Pan-viral serology uncovers distinct virome patterns as risk predictors of hepatocellular carcinoma and intrahepatic cholangiocarcinoma.

This study evaluates the pan-serological profiles of hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA) compared to several diseased and non-diseased control populations to identify risk factors and biomarkers of liver cancer. We used phage immunoprecipitation sequencing, an anti-viral antibody screening method using a synthetic-phage-displayed human virome epitope library, to screen patient serum samples for exposure to over 1,280 strains of pathogenic and non-pathogenic viruses. Using machine learning methods to develop an HCC or iCCA viral score, we discovered that both viral scores were positively associated with several liver function markers in two separate at-risk populations independent of viral hepatitis status. The HCC score predicted all-cause mortality over 8 years in patients with chronic liver disease at risk of HCC, while the viral hepatitis status was not predictive of survival. These results suggest that non-hepatitis viral infections may contribute to HCC and iCCA development and could be biomarkers in at-risk populations.

Gut dysbiosis in Thai intrahepatic cholangiocarcinoma and hepatocellular carcinoma.

Primary liver cancer (PLC), which includes intrahepatic cholangiocarcinoma (iCCA) and hepatocellular carcinoma (HCC), has the highest incidence of all cancer types in Thailand. Known etiological factors, such as viral hepatitis and chronic liver disease do not fully account for the country's unusually high incidence. However, the gut-liver axis, which contributes to carcinogenesis and disease progression, is influenced by the gut microbiome. To investigate this relationship, fecal matter from 44 Thai PLC patients and 76 healthy controls were subjected to whole-genome metagenomic shotgun sequencing and then analyzed by marker gene-based and assembly based methods. Results revealed greater gut microbiome heterogeneity in iCCA compared to HCC and healthy controls. Two Veillonella species were found to be more abundant in iCCA samples and could distinguish iCCA from HCC and healthy controls. Conversely, Ruminococcus gnavus was depleted in iCCA patients and could distinguish HCC from iCCA samples. High Veillonella genus counts in the iCCA group were associated with enriched amino acid biosynthesis and glycolysis pathways, while enriched phospholipid and thiamine metabolism pathways characterized the HCC group with high Blautia genus counts. These findings reveal distinct landscapes of gut dysbiosis among Thai iCCA and HCC patients and warrant further investigation as potential biomarkers.

Prognostic parameters on baseline and interim [ 18 F]FDG-PET/computed tomography in diffuse large B-cell lymphoma patients.

OBJECTIVE: 2-[ 18 F]fluoro-2-deoxy- d -glucose PET/computed tomography ([ 18 F]FDG-PET/CT) is a widely used imaging method in the management of diffuse large B-cell lymphomas (DLBCL). Our aim was to investigate the prognostic performance of different PET biomarkers in a multicenter setting. METHODS: We investigated baseline volumetric values [metabolic tumor volume (MTV) and total lesion glycolysis (TLG), also normalized for body weight] segmented with three different methods [>SUV4 (glob4); 41% isocontour (41pc), and a gradient-based lesion growing algorithm (grad)] and interim parameters [Deauville score, maximal standardized uptake value (ΔSUVmax), modified qPET, and ratio PET (rPET)] alongside clinical parameters (stage, revised International Prognostic Index), using 24-month progression-free survival as the clinical endpoint. Receiver operating characteristics analyses were performed to define optimal cutoff points for the continuous PET parameters. RESULTS: A total of 107 diffuse large B-cell lymphoma patients were included (54 women; mean age: 53.7 years). MTV and TLG calculations showed good correlation among glob4, 41pc, and grad methods; however, optimal cutoff points were markedly different.Significantly different PFS was observed between low- and high-risk groups according to baseline MTV, body weight-adjusted (bwa) MTV, TLG, bwaTLG, as well as interim parameters Deauville score, ΔSUVmax, mqPET, and rPET. Univariate Cox regression analyses showed hazard ratios (HRs) lowest for bwaMTVglob4 (HR = 2.3) and highest for rPET (HR = 9.09). In a multivariate Cox-regression model, rPET was shown to be an independent predictor of PFS ( P = 0.041; HR = 9.15). Combined analysis showed that ΔSUVmax positive patients with high MTV formed a group with distinctly poor PFS (35.3%). CONCLUSION: Baseline MTV and TLG values and optimal cutoff points achieved with different segmentation methods varied markedly and showed a limited prognostic impact. Interim PET/CT parameters provided more accurate prognostic information with semiquantitative 'Deauville-like' parameters performing best in the present study.

Combined Visual and Semiquantitative Evaluation Improves Outcome Prediction by Early Midtreatment 18F-FDG PET in Diffuse Large B-Cell Lymphoma.

The purpose of this study was to assess the predictive and prognostic value of interim FDG PET (iPET) in evaluating early response to immunochemotherapy after 2 cycles (PET-2) in diffuse large B-cell lymphoma (DLBCL) by applying 2 different methods of interpretation: the Deauville visual 5-point scale (5-PS) and a change in SUV (ΔSUV) by semiquantitative evaluation. Methods: In total, 145 patients with newly diagnosed DLBCL underwent pretreatment PET and PET-2 assessment. PET-2 was classified according to both 5-PS and percentage ΔSUV. Receiver-operating-characteristic analysis was performed to compare the accuracy of the 2 methods for predicting progression-free survival. Survival estimates, based on each method separately and combined, were calculated for iPET-positive (iPET+) and iPET-negative (iPET-) groups and compared. Results: Both with 5-PS and with ΔSUV-based evaluations, significant differences were found between the progression-free survival of iPET- and iPET+ patient groups (P < 0.001). Visually, the best negative predictive value (NPV) and positive predictive value (PPV) occurred when iPET was defined as positive if the Deauville score was 4-5 (89% and 59%, respectively). Using the 66% ΔSUV cutoff reported previously, NPV and PPV were 80% and 76%, respectively. ΔSUV at the 48.9% cutoff, reported for the first time here, produced 100% specificity along with the highest sensitivity (24%). The 5-PS and a semiquantitative ΔSUV of less than 48.9% for each PET-2 gave the same PET-2 classification (positive or negative) in 70% (102/145) of all patients. This combined classification delivered NPV and PPV of 89% and 100%, respectively, and all iPET+ patients failed to achieve or remain in remission. Conclusion: In this large consistently treated and assessed series of DLBCL patients, iPET had good prognostic value interpreted either visually or semiquantitatively. We determined that the most effective ΔSUV cutoff was 48.9% and that when combined with 5-PS assessment, a positive PET-2 result was highly predictive of treatment failure.

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.

Ultrasound surveillance for cholangiocarcinoma in an endemic area: A prove of survival benefits.

BACKGROUND AND AIM: Cholangiocarcinoma (CCA) is an aggressive malignancy with rapid progression and poor prognosis. Abdominal ultrasound surveillance may detect early-stage malignancy and improve surgical outcome. However, little data exist on the benefits of abdominal ultrasound surveillance in populations at high risk for CCA development in an endemic area. This study compared survival outcomes of CCA patients recruited through abdominal ultrasound surveillance program and those presented to the hospital independent of surveillance. METHODS: The surveillance population-based cohort was 4225 villagers in Northern Thailand, aged 30-60 years, who consented to a 5-year abdominal ultrasound surveillance program, which included interval ultrasound examinations every 6 months. The non-surveillance cohort was hospital-based CCA patients diagnosed during April 2007 to November 2015. Numbers of operable tumors, percentages of R0 resection, and survival analyses were compared between the two cohorts. RESULTS: There were 48 and 192 CCA patients in the surveillance and the non-surveillance cohorts, respectively. Of these, 37/48 (77.1%) and 22/192 (11.5%) were in an operable stage and R0 resections performed in 36/48 (97.3%) and 14/192 (63.6%), respectively. The median survival in each group was 31.8 and 6.7 months, respectively (with correction of lead time bias) (P < 0.0001). By multivariate analysis, abdominal ultrasound surveillance (hazard ratio [HR] = 0.41; P = 0.012), operable stage (HR = 0.11; P < 0.001), and serum albumin ≥ 3.5 g/dL (HR = 0.42; P < 0.001) were significantly associated with decreased mortality, whereas size of CCA (HR = 1.11; P < 0.001), serum alanine aminotransferase > 40 IU/L (HR = 1.71; P = 0.017), and tumor recurrence (HR = 4.86; P = 0.017) were associated with increased mortality. CONCLUSION: Abdominal ultrasound surveillance provided survival benefits and should be considered in areas highly endemic for CCA to reduce mortality.

Common Molecular Subtypes Among Asian Hepatocellular Carcinoma and Cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) and hepatocellular carcinoma (HCC) are clinically disparate primary liver cancers with etiological and biological heterogeneity. We identified common molecular subtypes linked to similar prognosis among 199 Thai ICC and HCC patients through systems integration of genomics, transcriptomics, and metabolomics. While ICC and HCC share recurrently mutated genes, including TP53, ARID1A, and ARID2, mitotic checkpoint anomalies distinguish the C1 subtype with key drivers PLK1 and ECT2, whereas the C2 subtype is linked to obesity, T cell infiltration, and bile acid metabolism. These molecular subtypes are found in 582 Asian, but less so in 265 Caucasian patients. Thus, Asian ICC and HCC, while clinically treated as separate entities, share common molecular subtypes with similar actionable drivers to improve precision therapy.

Role of quantitative hepatitis B surface antigen in predicting inactive carriers and HBsAg seroclearance in HBeAg-negative chronic hepatitis B patients.

To evaluate quantitative hepatitis B surface antigen (qHBsAg) as a diagnostic marker for inactive carriers (ICs) and hepatitis B surface antigen (HBsAg) seroclearance in hepatitis B e antigen (HBeAg)-negative chronic hepatitis B (CHB) patients. We retrospectively studied 300 HBeAg-negative CHB patients with initial serum hepatitis B virus (HBV) Deoxyribonucleic acid (DNA) levels <2000 IU/mL. Serum HBV DNA and alanine aminotransferase (ALT) levels were monitored every 6 months for 24 months. ICs were identified as having persistent HBV DNA levels <2000 IU/mL and normal ALT levels, whereas active carriers (ACs) were identified as having HBV DNA levels ≥2000 IU/mL, with or without elevated ALT levels. The serum qHBsAg level was defined at baseline and evaluated as a diagnostic predictor using a receiver-operating characteristic curve. The study group comprised 134 men and 166 women with a median age of 41.5 years. At baseline, 200 ICs displayed lower levels of qHBsAg (1492 IU/mL) compared with 100 ACs (2936 IU/mL) (P = 0.005). The qHBsAg level was independently associated with the IC state and HBsAg seroclearance. Baseline qHBsAg levels <1000 IU/mL and HBV DNA levels <2000 IU/mL, when detected simultaneously, allowed for identification of ICs with 41% sensitivity and 72% specificity. Fifteen patients (5%) displayed HBsAg seroclearance after 24 months. A qHBsAg cutoff value of <50 IU/mL provided 100% sensitivity and 92% specificity in predicting HBsAg seroclearance. The qHBsAg level at a single timepoint among HBeAg-negative CHB patients with low HBV DNA levels at baseline was not a predictive marker for ICs; however, it accurately predicted spontaneous HBsAg seroclearance at 24 months.

Hepatocellular carcinoma screening and surveillance in 2293 chronic hepatitis B patients in an endemic area.

AIM: To determine the role of screening and surveillance of hepatocellular carcinoma (HCC) in treatment-naïve chronic hepatitis B (CHB) patients. METHODS: We recruited 2293 CHB patients (both males and females; aged 20-65 years). All patients were screened and underwent surveillance using abdominal ultrasonography (AUS) and serum alpha-fetoprotein (AFP) assay every 6 mo. The diagnosis, staging and treatment of HCC followed the American Association for the Study of Liver Diseases practice guidelines and the Barcelona Clinic Liver Cancer guidelines. The exclusion criteria included: decompensated cirrhosis; a history of any cancer in the last 5 years; previous antiviral treatment for CHB; concurrent infection with hepatitis C virus or human immunodeficiency virus; a Karnofsky Performance Status score < 60%; or any medical condition preventing eligibility to complete the protocol. The prevalence and incidence rates of HCC were determined; survival rates were calculated at 3-year post HCC diagnosis. The sensitivity and specificity were calculated on a per-patient basis. RESULTS: Among 2293 treatment-naïve CHB patients, seven cases had HCC at initial screening, giving a prevalence rate of 305 per 100000 persons; 3.3% were diagnosed with liver cirrhosis, all of which were Child-Pugh class A. With a median follow-up time of 42 (range, 3-48) mo, 10 additional cases were diagnosed with HCC, resulting in an incidence rate of 143 per 100000 persons per year. This burden was as high as that reported in other studies from East Asian countries. All HCC patients were aged ≥ 40 years. Most were at an early stage (Stage 0, A or B); 14/17 cases were successfully treated with surgical resection or radiofrequency ablation, with a high 3-year survival rate of 90%. Hemangioma was the most common focal liver lesion in CHB patients detected by AUS; the main causes of AFP elevation at the initial screening were cirrhosis, increased alanine aminotransferase level and HCC. AUS detected 16/17 HCC cases whereas AFP levels ≥ 20 µg/L at diagnosis were observed in only 7/17 patients, most with a tumor size > 5 cm. For HCC screening and surveillance, AUS had a sensitivity and specificity of 94% and 82%, respectively, whereas the sensitivity and specificity of AFP at a cut-off value of ≥ 20 µg/L were 41% and 98%, respectively. Combined use of AUS and AFP assay did not improve effectiveness. CONCLUSION: Implementation of active screening and surveillance using AUS to detect early-stage HCC in naïve CHB patients aged ≥ 40 years in an endemic area is of benefit.

High prevalence of advanced colorectal neoplasia in the Thai population: a prospective screening colonoscopy of 1,404 cases.

BACKGROUND: Increasing morbidity and mortality from colorectal cancer is evident in recent years in the developing Asian nations. Particularly in Thailand and most neighbouring low-income countries, screening colonoscopy is not yet recommended nor implemented at the national policy level. METHODS: Screening colonoscopy was offered to 1,500 healthy volunteers aged 50-65 years old who were registered into the program between July 2009 and June 2010. Biopsy and surgery was performed depending on the identified lesions. Fecal immunochemical tests (FIT) were additionally performed for comparison with colonoscopy. RESULTS: There were 1,404 participants who underwent colonoscopy. The mean age of the cohort was 56.9 ± 4.2 years and 69.4 % were females. About 30 % (411 cases) of all colonoscopies had abnormal colonoscopic findings, and of these, 256 cases had adenomatous polyps. High risk adenomas (villous or tubulovillous or high grade dysplasia or size > 1 cm or > 3 adenomatous polyps) were found in 98 cases (7 %), low risk adenoma in 158 cases (11.3 %), and hyperplastic polyps in 119 cases (8.5 %). Eighteen cases (1.3 %) had colorectal cancer and 90 % of them (16 cases) were non-metastatic including five stage 0 cases, seven stage I cases, and four stage IIA cases. Only two cases had metastasis: one to regional lymph nodes (stage IIIB) and another to other organs (stage IVA). The most common cancer site was the distal intestine including rectum (7 cases, 38.9 %) and sigmoid colon (7 cases, 38.9 %). Ten colorectal cancer cases had positive FIT whereas 8 colorectal cancer cases were FIT-negative. The sensitivity and specificity of FIT was 55.6 % and 96.2 %, respectively, while the positive predictive value was 16.4 % and negative predictive value was 99.4 %. The overall survival of colorectal cancer cases at 5-year was 83.3 %. CONCLUSION: High prevalence of colorectal cancer and high-risk adenoma was found in the Thai population aged 50-65 years old by screening colonoscopy. FIT was not sensitive enough to detect colorectal cancer in this asymptomatic cohort. Integration of screening colonoscopy into the national cancer screening program should be implemented to detect early cases of advanced colorectal neoplasia and improve survival of colorectal cancer patients in Thailand.

Intracranial Hemorrhage in Patients with Hematologic Disorders: Prevalence and Predictive Factors.

BACKGROUND: Intracranial hemorrhage (ICH) is an uncommon complication in patients with hematologic disorders although high fatality rates have been shown in these patients. At present, no epidemiological data regarding ICH in patients with hematologic disorders has been collected and/or reported in Thailand. OBJECTIVE: The purpose of this study was to determine the incidence of ICH in hospitalized patients with hematologic disorders and to identify predictive factors associated with ICH in these patients. MATERIAL AND METHOD: The medical records of all patients with hematologic disorders admitted to Siriraj Hospital (Bangkok, Thailand) between January 2002 and September 2011 were reviewed. Patients with ICH were identified and factors associated with ICH were investigated using a retrospective case-control design. RESULTS: Of 9,62 patients identified with hematologic disorders, ICH was diagnosed in 106 (1.1%). The ICH rate was higher in acute myeloid leukemia (AML) patients than in patients with other hematologic malignancies (4.29% vs. 0.78%; p<0. 001) and higher in aplastic anemia (AA) patients than in patients with other benign hematologic disorders (4.00% vs. 0.97%; p<0.001). Cortical hemorrhage was the main presentation in all hematologic disorders, with a single lesion in the parietal area as the most common site. The overall mortality rate was 85% with most patients succumbing within two days of onset. The independent predictors of ICH were hyperleukocytosis and a low platelet count in AML patients, and ecchymosis, upper gastrointestinal hemorrhage, hematuria, and a low platelet count in AA patients. CONCLUSION: AML and AA patients had the highest risk of ICH compared with other hematologic disorders and several predictive factors for ICH were identified.

Cholinergic activation enhances retinoic acid-induced differentiation in the human NB-4 acute promyelocytic leukemia cell line.

The non-neuronal cholinergic system (NNCS) has been shown to play a role in regulating hematopoietic differentiation. We determined the expression of cholinergic components in leukemic cell lines by Western blotting and in normal leukocyte subsets by flow cytometry and found a heterogeneous expression of choline acetyltransferase (ChAT), acetylcholinesterase (AChE), choline transporter (CHT), M3 muscarinic acetylcholine receptor (M3-mAChR) and α7 nicotinic acetylcholine receptor (α7-nAChR). We then evaluated NNCS role in differentiation of human NB-4 acute promyelocytic leukemia cell line and discovered a dramatic induction of M3-mAChR after all-trans retinoic acid (ATRA) treatment (p<0.0001). Adding carbachol which is a cholinergic agonist to the ATRA treatment resulted in an increase of a granulocytic differentiation marker (CD11b) as compared with ATRA treatment alone (p<0.05), indicating that cholinergic activation enhanced ATRA in inducing NB-4 maturation. The combination of carbachol and ATRA treatment for 72h also resulted in decreased viability and increased cleaved caspase-3 expression when compared with ATRA treatment alone (p<0.05). However, this combination did not cause poly (ADP-ribose) polymerase (PARP) cleavage. Overall, we have shown that NB-4 cells expressed M3-mAChR in a differentiation-dependent manner and cholinergic stimulation induced maturation and death of ATRA-induced differentiated NB-4 cells.

Ultrasound screening for cholangiocarcinoma could detect premalignant lesions and early-stage diseases with survival benefits: a population-based prospective study of 4,225 subjects in an endemic area.

BACKGROUND: Thailand has a high incidence of cholangiocarcinoma (CCA), particularly in the north and northeastern regions. Most CCA patients come at a late, unresectable stage and presently no optimal screening test for CCA has been established. We determined the prevalence of CCA in a remote northern village and explored if screening could lead to early detection and survival benefits. METHODS: A 5-year population-based study was started in October, 2011 for consented Thai individuals, aged 30-60 years. The screening program comprised blood testing, stool examination and serial ultrasonography every 6 months. RESULTS: During the first 3 years, 4,225 eligible individuals were enrolled. CCA was detected in 32 patients, with a mean age of 51.9 years (41-62 years), and 21/32 cases were at a curative resectable stage. The prevalence rate of CCA was 165.7 per 100,000 and one- and two-year incidence rate was 236.7/100,000 and 520.7/100,000, respectively. One- and 2-year overall survival rates of CCA patients were 90.9 and 61.5 %, respectively. Prognosis was better in resectable cases with 100 % 1-year and 77.8 % 2-year survival rates. Interestingly, premalignant pathological lesions (stage 0) were identified in 11 cases with 100 % 3-year survival rate. Serum biomarkers and alkaline phosphatase were not sufficient to detect early-stage disease. In 22 patients, stool samples were positive for Opistorchis viverrini, based on polymerase chain reaction. CONCLUSION: Detection of premalignant lesions and early-stage resectable CCA by ultrasonography resulted in improved clinical outcome. Ultrasonography should be offered as a first screening tool for CCA in an endemic area until other useful biological markers become available.

Clinical Features and Outcomes of Patients with Non-Iron Nutritional Deficiency Anemia in an In-Patient Setting at Siriraj Hospital: A 10-Year Retrospective Study

Background: Anemia is a major public health issue despite preventive interventions. Data on non-iron nutritional deficiency anemia in hospitalized patients are limited. Objective: This study explored the incidence, clinical features, and outcomes of hospitalized patients diagnosed with non-iron nutritional deficiency anemia at a major teaching hospital in Thailand. Material and Method: Medical records of in-patient departments dated between January 2001 and June 2011 were retrospectively reviewed. Results: One hundred and two cases were identified, including 40 patients with vitamin B12 deficiency, 46 with folate deficiency, and 16 with other nutritional deficiency anemias; corresponding incidence rates were 0.4, 0.6, and 0.2 cases per 100,000 per year, respectively. Patients with vitamin B12 deficiency were mostly female, while patients with folate deficiency were preponderantly male. Glossitis and pancytopenia were common characteristics of vitamin B12 deficiency cases, whereas alcohol abuse and cirrhosis were more frequent in folate deficiency cases, as expected. Serum ferritin levels were relatively high across all categories. A significant proportion of anemia cases across all subgroups presented concomitantly with anorexia or poor food intake, which indicates underlying nutritional problems in these patients. Survival of patients with folate and other types of nutritional deficiency anemia was lower than for patients with vitamin B12 deficiency anemia (hazard ratio [HR] and p-values were 2.65, 0.001 and 2.35, 0.023, respectively). Hemoglobin normalization in patients with vitamin B12 deficiency anemia could be achieved by intramuscular injection and oral vitamin B12 treatment in 55.56% and 33.33% (p = 0.248), with a median response time of 9 and 86 weeks (p = 0.151), respectively. Conclusion: Non-iron nutritional deficiency anemia was not common in hospitalized patients in this study. Vitamin B12 injections resulted in faster responses, but with similar efficacy compared with oral treatments. Survival of patients with vitamin B12 deficiency anemia was significantly better than that of those with folate or other types of nutritional anemia.

Acquired somatic mutations of isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2) in preleukemic disorders.

Mutations of isocitrate dehydrogenase isoform 1 and 2 (IDH1 and IDH2) genes have been identified in glioblastoma and acute myeloid leukemia (AML). However, little is known about the molecular alterations of IDH genes in preleukemic disorders with a propensity to transform to AML. We performed polymerase chain reaction-denaturing high performance liquid chromatography (PCR-DHPLC) followed by direct sequencing to detect IDH mutations in 237 patients with myeloproliferative neoplasms (MPNs; n=108), myelodysplastic syndrome (MDS; n=22), paroxysmal nocturnal hemoglobinuria (PNH; n=41), and aplastic anemia (AA; n=66). No IDH1 R132 and IDH2 R172 mutations were identified in the entire cohort, whereas IDH1 G105G allele was detected in 4/108 MPN (3.70%), 2/22 MDS (9.09%), and 2/41 PNH (4.88%) patients. Three IDH2 R140Q mutations were found in 2/108 MPN (1.85%) and 1/22 MDS (4.54%) patients, while one IDH2 G145G allele was found in 0.92% (1/108) of MPN patients. Overall, our data suggest that IDH mutations are rare in the preleukemic disorders and may not be the major initial step in AML leukemogenesis.

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.

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.

BCR-ABL kinase domain mutations in tyrosine kinase inhibitors-naïve and -exposed Southeast Asian chronic myeloid leukemia patients.

BCR-ABL kinase domain (KD) mutation is the main mechanism associated with resistance to tyrosine kinase inhibitors (TKI) in chronic myeloid leukemia (CML) patients. This study targeted a large cohort of CML (n=171) comprising 80 naïve CML cases without prior TKI exposure as well as 91 cases undergoing 1st generation (imatinib) and/or 2nd generation (nilotinib/dasatinib) TKI therapy. KD mutations were analyzed by denaturing high performance liquid chromatography followed by direct sequencing. Twenty-one types of mutations were found in 37 patients including 13 known mutations and 8 previously unidentified mutations. Thirty cases had a single mutation while 7 cases had multiple mutations. Twenty-three percent of patients receiving first-line imatinib, 69% of imatinib-resistant patients receiving 2nd generation TKI, and 75% of advanced phase patients treated with front-line 2nd generation TKI had KD mutations. Interestingly, 9% of TKI-naïve CML cases were also discovered to carry the KD mutations including 5 novel variants. Patients who received hydroxyurea had a 2-fold increase in KD mutations as compared to newly diagnosed patients but they still had a lower mutation frequency than TKI-exposed cases. Mutations in the naïve cases were mainly localized in the C-helix domain and SH3 contact site whereas in exposed cases predominantly in the drug contact site, P-loop, and catalytic domain. T315I resistant mutation was identified only in TKI-exposed cases. In conclusion, several known and novel BCR-ABL KD mutations were discovered in the TKI-naïve and -exposed Southeast Asian CML patients, supporting the concept that naturally occurring KD mutations were present in leukemic cells prior to drug exposure. T315I resistant mutation was completely undetectable in this naïve Southeast Asian cohort; its incidence, however, increases with drug exposure.

Diffuse large B cell lymphoma presenting as Horner's syndrome in a patient diagnosed with neurofibromatosis type 1: a case report and review of the literature.

INTRODUCTION: Horner's syndrome has a variety of etiologies ranging from benign to serious life-threatening conditions and has been infrequently reported as a presenting symptom of patients with lymphoid neoplasms. Only one case of Burkitt's lymphoma presenting with toothache, paresthesia, and Horner's syndrome has been described and no case reports of diffuse large B-cell lymphoma as the etiology of Horner's syndrome currently exist in the literature. In addition, lymphoid neoplasms have rarely been reported to occur in patients with neurofibromatosis type 1 despite an increased risk of many types of cancer in such cases. CASE PRESENTATION: A 28-year-old Thai man presented with a progressively enlarged left supraclavicular mass together with a significant weight loss and night sweating for four months. He also noticed hoarseness and ptosis of his left eye associated with double vision for two months. Physical examination revealed large supraclavicular lymphadenopathy and Horner's syndrome (ptosis, miosis, and anhydrosis) on the left side of his face. A large mediastinal mass was clearly detected by chest X-ray and computed tomography and subsequent lymph node biopsy provided a diagnosis of diffuse large B-cell lymphoma. Interestingly, the patient was also definitely diagnosed with neurofibromatosis type 1 from multiple café au lait macules, axillary freckles, three neurofibromas, multiple Lisch nodules, and a history of affected family members. He subsequently received chemotherapy with a good response. Twenty-seven cases of various types of lymphoid neoplasms previously reported to occur in neurofibromatosis type 1 patients were also extracted from the literature. All cases were non-Hodgkin lymphoma and the major subtype was T-cell. Only nine cases were B-cell lymphoma. The majority of cases were young with a median age at lymphoma diagnosis of 9.4 years (range 1.1 to 77 years). Two-thirds of the cases were boys or men. Other concomitant malignancies were brain tumor, colorectal cancer, pheochromocytoma, and acute lymphoblastic leukemia. CONCLUSIONS: We describe for the first time a case of diffuse large B-cell lymphoma that occurred in a neurofibromatosis type 1 patient with Horner's syndrome. Horner's syndrome can be an initial manifestation of diffuse large B-cell lymphoma. Patients who present with a classical triad of Horner's syndrome should always be fully investigated for lymphomatous involvement, especially in the thorax. The exact molecular mechanism for diffuse large B-cell lymphoma development in neurofibromatosis type 1 cases remains to be elucidated.