Cytogenetic Analysis Of Bone Marrow Research Articles

Feasibility, accuracy, and usability analysis of MapAML, a first-in-class app for integrated diagnosis in acute myeloid leukemia.

Performing a comprehensive diagnosis of acute myeloid leukemia (AML) is complex and involves the integration of clinical information, bone marrow morphology, immunophenotyping, cytogenetic, and molecular analysis, which can be challenging to the general hematologist. The aim of this study was to evaluate the usability and accuracy of MapAML, a smartphone app for integrated diagnosis in AML, created to aid the hematologist in its clinical practice. App performance was evaluated in dedicated sessions, in which 21 hematologists or fellows in hematology performed an integrated diagnosis of deidentified real-world clinical AML cases, first without and posteriorly with MapAML use. Diagnosis accuracy increased after MapAML utilization, with the average score going from 7.08 without app to 8.88 with app use (on a scale from 0 to 10), representing a significant accuracy improvement (p = .002). Usability evaluation was very favorable, with 81% of users considering the app very or extremely simple to use. There was also a significant increase in confidence to perform a complete and accurate diagnosis in AML after app use, with 61.9% of the participants willing to use the app in their clinical practice. In this study, MapAML increased accuracy with excellent usability for integrated diagnosis in AML.

Usefulness of Peripheral Blood Samples in Diagnosis and Prognosis Assessment of Myelodysplastic Syndromes and Chronic Myelomonocytic Leukemia Low Risk Patients Using Next Generation Sequencing

Introduction: myelodysplastic syndromes (MDS) are a heterogeneous group of hematological diseases. Morphologic bone marrow (BM) examination, cytogenetic analysis and molecular techniques are indispensable for accurate diagnosis and disease classification. BM constitutes the most suitable sample for molecular studies, however, peripheral blood (PB) can be obtained by minimally invasive venipuncture and would be ideal for comprehensive sequential monitoring of MDS over time. Aim: to assess whether targeted deep sequencing (TDS) allows the detection of somatic variants from diagnosis and follow-up PB samples and if the molecular profiles are comparable to those from BM samples. Methods: preliminary results include the study of 32 low risk patients (28 MDS and 4 chronic myelomonocytic leukemia, CMML). At least one sample, either BM or PB, was obtained at the moment of diagnosis and whenever possible, paired samples obtained the same day were collected. Over the course of the disease only PB samples were obtained. Libraries were prepared for all available samples using DNA from whole BM or whole PB using a custom hybridization-probe based panel (KAPA HyperCap, Roche), including selected exons of 50 myeloid-related genes. TDS was performed on Illumina MiSeq instrument at a mean coverage of 1000x. Data were analyzed using local bioinformatic pipeline. Only those variants with ≥100x locus coverage and ≥25 reads for the alternative allele (quality criteria) were considered for downstream analysis. Variants were filtered according to variant type, location, read depth (>100x) and population frequency (<1%). Results: patients´ characteristics are described in table 1. The combination of cytogenetics and TDS identified genetic alterations in 29/32 patients (91%). TDS allowed the identification of somatic variants in 27/32 patients (85%), being TET2 (n=23, 29%), SF3B1 (n=12, 15%) and ASXL1 (n=8, 10%) the most frequently mutated genes. Considering molecular information, restratification of IPSS-R to IPSS-M risk groups of MDS patients (n=28) was performed (Table 1). As previously described (Sauta et al, J Clin Oncol, 2023), we performed a five-to-five comparison of IPSS-R and IPSS-M patients' distribution (merging moderate low and moderate high to moderate in IPSS-M). This resulted in the restratification of 36% of MDS patients (10/28) of which 6% (n=2) were downstaged and 25% (n=8) were upstaged. Suitability of PB samples for NGS analysis was evaluated in 21 patients in which paired BM and PB samples were obtained at diagnosis. Overall, 65 variants were found, and 63 of them were detectable in both BM and PB samples. Despite the other two variants were also detected in PB samples at low variant allele frequency (VAF) values ( TET2 and NRAS, VAF=1,5% and 0,99%, respectively), they strictly did not meet our quality criteria (≥25 reads for the alternative allele). Nevertheless, a strong correlation was identified between VAF values for the individual variants detected in BM vs. PB (Figure 1). As part of this project, molecular profile during patients' follow-up is being studied using PB samples. To date, at least one follow-up sample of each patient was studied. The mean time between diagnosis and the first follow-up sample was 28 months (range 6-84 months). After this time, and despite molecular profiles are heterogeneous, variations could be detected in up to 72% of patients (23/32) using PB samples. The most commonly detected variation was an increasing in VAF values (9/23; defined by a difference >5% in relation to the VAF detected in the previous sample), followed by a new variant detection + increasing in VAF values (7/23), new variant detection only (6/23) and a single case where simultaneous decreasing VAF and disappearance of subclones was detected (single patient receiving treatment with hypomethylating agent). Conclusion: molecular testing has become increasingly important for accurate diagnosis and risk assessment in MDS and CMML patients, specially with the new classifications and prognostic scoring system. Here we demonstrate that the molecular analysis from PB cells constitutes an appropriate alternative for the detection and quantification of somatic variants in MDS patients at diagnosis and during disease course. Funding: Bristol Myers Squibb (PI-13279-IIT), FEHH and 2021 SGR 00560 (GRC).

Minimal Diagnostic Value of Karyotyping for Suspected Myelodysplastic Syndrome in the Absence of Bone Marrow Dysplasia

BACKGROUND: Myelodysplastic syndrome (MDS) is a group of hematopoietic stem cell disorders characterized by ineffective hematopoiesis, bone marrow dysplasia, and cytopenias. Marrow morphology, cytogenetics, and sequencing panels are important for the diagnosis and prognosis of MDS and are often obtained in parallel for clinical workup. The diagnostic utility of time-consuming cytogenetic analyses in cases without any significant dysplasia is uncertain. OBJECTIVE: We sought to determine the diagnostic utility of cytogenetic analysis of bone marrow aspirate/biopsies obtained for the indication of suspected MDS in the absence of morphological dysplasia. METHODS: We identified bone marrow aspirate/biopsy cases obtained for the indication of suspected MDS between 2021-01-01 and 2022-06-22 from the Greater Vancouver Area, Canada that underwent cytogenetic analysis at clinical request. Anemia, neutropenia, and thrombocytopenia were defined as peripheral blood counts of hemoglobin <120 g/L for women or <130 g/L for men, neutrophils <2 x109/L, and platelets <150 x109/L, respectively. RESULTS: Fifty-four cases met criteria for review (55.5% male sex; mean age = 62.6 years [SD = 16.6 years]). The mean (SD) peripheral blood count parameters for the cohort were: hemoglobin 108 g/L (22 g/L), mean corpuscular volume 94 fL (8 fL), neutrophil count 2.84 x109/L (2.08 x109/L), and platelet count 175 x109/L (132 x109/L). Anemia, neutropenia, thrombocytopenia, pancytopenia, and any cytopenia were present in 41 (75.9%), 23 (42.6%), 26 (48.1%), 13 (24.1%), and 48 cases (88.9%), respectively. For cases with anemia, neutropenia, or thrombocytopenia, the corresponding mean (SD) peripheral blood counts were: 99 g/L (18 g/L), 1.25 x109/L (0.56 x109/L), and 77 x109/L (44 x109/L), respectively. Marrow was hypocellular and hypercellular for age in 11 (20.4%) and 9 (16.7%) of the cases, respectively. The only abnormal karyotypes were 2 cases of loss of chromosome Y (3.7%; 70-year-old with 18/20 metaphases and 84-year-old with 14/20 metaphases). Both cases had no evidence of hematologic neoplasia, which is in keeping with an age-related finding. Additionally, targeted myeloid panel next-generation sequencing was performed for 8/54 cases for suspected myeloid neoplasm. There were: 3 cases with no reportable variants, 3 cases with variants of uncertain significance, and 2 cases with pathogenic TET2 variants. None of the cases with reportable variants identified were found in cases showing loss of chromosome Y. CONCLUSION: This study suggests that in the absence of myeloid dysplasia of the bone marrow, there is minimal diagnostic value of cytogenetic analysis for the workup of suspected MDS. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

HOW IMPORTANT IS THROMBOCYTOSIS FOR PROGNOSIS OF MDS WITH 3Q21Q26 SYNDROME?

The structural changes involving the long arm of chromosome 3 at bands 3q21 and 3q26.2 in the form of inversion are named paracentric inversion inv(3)(q21q26.2) and in myeloid neoplasms have long been recognized, but are rare. The 3q21q26 syndrome usually occurs in a high-risk myelodysplastic syndrome (MDS) or the setting of acute myeloid leukemia (AML) and is most commonly reported as inv(3)(q21q26.2). Myeloid neoplasms with inv(3) are rare disorders with an incidence of 1% in MDS and AML. Thus, this report aims to show a patient with MDS and high platelets count who presented inv(3)(q21q26.2). A 72-year-old woman looked for medical attention due to fatigue and weakness. The patient reported a history of smoking for 41 years and denied any exposure to toxic agents. At physical examination, only pale was detected. A complete blood count revealed hemoglobin 7g/dL, MCV = 94 fL, leukocytes 5,600/mm3, neutrophils 2,242/mm3 and thrombocytosis with a platelet count of 514,000/mm3. Bone marrow aspirate showed dyserythropoiesis in 30% of cells and 6,5% blasts. The bone marrow cytogenetic analysis showed 46,XX,inv(3)(q21q26.2)[16]/46,XX[4]. The diagnosis of MDS with excess blasts - 1 was established according to the 2016 World Health Organization classification and International Prognostic Scoring System was very high. While waiting for beginning treatment, the patient died of respiratory failure due to COVID-19. Myelodysplastic syndrome with inv(3)(q21q26.2) is a rare aggressive disorder that occurs in less than 1% of all MDS cases and has been associated with a poor outcome: chemoresistance, high risk of leukemic transformation and short survival. Our case showed thrombocytosis with a platelet count of 514,000/mm3. The incidence of thrombocytosis in MDS has been reported in 8% of cases with platelets > 400 ×109/L. The major report which evaluated thrombocytosis in MDS studied 2,042 cases, detecting high platelets count in 5% of cases (102/2,042). It appears that thrombocytosis does not adequately capture the aggressive nature of inv(3)(q21q26.2) in MDS but still plays an important role in the pathogenesis of this heterogeneous and dynamic disease. Our patient reported herein showed dyserythropoiesis in 30% of cells and 6,5% blasts, but nothing was detected regarding megakaryocytic lineage. Our patient died very soon after diagnosis due to viral infection. Thrombocytosis is an unusual clinical feature in MDS associated with inv(3)(q21q26.2) and the unfavorable prognosis of inv(3) is independent of thrombocytosis.

The Dawn of next generation DNA sequencing in myelodysplastic syndromes- experience from Pakistan

BackgroundMyelodysplastic syndromes (MDS) are clonal disorders of hematopoietic stem cells exhibiting ineffective hematopoiesis and tendency for transformation into acute myeloid leukemia (AML). The available karyotyping and fluorescent in situ hybridization provide limited information on molecular abnormalities for diagnosis/prognosis of MDS. Next generation DNA sequencing (NGS), providing deep insights into molecular mechanisms being involved in pathophysiology, was employed to study MDS in Pakistani cohort.Patients and methodsIt was a descriptive cross-sectional study carried out at National institute of blood diseases and bone marrow transplant from 2016 to 2019. Total of 22 cases of MDS were included. Complete blood counts, bone marrow assessment and cytogenetic analysis was done. Patients were classified according to revised WHO classification 2016 and IPSS score was applied for risk stratification. Baseline blood samples were subjected to analysis by NGS using a panel of 54 genes associated with myeloid malignancies.ResultsThe median age of patients was 48.5 ± 9.19 years. The most common presenting complaint was weakness 10(45.45%). Cytogenetics analysis revealed abnormal karyotype in 10 (45.45%) patients. On NGS, 54 non-silent rare frequency somatic mutational events in 29 genes were observed (average of 3.82 (SD ± 2.08) mutations per patient), including mutations previously not observed in MDS or AML. Notably, two genes of cohesin complex, RAD21 and STAG2, and two tumor suppressor genes, CDKN2A and TP53, contained highest number of recurrent non-silent somatic mutations in the MDS. Strikingly, a missense somatic mutation p.M272Rof Rad21 was observed in 13 cases. Overall, non-silent somatic mutations in these four genes were observed in 21 of the 22 cases. The filtration with PharmGKB database highlighted a non-synonymous genetic variant rs1042522 [G > C] located in the TP53. Genotype GG and GC of this variant are associated with decreased response to cisplatin and paclitaxel chemotherapy. These two genotypes were found in 13 cases.ConclusionSequencing studies suggest that numerous genetic variants are involved in the initiation of MDS and in the development of AML. In countries like Pakistan where financial reservation of patients makes the use of such analysis even more difficult when the availability of advanced techniques is already a prevailing issue, our study could be an initiating effort in adding important information to the local data. Further studies and large sample size are needed in future to enlighten molecular profiling and ultimately would be helpful to compare and contrast the molecular characteristics of Asian versus global population.

Long-term outcome of tyrosine kinase inhibitor treatment in children and adolescents with newly diagnosed chronic myeloid leukemia in chronic phase.

Long-term outcome of tyrosine kinase inhibitor treatment in children and adolescents with newly diagnosed chronic myeloid leukemia in chronic phase.

Pediatric Chronic Myeloid Leukemia with Megakaryocytic Blast Crisis as Initial Presentation: Case Report and Review of Literature

Abstract: Background: Pediatric Chronic Myeloid Leukemia (CML) is a rare entity accounting for 2-3% of pediatric malignancies. CML rarely presents as Blast Crisis (BC) at the time of diagnosis, and megakaryocytic blast crisis is even rarer. Case Presentation: We herein, report a case of a young female, 10-year-old who presented with anemia, leukocytosis and massive splenomegaly. Clinical features, peripheral film and bone marrow findings were consistent with CML in megakaryocytic blast crisis. Bone marrow cytogenetic analysis revealed karyotype of 46, XX, t(9:22)(q34;q11.2) in 20 metaphases and BCR-ABL P210 by PCR was detected with transcript level of 83%, which further confirmed our diagnosis. Conclusion: De novo presentation of chronic myeloid leukemia with megakaryocytic blast crisis is rarely observed in pediatric population with very few cases published till now. We are presenting this case because of its rarity, likelihood of misdiagnosis as AML (M7) and poor prognosis, if not treated precisely. Keywords: Chronic Myeloid Leukemia (CML), Acute Myeloid Leukemia (AML), Blast Crisis (BC), Acute Megakaryocytic Leukemia (AMKL), Chronic Phase (CP), Accelerated Phase (AP), Tyrosine Kinase Inhibitor (TKI).

Sole trisomy 6 an uncommon finding in pediatric acute myeloid leukemia, probably associated to bad prognosis

BackgroundAcute leukemias represent the main malignancies occurring among children under the age of 15 years. Around 17% corresponds to acute myeloid leukemia (AML). The cytogenetic analysis of bone marrow complements the diagnosis of hematological malignancies, therefore finding chromosomal aberrations provides a more reliable prognosis of the disease. Among the cytogenetic aberrations, sole trisomy is frequent in malignant neoplasias, but few cases related to AML have been reported.Case presentationWe report a sole trisomy 6 in a pediatric patient diagnosed as AML M4 and poor progression. We carried out a literature review of AML patients with sole trisomy 6 and compared their evolution against AML patients with normal karyotype.ConclusionsThis is the first case of pediatric AML M4 with this cytogenetic finding. Sole trisomy 6 is infrequently reported in AML but scarce in pediatric cases. Based on overall survival analysis, we suggest that sole trisomy 6 could be associated with poor prognosis, in both, adult as well as pediatric AML.

Retrospective evaluation of chromosome 1 anomalies in hematologic malignancies: A single center study

Various anomalies of chromosome 1 which is the largest chromosome of the human karyotype were found in various hematologic diseases. In this retrospective study, clinical features and cytogenetic anomalies of 35 hematological patients with various chromosome 1 anomalies were correlated. Also the effect of chromosome 1 anomalies to the disease prognosis of those patients was discussed. Conventional cytogenetic analysis of those patients was performed by investigating metaphases of 24 hours stimulated bone marrow samples. After cell culturing, the samples were treated with trypsin and stained with Giemsa (GTG Banding). Analyses were performed on image analysis system. Chromosome 1 anomalies were determined in 35 patients (0.5 %) among 6865 samples having done their conventional bone marrow cytogenetic analysis in our center between January 2008 and March 2016. The ratio of chromosome 1 anomalies of totally 701 anomalies among 6865 patients was 4.9%. Chromosome 1 anomalies were found mostly in patients with Multiple Myeloma (MM), Myelodysplastic syndrome (MDS) and Acute Myeloid Leukemia (AML) in our study group. The most common anomaly was deletion 1 which was seen in 16 (37%) patients. Second most common anomaly was derivation 1 which was seen in 13 (30%) patients. Also translocations between chromosome 1 and other chromosomes were observed. The genetic aberration formed as a result of chromosomal anomalies result in the formation of hematologic malignancies. The effect on disease pathogenesis and prognosis of some of those anomalies are unknown and have to be investigated and determined.

Analysis of FLT3-ITD and FLT3-ASP835 Mutations in De Novo Acute Myeloid Leukemia: Evaluation of Prevalence, Distribution Pattern and Correlation with FAB Subtype, and Cytogenetics from a Tertiary Referral Hospital in India

Introduction: After introduction of Midostaurin, the importance of FLT3 mutation analysis in AML is irreplaceable. In this study we tried to find the incidence of this mutation in Indian Population. Aims and Objective: -To evaluate clinical parameters like age, sex, clinical presentation and duration of symptoms in atleast 60 de novo AML patients. -Perform bone marrow examination, FAB subtyping and cytogenetic analysis in these cases. -To estimate the prevalence of FLT3-ITD and FLT3-Asp835 mutations by DNA-PCR in these cases. -To compare the FLT3-ITD and FLT3-Asp835 mutations status with clinical parameters and cytogenetics data. Methods: We Included: -All cases of primary de novo Acute Myeloid Leukemia cases, who have not received any kind of chemotherapy for the same. -Cases in whom adequate representative sample was available for cytogenetic analysis and PCR studies. We excluded: -All secondary Acute myeloid leukemia cases. -Children less than 2 years of age. -Patients who have received any chemotherapy before. We did cytogenetics, Flowcytometry and RT-PCR based detection of FLT-3 as per standard protocol. Results and Discussion: In the time period between January 2015 to September 2016, a total of 68 Acute Myeloid Leukemia (AML) patients were admitted to our Institute and included in the study. In the current study, the following observations were noted: -Around 30% of patients referred to us with a provisional diagnosis of AML are not affording any further workup or treatment. -The median age of occurrence of AML is much lower than in western countries. -De novo cases are much more frequent. -More than 80% of our patients presented with some form of bleeding with infections of variable severity in nearly 90% of patients. -According to FAB classification AML-M2 was the most prevalent subtype in our study. -Nearly 67% of our patients belong to Normal Karyotype followed by inv (16) and t(8:21) in around 10% of cases each. -FLT3-ITD was present in 24% of cases and FLT3-Asp835 mutations were present in 6% of cases in concordance with reported literature. -NPM 1 mutations were present in 24% of cases. -NPM 1 overlapped with FLT3-ITD in 8% of the cases with one case showing overlap of NPM 1 AND FLT3-Asp835 mutation. -At presentation FLT3-ITD positive patients reveal significantly high total count, low hemoglobin, low platelets and high Serum LDH values. -All t(8:21) positive were negative for FLT3 mutations in this study. -FLT3-ITD positivity correlated significantly with dim or no expression of immaturity markers namely CD34 and HLA-DR in Immunophenotyping. -In our study 65.7% of patients achieved complete remission following standard induction -Primary resistant disease was noted in 8% of cases (20% induction failure cases following one cycle of induction). -2% of patients succumbed to induction related mortality with neutropenic sepsis being the major contributor. -Induction failure significantly correlated with FLT-ITD positivity but no significant differences were noted in induction death. -A 42% relapse rate is noted in our patients with &gt;70% of them occurring within a year of therapy completion. -4 % of our treated patients are alive during evaluation with median complete remission duration of 11 months. -Most patients who had a relapse did not undergo further therapy and were referred back to their primary physicians for supportive care only. Since a meaningful survival analysis was not possible with most of these patients being lost to follow up, the overall survival data has not been reported. -Only two patients underwent Allogeneic transplant and were alive during data analysis. -Midostaurin was not available during study period and 30% of patients were put on Sorafenib. Conclusion: In conclusion the prevalence of FLT3 mutations in our population is similar to reported literature. Since patients who harbour FLT3/ITD have dismal outcome with standard therapy and considering the fact in India most patients are unable to afford treatment, patients should be encouraged to be a part of a clinical trial wherever possible. Although the complete response rates in our study are comparable to published literature, even patients who did not have any mutations had a poorer disease free survival compared to patients reported previously who were treated on a similar protocol without stem cell transplantation. Hence there is a need to study the biology of these patients looking at a wider range of genes which can have an impact on the outcomes. Figure Disclosures No relevant conflicts of interest to declare.

Myelodysplastic Syndrome (MDS) in a Child who Transformed to Acute Myeloid Leukaemia with Myelodysplasia-Related Changes (AML-MRC)

Background: Myelodysplastic syndromes (MDS) are a group of clonal hematopoietic stem cell diseases, accounting for &lt; 5% of all hematologic malignancies in children.  MDS is characterized by cytopenia, dysplasia in one or more of the major myeloid lineages, ineffective hematopoiesis, and recurrent genetic abnormalities. Approximately 30% of patients with MDS show progression to AML within a few months up to several years. This is an MDS case report in children who have undergone Acute Myeloid Leukaemia transformation with Myelodysplasia-Related Changes (AML-MRC).Case Report: A 16-year-old boy came to the Emergency Department of Sanglah General Hospital with a complaint of pain and swelling in both knees and joints since last month. Fatigue and fever happened a week before admission. Previously, he had been hospitalized twice with the same symptoms. Physical examination revealed pale conjunctiva with pancytopenia in the laboratory result. A blood smear showed normochromic normocytic anemia, neutropenia, and thrombocytopenia. Bone marrow aspiration morphology showed hypercellularity with blast presentation &gt; 10% and multilineage dysplasia &gt; 50% supporting MDS diagnosed transformation to Acute Myeloid Leukaemia with Myelodysplasia-Related Changes (AML-MRC). Leukemia phenotyping showed positive results on CD 33, CD 36 and HLA-DR presenting Monocytic Lineage.                                                                                                            Conclusion: High degree dysplasia and blast percentage in blood and bone marrow increase tendency to transform into acute myeloid leukemia (AML). The diagnose of MDS requires analysis of blood and bone marrow morphology and cytogenetic analysis as a recommendation by WHO classification.

Philadelphia chromosome-negative myeloid neoplasms in patients with Philadelphia chromosome-positive chronic myeloid leukemia during tyrosine kinase inhibtor-therapy

目的探讨Ph阳性慢性髓性白血病（Ph+CML）患者在酪氨酸激酶抑制剂（TKI）治疗中出现Ph阴性克隆性染色体异常（CCA/Ph−）后发生Ph−骨髓增生异常综合征或急性髓系白血病（MDS/AML）患者的临床特征。方法回顾性分析2001年5月至2017年12月收治的出现CCA/Ph−的CML患者资料。结果共收集63例在TKI治疗过程中检出CCA/Ph−的Ph+CML患者，其中7例发生Ph−MDS/AML，56例疾病稳定。与疾病稳定组相比，首次检出CCA/Ph−时，Ph− MDS/AML组患者血红蛋白（P=0.007）和PLT（P=0.006）水平更低，外周血原始细胞比例（P<0.001）更高；当发生MDS或AML时，Ph−MDS/AML组患者的CCA/Ph−以“−7”为主（5/7，71.4%），而Ph−疾病稳定组以“+8”为多见（32/56，57.1%）。Ph−MDS/AML组患者总体预后不良，而Ph−疾病稳定组多数CCA/Ph−持续存在或有变化，大部分患者分子学反应良好，疾病稳定。结论CML患者接受TKI治疗中，少数会发生CCA/Ph−，其中大部分病情稳定，极少数会发生Ph−MDS/AML。当出现不明原因血细胞减少或CCA/Ph−为“−7”时，应重视外周血涂片、骨髓细胞遗传学和骨髓细胞形态学检测。

Chronic Myeloid Leukemia in Adolescents and Young Adults Treated with Generic Form of Imatinib in Resource Limited Setting: A Single Center Experience from Myanmar

Chronic myeloid leukemia (CML) is an acquired myelo-proliferative disorder characterized by the presence of BCR-ABL1 fusion transcript with deregulated tyrosine kinase activity. Leukemias are one of the leading causes of cancer related deaths in adolescents and young adults (AYA) particularly in resource limited countries. Recent democratic government in Myanmar holds supportive policies for treatment of cancers in younger population and subsidizes some therapies including imatinib mesylate, a selective BCR-ABL 1 tyrosine kinase inhibitor, although there is still limitation for cytogenetic and molecular monitoring of therapy. This study on response to imatinib in newly diagnosed chronic phase CML was carried out during January 2016 to February 2018 at the North Okkalapa General and Teaching Hospital of Yangon, Myanmar where molecularly confirmed new CML in chronic phase were treated with generic form of imatinib bought by hospital tender system, Unitinib (United Biotech (P) ltd. India) followed by cytogenetic analysis of bone marrow and molecular detection of BCR-ABL1 transcript from peripheral blood by in house real time PCR machine at one year. They are divided into AYA (aged 15-39 years) and adult age groups (40 years and older) and clinical parameters and response to treatment with imatinib 400mg per day were compared. Among 56 cases (median age of 39.5 years, male to female ratio of 1.5:1), half were AYA (28 of 56) with median age of 28.5 years compared to 50 years in adult group with higher male to female ratio of 3.6:1 in AYA group. Patients in AYA had larger spleen size (11 vs 5.5 cm) and higher median white cell count compared to adults (366 x 109/l vs 224 x 109/l) although initial platelet count, peripheral blood eosinophil, basophil and blast percent and Sokal score were comparable. Additional chromosomal abnormalities were detected in 2 AYA and 3 adults with CML. There was no difference in complete haematologic response at 3 and 12 months between AYA and adults (96.4% vs 96.4% and 92.9% vs 89.3% respectively). At 12 months, complete cytogenetic response of AYA at 67.9% was less than 75.0% of adults, it was statistically not significant. Using in house molecular method although not standardized, 14.3% of AYA and 32.1% of adults were molecularly undetectable at one year. The response of AYA to imatinib in this study was comparable to adults despite having adverse prognostic features and receiving only generic forms of treatment in resource limited setting and it would further help support from authorities for leukemia in younger population. DisclosuresNo relevant conflicts of interest to declare.

Protective Effects of Fullerene C60 Nanoparticles and Virgin Olive Oil against Genotoxicity Induced by Cyclophosphamide in Rats.

The potential effects of the fullerene C60 nanoparticle (C60) as well as virgin olive oil (VOO) against the cyclophosphamide- (CP-) induced cytotoxic and mutagenic effects were evaluated by two main methods: molecular intersimple sequence repeat (ISSR) assay and cytogenetic biomarkers. Thirty adult male rats were divided to five groups (control, CP, C60, CP + C60, and CP + VOO). CP was i.p. injected with a single dose of 200 mg/kg; C60 and VOO were given orally (4 mg/kg dissolved in VOO and 1 ml, resp.) in alternative days for 20 days. The ISSR analysis revealed an increased in the DNA fragmentation level for liver and heart tissues represented by 21.2% and 32.6%, respectively, in the CP group. The DNA polymorphism levels were modulated and improved in CP + C60 (8.9% and 12%) and CP + VOO (9.8% and 12.7%) for hepatic and cardiac tissues, respectively. The bone marrow cytogenetic analysis revealed that C60 and VOO had significantly decreased the frequency of CP-induced chromosomal aberrations (chromosomal ring, deletion, dicentric chromosome, fragmentation, and polyploidy). Fullerene C60 and VOO have ability to reduce DNA damage and decrease chromosomal aberrations. In conclusion, fullerene C60 and VOO have protective effects against the CP-induced mutagenicity and genotoxicity. Fullerene C60 and VOO open an interesting field concerning their potential antigenotoxic agents against deleterious side effects of chemotherapeutics.

Juvenile myelomonocytic leukemia in an infant with congenital human immunodeficiency virus and cytomegalovirus infection

An 11-month-old boy was found to have bicytopenia and leukocytosis on routine attendance at a pediatric infectious diseases clinic. The child was under review for congenital co-infection with the human immunodeficiency virus (HIV) and cytomegalovirus (CMV). He was on antiretroviral therapy and had previously been treated with valganciclovir. His foster mother had no new concerns, and he had not had any fever recently. No significant abnormality was found on physical examination. His blood count showed a normocytic normochromic anemia (hemoglobin concentration 95 g/L), white cell count 26.5 × 109/L, and platelet count 47 × 109/L. There was monocytosis (7.7 × 109/L) and basophilia (0.3 × 109/L). In addition his blood film showed nucleated red blood cells, dysplastic basophils and eosinophils, granulocyte precursors, and occasional blast cells (images). High performance liquid chromatography showed hemoglobin F of 37%. Opinion was divided as to whether the abnormalities were secondary to CMV infection or malignancy. A diagnosis of juvenile myelomonocytic leukemia (JMML) was favored and further investigations were therefore performed. Abdominal ultrasound examination showed the spleen to measure 9.5 cm. A bone marrow aspirate showed active erythropoiesis with reduced granulopoiesis and megakaryocytes. Blast cells were 7% on cytology and 10% on flow cytometry; they expressed CD45 (moderate), CD33, CD117, CD7, and CD56. Monocytic markers were expressed by 32% of cells: CD45 (strong), CD4 (weak), CD64, CD11b, CD13, CD14, CD36, and CD56. Fluorescence in situ hybridization showed monosomy 7 in 64% of cells. Molecular analysis showed an NRAS Q61K mutation (PTPN11 and KRAS were wild type). Subsequently he was found to have a negative PCR for CMV, a low HIV viral load and a CD4-positive lymphocyte count of 2770 × 106/L. CMV infection in infants can cause a leukemoid reaction that resembles JMML.1 However an increased basophil count, dysplastic eosinophils and basophils, and an increased hemoglobin F would not be expected. Careful examination of the blood film suggested the correct diagnosis, which was confirmed by bone marrow examination and cytogenetic analysis. None.

Clinicopathologic Characteristics and Outcome of Isolated Ovarian Relapse in Adolescent with Acute Lymphoblastic Leukemia

To investigate the clinicopathologic characteristics,diagnosis and treatment of isolated ovarian relapse of acute lymphoblastic leukemia(ALL). A 16-year-old girl presented with complaints of bone and joint pain. The peripheral blood and bone marrow(BM) smears showed 32% and 72% blasts, respectively, which were myeloperoxidase-negative. The blasts were positive for HLA-DR, TdT, CD10, CD19, CD22 and cCD79a and negative for CD34, CD5, CD7, CD13, CD33, CD56 and MPO detected by flow cytometry. BM cytogenetic analysis and fusion gene screening revealed t(1;19)(q23;p13) and E2A/PBX1. She was diagnosed as B-cell acute lymphoblastic leukemia (B-ALL) and was treated with CALGB8811 protocol. She presented lower abdominal pain with intermittent colick at 7 months after complete remission. The pelvic ultrasound showed a lobulated mixed echogenic mass in the right ovary, and an exploratory laparotomy was performed. Pathologic examination and immunohistochemistry of resected ovarian tumor revealed extensive infiltration by lymphoblasts with positive for TdT, CD20, CD43 and CD79a. Further investigations failed to reveal any other extramedullary involvement. Hemogram, peripheral blood and bone marrow smear examination were unremarkable at the same time. The isolated extramedullary ovarian relapse of ALL was confirmed. Simultaneous, the detection of minimal residual disease by multiparametric flow cytometry showed positive with 5.0×10-4. The reinduction chemotherapy including a high-dose methotrexate and cytarabine was given to the patients. She experienced the second ovarian relapse after 1 year and refused further treatment. Although uncommon, ovarian recurrence after chemotherapy for ALL should be considered in the patients with suggestive symptoms. Screening by pelvic ultrasonography may be valuble for early detection of pelvic disease in ALL.

Monoclonal B-cell lymphocytosis: from literature to laboratory practice

Monoclonal B-cell lymphocytosis (MBL) is defined as an asymptomatic condition characterized by the presence of less than 5,000 monoclonal B-cells per microliter and the absence of clinical signs or symptoms of a B-cell lymphoproliferative disorder. Most MBL cases involve B cells presenting an identical phenotype to CLL (CLL-like MBL) with a Catovsky-Matutes score of 3 to 5 and share the same chromosomal abnormalities than CLL. Depending on the absolute B cell count, one may distinguish low-count CLL-like MBL (<500 B cells/μL) which have no evidence of progression, no reduction in overall survival, no increase in infection risk and do not require any specific follow-up. Patients with clinical CLL-like MBL (>500 B cells/μL) have a 1% to 2% per year risk of progression to CLL requiring therapy, a higher risk of infectious complications and mortality implicating an annual follow-up by hematologist. MBL may also express other less common phenotypes and are named atypical MBL in case of CD5 antigen expression (Catovsky-Matutes score: 1-2) and non-CLL-like MBL for CD5 negative cases (Catovsky-Matutes score: 0-2). Their poorer prognosis implicates imaging studies, bone marrow biopsy and cytogenetic analysis in addition to physical examination in order to rule out non-hodgkinien lymphoma, and require a more frequent follow-up. This review focuses on key concepts in the classification, diagnosis, monitoring and biology of MBL in laboratory practice.

Respiratory Failure Associated with Ascariasis in a Patient with Immunodeficiency.

In industrialized countries, risk groups for parasitic diseases include travelers, recent immigrants, and patients with immunodeficiency following chemotherapy and radiotherapy and AIDS. A 66-year-old Polish male was admitted in December 2012 to the Department of Haematology in a fairly good general condition. On the basis of cytological, cytochemical, immunophenotypic, and cytogenetic analysis of bone marrow, the patient was diagnosed with acute myeloblastic leukemia. On the 7th day of hospitalization in the Department of Haematology, patient was moved to the Intensive Care Unit (ICU) due to acute respiratory and circulatory failure. In March 2013, 3 months after the onset of respiratory failures, a mature form of Ascaris spp. appeared in the patient's mouth. This report highlights the importance of considering an Ascaris infection in patients with low immunity presenting no eosinophilia but pulmonary failure in the central countries of Europe.

JAK2 V617F Analysis in Indonesian Myeloproliferative Neoplasms Patients

Background : Three subtypes of myeloproliferative neoplasms (MPNs): Polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) showed overlapping phenotype. There has been no specific cytogenetic marker identified in these subtypes. JAK2 V617F mutation prevalence in Caucasian MPNs was first reported as 97% in PV, 57% in ET, and 50% in PMF.Objective: This study was done to define the prevalence of JAK2 V617F mutation and to identify cytogenetic markers in MPNs.Methods : The study design was cross-sectional. Patients who were admitted to Dr. Kariadi Hospital with clinical diagnosis of MPNs were referred for bone marrow cytogenetic analysis in Telogorejo Hospital. JAK2 V617F mutation was tested for using Amplification Refractory Mutation System Polymerase Chain Reaction (ARMS-PCR) from peripheral blood vein. Clinical data were secondary data retrieved from hospital medical records.Results : There was no cytogenetic abnormality found in all MPNs patients. The prevalence of JAK2 V617F mutation in MPNs patients was 73,68%. Mutation prevalence distribution in each subtypes were 100% in PV, 63,6% in ET and 100% in PMF. Conclusion : Chromosomal abnormality was not found using conventional cytogenetic analysis. More sensitive methods might elucidate submicroscopic chromosomal abnormalities in these patients. The prevalence of JAK2 V617F mutation was comparable with studies in Caucasian. It is recommended that JAK2 V617F testing should be incorporated in the management therapy of MPNs in Indonesia.

Atypical Presentation of Childhood Leukemia.

To, The Editor, Leukemia is the most common childhood cancer worldwide [1]. Acute lymphoblastic leukemia (ALL) usually manifests with pallor, hepatosplenomegaly, lymphadenopathy, fever, bone pain, and bleeding [2]. Presence of frank osteolytic lesions with hypercalcemia is infrequent [3].We describes here a case of paediatric ALL who presented with multiple lytic lesions, hypercalcemia and absence of blasts in the peripheral blood film. A 10-year-old boy presents with generalised weakness, pain in right hypochondriac region which was non radiating, aggravated by coughing, associated with vomiting which was non bilious and non projectile for last 15 days. On examination, mild pallor was present with no icterus and lymphadenopathy. Liver was palpable 2 cm below the right costal margin. On investigations, complete hemogram showed Hb of 9.2g/ dl, WBC count of 8.0×109 /L with neutrophils 54%, lymphocyte 36%, monocyte 7%, eosinophil 2%, myelocyte 1% and platelet count 271 ×109 /L. Peripheral blood film showed mild anisocytosis with microcytes and hypochromia. His serum biochemistry studies were as follows: sodium: 134.8 mmol/L, potassium: 4.2 mmol/L, Crealinium: 0.93mg/dl, Calcium: 14.2 mg/dl, Phosphorous 6.4 mg/dl, uric acid: 7.6 mg/dl, LDH: 654 IU/L, Alkaline phosphatase: 110U/L, Albumin: 3.6g/dl. His ESR was 100 mm/h. Serum vitamin D3 level was 16.3 (15-30ng/ml). His serum parathyroid hormone (PTH) level was 7.2 (13.7-77.2 pg/ml). His abdominal ultrasound was normal except for hepatomegaly. The skeletal survey showed multiple lytic lesions in skull [Table/Fig-1] and ill defined translucent areas in pelvic bone. In view of anemia and multiple lytic lesions, bone marrow examination was done. Bone marrow examination revealed hypercellular marrow with 55% blasts which were positive for CD 10, CD 19, CD 22 and negative for myeloperoxidase confirming to diagnosis of precursor B acute lymphoblastic leukemia. Cytogenetic analysis of bone marrow showed 46XY karyotype. Molecular analysis for t (9; 22), t (12; 21), t (1; 19), and t (4; 11) using PCR method were negative. Patient was started on aggressive hydration, bisphosphonate and frusemide with monitoring of tumour lysis markers. For ALL, he was started with chemotherapy as per BFM 2002 protocol. His serum calcium levels decreased to 11.0 mg/dl after 48 hrs. Currently he is undergoing chemotherapy and his Day +15 marrow was hypocellular with 3% blasts (M1 marrow). Hypercalcemia is a rare finding in paediatric ALL ranging in frequency from 0.6- 4.8% [4]. Hypercalcemia of malignancy can occur due to two mechanisms. First is localized bone destruction by invasive cancer cells and second mechanism involves osteoclastic bone resorption after the release of humoral derived factors from tumour cells. Although hypercalcemia is assumed to be linked with high tumour bulk, a series of 22 patients showed no difference in event free survival in children with hypercalcemia at presentation. It has been reported that t (17;19), which is associated with poor prognosis, is frequently seen in patients with hypercalcemia due to humoral factors [5]. In our patient, hypercalcemia was due to direct bone invasion and cytogenetics was normal. The presence of multiple lytic lesions and hypercalcemia with no blasts in the peripheral blood are uncommon findings in ALL, which has prompted us to report this case. [Table/Fig-1]: X-ray Skull showing multiple lytic lesions