Clinico-haematologic association and prognostic relevance of NPM1 and FLT3-ITD mutations in acute myeloid leukaemia

Background and Objectives: Molecular genetic abnormalities have a significant role not only in diagnosis but also in determining the clinical course and prognosis. Nucleophosmin-1 [NPM-1] is associated with good prognosis while internal tandem duplication of the fms-like tyrosine kinase-3 gene [FLT3-ITD] confers a poor prognosis. Knowledge of the status of these mutations in AML patients not only guides treatment decisions but also helps in predicting response to frontline induction and consolidation chemotherapy as well as the risk of relapse and overall survival. Our objectives were to determine the prevalence, clinico-haematological features and immunophenotypic characteristics of AML patients with FLT3-ITD and NPM1 mutation and to evaluate the response to induction therapy [CR] and disease free survival [DFS] in this cohort of patients

Methods: Patients diagnosed as AML from March 2015 to March 2017 at Armed Forces Institute of Pathology Rawalpindi were included in the study. Clinico-haematologic and immunophenotypic parameters were noted and molecular analysis for FLT3-ITD and NPM1 mutation was performed. Any correlation with cytogenetics or other molecular markers was also studied. Response to standard induction chemotherapy and disease-free survival were assessed

Results: A total of 108 cases of AML were analyzed. Median age was 35 years and 64.8% were males. The median age of the study group was 35 years. Of these, 70 [64.8%] were males while 38 [35.2%] were females. Twenty-nine [26.9%] patients were NPM1 positive, twelve [11.1%] were FLT3-ITD positive while eight [7.4%] were positive for both mutations. Patients with NPM1 mutations were associated with female gender, higher haemoglobin level and platelet counts while those with FLT3-ITD mutations were predominantly seen in male patients and had significantly higher WBC counts, bone marrow blasts, biopsy cellularity and LDH levels. CR rates of NPM1 positive, FLT3-ITD positive and both mutation positive groups were 72%, 60% and 71%, respectively. The median disease-free survival was significantly lower in the FLT3-ITD positive group [7.1 months] as compared to the NPM1 positive group [16.1 months]. The median disease-free survival was 12 months and 11.9 months in the NPM1 positive/FLT3-ITD positive and the NPM1 negative/FLT3-ITD negative groups, respectively

Conclusion: AML patients harbouring NPM1 and FLT3-ITD mutations have distinct clinical and haematological characteristics. NPM1 mutations have a better CR and DFS as compared to FLT3-ITD group.

3 Isoforms of PML RAR [Alpha] in acute Promyelocytic Leukaemia

Objective: To determine the frequencies of three isoforms of PML RAR[alpha] fusion gene in APL

Study Design: Descriptive cross sectional study

Place and Duration of Study: Department of Hematology, Armed Forces Institute of Pathology [AFIP], Rawalpindi Pakistan, from Apr 2015 to Oct 2015

Material and Methods: This study involved 97 newly diagnosed cases of APL, aged between 15-70 years from both genders. Double nested PCR was carried out by Applied Biosystems 2720 Thermal Cycler in every patient to determine the isoform of PML RAR[alpha] fusion gene. A written informed consent was obtained from every patient

Results: The age of the patients ranged from 17 years to 69 years with a mean of 37.87 +/- 12.89 years. There were 68 [70.1 percent] male and 29 [29.9 percent] female patients in the study group. The bcr1 isoform was found in 9 [9.3 percent] patients. There was no significant difference in the frequency of bcr1 isoform across age groups; 17-34 years vs. 35-52 years vs. 53-69 years [14.6 percent vs. 7.0 percent vs. 0.0 percent; p=0.223] and genders; male vs. female [7.4 percent vs. 13.8 percent; p=0.317]. The bcr2 isoform was found in 26 [26.8 percent] patients. There was no significant difference in the frequency of bcr2 isoform across age groups; 17-34 years vs. 35-52 years vs. 53-69 years [19.5 percent vs. 27.9 percent vs. 46.2 percent; p=0.164] and genders; male vs. female [25.0 percent vs. 31.0 percent; p=0.539] while the bcr3 isoform was found in 62 [63.9 percent] patients. There was no significant difference in the frequency of bcr3 isoform across age groups; 17-34 years vs. 35-52 years vs. 53-69 years [65.9 percent vs. 65.1 percent vs. 53.8 percent; p=0.717] and genders; male vs. female [67.6 percent vs. 55.2 percent; p=0.242]

Conclusion: The most frequent isoform of PML-RAR[alpha] fusion gene was bcr3 which was observed in 62 [63.9 percent] cases followed by bcr2 [26.8 percent] and bcr1 [9.3 percent]. There was no significant difference in the frequency of isoforms with patient's age and gender

Spectrum of acute leukemias and aberrant markers expression based on flowcytometry in a tertiary care centre

Objective: To determine frequency of different types of leukemias and aberrant CD markers expression on these types. Study Design: Descriptive study. Place and Duration of Study: Department of Immunology, Armed Forces Institute of Pathology, Rawalpindi from Jul 2015 to Dec 2015

Material and Methods: All peripheral blood and bone marrow samples to confirm the suspicion of acute leukemia with flow cytometric immunephenoltyping were included in the study. Cells were stained with lineage specific monoclonal antibodies against cell specific CD markers through lyse wash procedure. Cell acquisition and analysis was done on Cell Quest software in multi parameter flow cytometer. Data was entered in SPSS v 20.0 to determine the frequencies of different types of leukemias and aberrant CD markers expression

Results: Over 6 months, 102 males and 49 females were tested with mean age 26 +/- 21 years. Commonest leukemia was AML M2. Among 69 pediatric cases with mean age 7.4 +/- 5.8 years, precursor B ALL was commonest. Among 82 adults with mean age 41.5 +/- 15.7 years, AML M2 was commonest leukemia. Total 32 cases [18 children and 12 adults] expressed cross lineage aberrant markers, CD13, CD33 and CD7

Conclusion: Aberrant CD markers expression must be kept in mind during lineage assignment of acute leukemias while performing flow cytometric immunophenotyping

Gastric intestinal metaplasia: an intermediate precancerous lesion in the cascade of gastric carcinogenesis

Gastric intestinal metaplasia, an intermediate lesion in the development of intestinal-type gastric cancer, is observed in the milieu of long standing non-atrophic gastritis and atrophic gastritis. Most patients with intestinal metaplasia remain asymptomatic unless cobalamin deficiency occurs secondary to loss of glands [that produce intrinsic factor and acid]. Genetic events that predispose to development of gastric intestinal metaplasia remains an enigma. Mechanisms leading to the progression of atrophy to metaplasia still needs to be comprehensively explored. Many studies in the literature describe a positive effect of typing intestinal metaplasia and concluded that intestinal metaplasia type III carries the highest risk for developing gastric cancer while others refute it. It is well established that Helicobacter pylori infection is the most important factor for the development of chronic gastritis, gastric intestinal metaplasia as well as gastric cancer. Countries with a higher prevalence of Helicobacter pylori infection and gastric cancer also have a high tendency of being prevalent for intestinal metaplasia. However, it remains elusive whether eradication of Helicobacter pylori infection tends to regress gastric intestinal metaplasia or reduce the subsequent risk of cancer development. Putting together, more prospective cohort studies should be designed to identify factors [antioxidants; anti-inflammatory drugs; food therapy] that may contribute in the regression of intestinal metaplasia, when used simultaneously with eradication therapy. Furthermore, molecular markers for evaluation of intestinal metaplasia, and the potential point-of-no-return should be further investigated. Consensus is also required to advocate a timeframe for surveillance of patients with gastric intestinal metaplasia

Delta-beta thalassaemia in a pathan family

Delta-beta-thalassaemia [macron beta-thalassaemia] is a rare type of thalassaemia which mostly results from deletion of macron and beta genes with preservation of gamma genes. Macron beta-thalassaemia is classified into [macron beta]+ and [macron beta] 0 types. The [macron beta] 0-thalassemia is further divided into G gamma A gamma [macron beta] 0-thalassaemia and G gamma [A gamma macron beta] 0-thalassaemia. In heterozygous state, [macronbeta] 0 mutations give rise to phenotype resembling beta-thalassaemia trait but with raised Hb-F, ranging from 5 to 20%, without a rise in Hb-A2. In homozygotes, the clinical picture is usually that of thalassaemia intermedia and the patients have 100% Hb-F. Workup of a 1-year child suffering from pallor, chronic ill health, and splenomegaly referred to our laboratory with the suspicion of beta-thalassaemia, ultimately resulted in a diagnosis on polymerase chain reaction as having homozygous inversion/deletion G gamma [A gamma macron beta] 0-thalassaemia. Her family members were also investigated

Immunophenotyping pattern in mixed phenotype acute leukaemias

Objective: To evaluate Immunophenotyping patterns in Mixed-Phenotype Acute Leukemias [MPAL]

Study Design: Descriptive study

Place and Duration of Study: This study was carried out in the department of Hematology, Armed Forces Institute of Pathology Rawalpindi, from 1st Jan 2013 to 31st Jan 2017

Material and Methods: After taking informed consent from the patients fulfilling the inclusion criteria, detailed history was taken and blood samples were drawn for blood complete picture. The patients suspected to have acute leukemia were subjected to bone marrow examination [aspiration and trephine biopsy] for further cytochemical staining [SBB] and Immunophenotyping

Results: Total 680 new cases of acute leukemias on initial workup of either gender age were included. Patients of other haematological disorders were excluded from the study. Among 680 new cases of acute leukaemia, 23[3.4%] cases were of MPAL immunophenotyping using scoring system proposed by EGIL [European Group for the Immunological Characterization of Leukemias] classification. Among MPAL, 19[83%] cases were Biphenotypic [13[57%] cases of My/B-ALL, 5[22%] cases of My/T-ALL, and 1[4%] case of T/B-ALL]. 4[17%] cases were Bilineage [My/B-ALL]. Most of the cases were diagnosed at less than 10 years of age

Conclusion: My/B-ALL is the most common immunophenotype followed by My/T-ALL. Therefore immunophenotyping is indispensable for diagnosis and for therapy decisions of MPAL]

Frequency of Gc-globin promoter 158 [C>T] XmnI polymorphism in patients with homozygous/compound heterozygous beta thalassaemia

BACKGROUND: Response to hydroxyurea therapy in homozygous or compound heterozygous beta thalassaemia [BT] has been reported as more favourable in the presence of XmnI polymorphism. The prevalence of XmnI polymorphism may vary with BT phenotypes and genotypes, and differs geographically in distribution. Prevalence of XmnI polymorphism is not known in northern Pakistan. Objective: To determine the frequency of Gc-globin promoter 158 [C>T] XmnI polymorphism [XmnI polymorphism] in patients with homozygous or compound heterozygous beta thalassaemia. MATERIALS: Polymerase chain reaction [PCR] for common beta thalassaemia mutations and Gc-globin promoter 158 [C>T] XmnI polymorphism was performed on 107 blood samples of transfusion dependent beta thalassaemia [BT] patients in Pakistan. One hundred samples of unrelated BT traits and 94 samples of healthy subjects as controls were also analysed for BT mutations and XmnI polymorphism. Results: Out of 301 DNA samples, XmnI polymorphism was detected in 71[24%]; in normal controls, XmnI polymorphism was detected in 34/94 [36%] subjects; while in homozygous/compound heterozygous BT, it was detected in 14/107[13%] patients [Fisher's exact test, p =. 0002]. In heterozygous BT group, XmnI polymorphism was detected in 23/100 subjects [Fisher's exact test, p =. 03 with normal controls, and p =. 049 with homozygous/compound heterozygous BT]. The most common BT genotype was Frame Shift [Fr] 8–9/Fr 8–9, and none of the patients with this genotype had XmnI polymorphism. The second most common genotype was IVSI-5/IVSI-5; 4/26 [15%]. Cases with this genotype had XmnI polymorphism. Conclusion: XmnI polymorphism in homozygous/compound heterozygous BT group is 13%. The most common genotype associated with XmnI polymorphism was IVSI-5/IVSI-5