Recent advances in diagnosis of acute myeloid leukaemia : a review.

Acute Myeloid Leukaemia is one of the commonest blood cancer in Malaysia. Recent advancements in molecular genetics have led to better understanding of AML pathogenesis which is crucial for patient stratification and therapeutic options. The advent of next generation sequencing (NGS) is now the vogue to interrogate acute myeloid leukaemia (AML) genome at single base pair resolution. In this review, we will discuss current approaches in the diagnosis of AML and the role of NGS in elucidation of genomic aberrations. Although NGS is anticipated to impact the new era of AML diagnosis, there are challenges in understanding mutations found in an AML genome and how this technology will be implemented in a diagnostic setting. NGS will pave the way for perso nalized medicine approach, with the hope of better response to tailored treatment regimen

Antigen expression pattern of acute promyelocytic leukaemia cases in Malaysia

Introduction: Acute Promyelocytic Leukaemia (APL) is associated with devastating coagulopathy and life threatening condition which requires immediate medical attention. It is crucial to establish an expedited diagnosis as early therapeutic intervention has led to optimal patient management. In this study, we assessed the type and frequency of antigen expressions in APL and correlated these findings with genetic studies. Methods: Multiparametric immunophenotyping was performed on 30 samples and findings were correlated with karyotypes, FISH for t(15;17) translocation and RT-PCR for PML-RARα for detection of breakpoint cluster regions (bcr1,bcr2 and bcr3). Results: On SSC/CD45, APL cells displayed high to moderate SSC, with the expression of CD33 (100%), CD13 (96.8%), cMPO (71%) but lacked CD34 (3.2%) and HLA-DR (9.7%). Aberrant expression of CD4 was seen in 12.9% and CD56 in 6.5% of the cases. A significant association between cumulative aberrant antigen expression and bcr1 were observed bcr1 (X2(2) =6.833,p.05) and (X2(2)=4.599,p>.05) respectively. Conclusions: Flow cytometry is a rapid and effective tool in detecting APL. It is interesting to note that there is significant association between cumulative aberrant antigen expression and genotype analysis. Further validation is required to corroborate this relationship

Genomic Alterations, Gene Expression Profiles and Functional Enrichment of Normal-Karyotype Acute Myeloid Leukaemia Based on Targeted Next-Generation Sequencing

Characterising genomic variants is paramount in understanding the pathogenesis and heterogeneity of normal-karyotype acute myeloid leukaemia (AML-NK). In this study, clinically significant genomic biomarkers were ascertained using targeted DNA sequencing and RNA sequencing on eight AML-NK patients’ samples collected at disease presentation and after complete remission. In silico and Sanger sequencing validations were performed to validate variants of interest, and they were followed by the performance of functional and pathway enrichment analyses for overrepresentation analysis of genes with somatic variants. Somatic variants involving 26 genes were identified and classified as follows: 18/42 (42.9%) as pathogenic, 4/42 (9.5%) as likely pathogenic, 4/42 (9.5%) as variants of unknown significance, 7/42 (16.7%) as likely benign and 9/42 (21.4%) as benign. Nine novel somatic variants were discovered, of which three were likely pathogenic, in the CEBPA gene with significant association with its upregulation. Transcription misregulation in cancer tops the affected pathways involving upstream genes (CEBPA and RUNX1) that were deregulated in most patients during disease presentation and were closely related to the most enriched molecular function gene ontology category, DNA-binding transcription activator activity RNA polymerase II-specific (GO:0001228). In summary, this study elucidated putative variants and their gene expression profiles along with functional and pathway enrichment in AML-NK patients.This research was funded by Universiti Sains Malaysia, grant number GIPS-PhD 311/PPSP/4404814 (23 April 2020). Universiti Sains Malaysia funded the APC

Microarray-based genomic analysis identifies germline and somatic copy number variants and loss of heterozygosity in acute myeloid leukaemia

Introduction: Insights into molecular karyotyping using comparative genomic hybridization (CGH) and single nucleotide polymorphism (SNP) arrays enable the identification of copy number variations (CNVs) at a higher resolution and facilitate the detection of copy neutral loss of heterozygosity (CN-LOH) otherwise undetectable by conventional cytogenetics. The applicability of a customised CGH+SNP 180K DNA microarray in the diagnostic evaluation of Acute Myeloid Leukaemia (AML) in comparison with conventional karyotyping was assessed in this study. Methods: Paired tumour and germline post induction (remission sample obtained from the same patient after induction) DNA were used to delineate germline variants in 41 AML samples and compared with the karyotype findings. Results: After comparing the tumour versus germline DNA, a total of 55 imbalances (n 5-10 MB = 21, n 10-20 MB = 8 and n >20 MB = 26) were identified. Gains were most common in chromosome 4 (26.7%) whereas losses were most frequent in chromosome 7 (28.6%) and X (25.0%). CN-LOH was mostly seen in chromosome 4 (75.0%). Comparison between array CGH+SNP and karyotyping revealed 20 cases were in excellent agreement and 13 cases did not concord whereas in 15 cases finding could not be confirmed as no karyotypes available. Conclusion: The use of a combined array CGH+SNP in this study enabled the detection of somatic and germline CNVs and CN-LOHs in AML. Array CGH+SNP accurately determined chromosomal breakpoints compared to conventional cytogenetics in relation to presence of CNVs and CN-LOHs

Microarray-based genomic analysis identifies germline and somatic copy number variants and loss of heterozygosity in acute myeloid leukaemia

Acute myeloid leukaemia (AML) is characterized by the overproduction of immature myeloid cells that accumulate in blood and bone marrow. While the specific cause of AML is usually unknown, several factors including chromosomal aberrations and genetic mutations have been implicated in the pathogenesis of this aggressive disease. Integration of genetic findings and clinicopathological information is crucial in establishing the diagnosis, prognosis and determining the therapeutic approach in the management of AML patients. The AML classification has evolved from morphology to cytogenetics/molecular genetics-based findings in recent years. Cytogenetic information is important in the detection of chromosomal abnormalities and has provided the framework for the diagnosis and risk-stratification in AML over the past decade. However, conventional cytogenetics is a technically demanding method. The success rate of chromosomal analysis is largely dependent on the availability of optimal and viable cells for culturing and the expertise with experience in identifying chromosomal aberrations at a limited resolution. Insights into molecular karyotyping using comparative genomic hybridization (CGH) and single nucleotide polymorphism (SNP) arrays enable the identification of copy number variations (CNVs) at a higher resolution and facilitate the detection of copy neutral loss of heterozygosity (CN-LOH) otherwise undetectable by conventional cytogenetics. The applicability of a customised CGH+SNP 180K DNA microarray with additional additional custom probes for 49 genes; every exon of eleven of these genes (TP53, DNMT3A, TET2, ASXL1, MLL,IKZF1, PAX5, EZH2, FLT3, NOTCH1 and ATM) was covered in the diagnostic evaluation of AML was assessed in this study. Paired tumour and germline (remission sample obtained from the same patient after induction) DNA were used to delineate germline variants in 41 AML samples. The prognosis based on karyotyping and molecular genetics was correlated with demographic (age, gender, ethnicity) and laboratory findings (WBC, aberrant antigen expression of CD2, CD4, CD7, CD19 and CD56). After comparing the tumour versus germline DNA, a total of 55 imbalances (n5-10 MB = 21, n 10-20 MB = 8 and n >20 MB = 26) were identified. Gains were most common in chromosome 4 (26.7%) whereas losses were most frequent in chromosome 7 (28.6%) and X (25.0%). CN-LOH was mostly seen in chromosome 4 (75.0%). Excellent agreements between the karyotype and CGH+SNP analyses were observed in 20 cases, with CGH+SNP analyses providing more precise breakpoint definition. Karyotype was not in agreement with CGH+SNP in 13 cases. In another three cases,array CGH+SNP detected aberrations which were missed by conventional karyotyping. Translocations were not detected by CGH+SNP in six cases. Correlation between prognosis on karyotyping and molecular genetics based on the clinical and laboratory findings showed statistically significant association between CD19 expression and a favourable prognosis. Statistically significant differences were observed between genders (P < 0.05 by Fisher‟s exact test); females had a more favourable prognosis compared to males. Chromosomal abnormalities with breakpoint coordinates were identified more accurately as compared to conventional cytogenetics with the use of the combined array CGH+SNP platform in this study. In summary, a combined platform of CGH+SNP provides invaluable insights into the elucidation of large spectrum of genomic aberrations in AML which may have prognostic implications

Neoteric Algorithm Using Cell Population Data (VCS Parameters) as a Rapid Screening Tool for Haematological Disorders

Hitherto, there has been no comprehensive study on the usefulness of cell population data (CPD) parameters as a screening tool in the discrimination of non-neoplastic and neoplastic haematological disorders. Hence, we aimed to develop an algorithm derived from CPD parameters to enable robust screening of neoplastic from non-neoplastic samples and subsequently to aid in differentiating various neoplastic haematological disorders. In this study, the CPD parameters from 245 subtypes of leukaemia and lymphoma were compared against 1103 non-neoplastic cases, and those CPD parameters that were vigorous discriminants were selected for algorithm development. We devised a novel algorithm: [(SD-V-NE*MN-UMALS-LY*SD-AL2-MO)/MN-C-NE] to distinguish neoplastic from non-neoplastic cases. Following that, the single parameter MN-AL2-NE was used as a discriminant to rule out reactive cases from neoplastic cases. We then assessed CPD parameters that were useful in delineating leukaemia subtypes as follows: AML (SD-MALS-NE and SD-UMALS-NE), APL (MN-V-NE and SD-V-MO), ALL (MN-MALS-NE and MN-LMALS-NE) and CLL (SD-C-MO). Prospective studies were carried out to validate the algorithm and single parameter, MN-AL2-NE. We propose these CPD parameter-based discriminant strategies to be adopted as an initial screening and flagging system in the preliminary evaluation of leukocyte morphology

Genetic Profiles and Risk Stratification in Adult De Novo Acute Myeloid Leukaemia in Relation to Age, Gender, and Ethnicity: A Study from Malaysia

Hitherto, no data describing the heterogeneity of genetic profiles and risk stratifications of adult acute myeloid leukaemia (AML) in Southeast Asia are reported. This study assessed genetic profiles, Moorman’s hierarchical classification, and ELN 2017-based risk stratifications in relation to age, gender, and ethnicity in Malaysian adult AML patients. A total of 854 AML patients: male (52%), female (48%) were recruited comprising three main ethnic groups: Malays (59%), Chinese (32%) and Indians (8%). Of 307 patients with abnormal karyotypes: 36% exhibited translocations; 10% deletions and 5% trisomies. The commonest genotype was FLT3-ITD-NPM1wt (276/414; 66.7%). ELN 2017 risk stratification was performed on 494 patients, and 41% were classified as favourable, 39% as intermediate and 20% as adverse groups. More females (47%) were in the favourable risk group compared to males (37%), whereas adverse risk was higher in patients above 60 (24%) of age compared to below 60 (18%) patients. We observed heterogeneity in the distribution of genetic profiles and risk stratifications between the age groups and gender, but not among the ethnic groups. Our study elucidated the diversity of adult AML genetic profiles between Southeast Asians and other regions worldwide

A Novel Algorithm Using Cell Population Data (VCS Parameters) as a Screening Discriminant between Alpha and Beta Thalassemia Traits

Thalassemia is one of the major inherited haematological disorders in the Southeast Asia region. This study explored the potential utility of red blood cell (RBC) parameters and reticulocyte cell population data (CPD) parameters in the differential diagnosis of &alpha; and &beta;-thalassaemia traits as a rapid and cost-effective tool for screening of thalassemia traits. In this study, a total of 1597 subjects (1394 apparently healthy subjects, 155 subjects with &alpha;-thalassaemia trait, and 48 subjects with &beta;-thalassaemia trait) were accrued. The parameters studied were the RBC parameters and reticulocyte CPD parameters derived from Unicel DxH800. A novel algorithm named &alpha;&beta;-algorithm was developed: (MN-LMALS-RET &times; RDW) &minus; MCH) to discriminate &alpha; from &beta;-thalassaemia trait with a cut-off value of 1742.5 [AUC = 0.966, sensitivity = 92%, specificity = 90%, 95% CI = 0.94&ndash;0.99]. Two prospective studies were carried: an in-house cohort to assess the specificity of this algorithm in 310 samples comprising various RBC disorders and in an interlaboratory cohort of 65 &alpha;-thalassemia trait, and 30 &beta;-thalassaemia trait subjects to assess the reproducibility of the findings. We propose the &alpha;&beta;-algorithm to serve as a rapid, inexpensive surrogate evaluation tool of &alpha; and &beta;-thalassaemia in the population screening of thalassemia traits in geographic regions with a high burden of these inherited blood disorders

Haematological Reference Intervals in a Multiethnic Population

Introduction: Similar to other populations, full blood count reference (FBC) intervals in Malaysia are generally derived from non-Malaysian subjects. However, numerous studies have shown significant differences between and within populations supporting the need for population specific intervals. Methods: Two thousand seven hundred twenty five apparently healthy adults comprising all ages, both genders and three principal races were recruited through voluntary participation. FBC was performed on two analysers, Sysmex XE-5000 and Unicel DxH 800, in addition to blood smears and haemoglobin analysis. Serum ferritin, soluble transferrin receptor and Creactive protein assays were performed in selected subjects. All parameters of qualified subjects were tested for normality followed by determination of reference intervals, measures of central tendency and dispersion along with point estimates for each subgroup. Results: Complete data was available in 2440 subjects of whom 56% (907 women and 469 men) were included in reference interval calculation. Compared to other populations there were significant differences for haemoglobin, red blood cell count, platelet count and haematocrit in Malaysians. There were differences between men and women, and between younger and older men; unlike in other populations, haemoglobin was similar in younger and older women. However ethnicity and smoking had little impact. 70% of anemia in premenopausal women, 24% in postmenopausal women and 20% of males is attributable to iron deficiency. There was excellent correlation between Sysmex XE-5000 and Unicel DxH 800. Conclusion: Our data confirms the importance of population specific haematological parameters and supports the need for local guidelines rather than adoption of generalised reference intervals and cut-offs

Incidence of anaemia, iron deficiency and iron deficiency anaemia.

<p>Incidence of anaemia, iron deficiency and iron deficiency anaemia.</p