countries regarding the biological characteristics of adult acute myeloid leukemia (AML). In this study, we performed a flow cytometric analysis of 267 Thai adult AML cases to delineate the pattern of leukemic cell surface antigens. Forty-eight cases (18%) were identified as acute promyelocytic leukemia (M3) and 219 cases as non-M3. The most frequent subtype of AML in Thailand was M1/M2 and the least frequent was M7. M3 immunophenotypes were characterized by their unique lack of expression of CD34 and HLA-DR as contrast to the high mean expression of 50% and 70%, respectively, in non-M3. Overall, 60% of cases expressed CD34. Aberrant lymphoid antigens were uniquely seen in specific subtypes of Thai AML, including CD19 (33% of non-M3 vs 23% of M3) and CD2 (12% of M3 vs 2% of non-M3). CD56 was frequently expressed in both M3 and non-M3 while CD16 appeared to be associated with M4/M5 (24% of cases) and CD7 with M1/M2 (21% of cases). Eighty-one percent of non-M3 expressed CD38 while only 53% of M3 did. We found that most Thai adult AML patients were on average 15-20 years younger than those of the West or Japan with only 25% of Thai cases over 60 years of age, although the immunophenotypes were not markedly different. Biological studies of acute leukemia in various countries should help to provide epidemiological clues that play a role in the pathogenesis of leukemia in different geographic regions of the world. Our study represents the largest series of AML ever investigated in the Southeast Asian region.