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.

Clinicohematological parameters and outcomes in a cohort of chronic lymphocytic leukemia patients with Deletion 17p from Pakistan

BACKGROUND: Chronic lymphocytic leukemia (CLL) exhibits profound heterogeneity in its clinical course. Its clinicohematological and cytogenetic features play a significant role in determining the clinical course and in predicting the treatment response and prognosis. In this context, 17p deletion is known to predict a poor prognosis, as these cases are refractory to conventional therapy. This study aimed to evaluate the clinicohematological characteristics, outcomes, and prognostic factors among CLL patients with and without del 17p in Pakistan. METHODS: This prospective observational study was conducted at the Department of Haematology, Armed Forces Institute of Pathology (Rawalpindi, Pakistan) between January 2013 and December 2017. Patients were diagnosed based on the International Workshop on Chronic Lymphocytic Leukaemia IWCLL criteria, their clinicohematological parameters were recorded, and cytogenetic analyses were performed. The time from diagnosis to treatment and the 2-year overall survival rate were also evaluated. RESULTS: We evaluated 130 CLL cases, including 24 patients (18.5%) with del 17p, who included 18 men (75%) and 6 women (25%). The median age was 68 years. Binet stage C was detected at the presentation in 16 patients (67%). Treatment was administered to 14 patients (70%) at a median interval of 11 months (range, 0–28 mo) after diagnosis. The overall response rate was 64.3%, the median event-free survival was 9 months (range, 1–23 mo), and the 2-year overall survival rate was 65%. CONCLUSION: Del 17p is relatively common in Pakistan, and patients harboring this deletion had poor treatment response and survival outcomes.

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

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 von willebrand disease in patients of heavy menstrual bleeding

Objective: To determine the frequency of Von Willebrand disease [vWD] in patients of heavy menstrual bleeding [HMB]

Study Design: Hospital based cross sectional study

Place and Duration of Study: Study was conducted at the Gynecology and Obstetrics department, Military Hospital, Rawalpindi in collaboration with Haematology Department of Armed Forces Institute of Pathology [AFIP] Rawalpindi, from Jul to Dec 2015

Material and Methods: Women presenting with HMB were enrolled in the study after informed consent. HMB was defined as cyclical bleeding at normal intervals but patient is using more than 5 pads per day or increase in duration 8/28 or more for at least last 06 months. Venous blood samples were taken and screened for the hemoglobin level [Hb], platelet count, prothrombin time [PT], activated partial thromboplastin time [aPTT] and Von Willebrand antigen [vWF:Ag] in addition to bleeding time [BT] at the Armed Forces Institute of Pathology [AFIP]. The demographic details [age, age at menarche], clinical features [menstrual history, quantity of bleeding] and laboratory findings were recorded on the study proforma

Results: A total of 200 patients were enrolled in this study with mean age of 32.3 +/- 8.5 years. Mean flow of menstrual blood was 9.8 +/- 2.5 pads / day. Mean Hb% was 8.1 +/- 1.4 g/dl. Twenty nine [14.5%] patients were having low level of vWF:Ag

Conclusion: There is high frequency of von Willebrand disease among females presenting with heavy menstrual bleeding in our set up. Therefore all patients with heavy menstrual bleeding except those with obvious causes like multiple fibroid should be screened for von Willebrand disease

Interferon and ribavirin associated depression in HCV patients and role of selective serotonin reuptake inhibitors

To determine the frequency and severity of depression associated with antiviral therapy of Hepatitis C Virus [HCV] infection and effect of selective serotonin reuptake Inhibitors [SSRIs] to treat these depressive symptoms. Type of Study: Observational Analytical study. Place of Study and Duration: The study was conducted at Psychiatry, Medicine and Pathology department of Combined Military Hospital Sialkot Pakistan from February 2009 to July 2010. All the patients in this study were suffering from HCV infection and were managed with Interferon [3 m.i.u. s/c thrice weekly] and Cap Ribavirin [400 mg bid] for six months. Patients were assessed by Hospital Anxiety and Depression Scale [HADS] - Urdu Version and Beck's Depressive Inventory [BDI] Scores after twelve weeks of antiviral therapy. Depressed patients were managed with selective serotonin reuptake inhibitors [SSRIs] for six weeks and again evaluated on HADS and BDI Scores. Response to SSRIs was defined as complete response, partial response and no response. A total of 105 patients were studied out of which 75 were male and 30 were female with mean age 29.4 years. Out of these 54 [51.43%] patients developed depression and this tendency to develop depression was not related with the age and sex of the patients. The mean HADS and BDI scores before and after treatments with SSRIs were compared for significance and it was quite significant. There was not a single patient who did not show response to SSRIs. Depression is frequently associated with antiviral therapy of HCV RNA viraemia with interferon and SSRIs have proved an effective and safe remedy in these patients

Outcome of allogeneic haematopoietic stem cell transplantation in aplastic Anaemia

To analyze factors associated with survival, rejection and graft versus host disease in aplastic anaemia patients undergoing allogeneic haematopoietic stem cell transplantation [SCT] from HLA matched sibling donors. Analytical study. Armed Forces Bone Marrow Transplant Centre, Rawalpindi, Pakistan from July 2001 to June 2010. Consecutive aplastic anaemia [AA] patients undergoing haematopoietic stem cell transplantation from HLA-matched sibling donors at this centre were included in this study. Potential factors affecting overall survival, rejection, disease-free survival and graft versus host disease were analyzed. Survival analysis was done by Kaplan-Meier method. Cox regression model was applied for multivariate analysis. Ninety male and thirty-five female patients with AA were included in the study. Median age was 18 years. Conditioning regimens used were cyclophosphamide [Cy] plus antilymphocyte globulin [ALG] or antithymocyte globulin [ATG], fludarabine [FLU] +Cy+ATG, Campath 1-H +Cy in 89, 30 and 6 cases respectively. GVHD prophylaxis used was ciclosporin [CSA] plus prednisolone and short methotrexate in 81 while 44 received CSA plus prednisolone. At a median follow-up of 1185 days OS and DFS were 84% and 78% respectively. Factors associated with better OS were male sex, Flu/Cy/ATG conditioning and use of bone marrow as stem cell source. Flu/Cy/ATG conditioning regimen, bone marrow as stem cell source and CSA, prednisolone and short methotrexate regimen were associated with better survival in AA

Tinnitus matching

To study the pitch and loudness of tinnitus and to find minimal masking level. Descriptive analytical study. This study was conducted at Combined Military Hospital Rawalpindi, the tertiary referral center for Pakistan Armed Forces from 1998 to 2001. Patients with tinnitus were included in the study. Exclusion criterion was transient tinnitus settling on removal of offending agent like foreign body and not having tinnitus as major presenting feature. Patients were thoroughly interviewed and tinnitus card [questionnaire] was filled. Thorough general physical and Ear Nose Throat examination was carried out including relevant tests to elucidate any cochleovestibular pathology. All patients had pure tone audiometery and tinnitus testing and sophisticated investigations were carried out whenever indicated. Most patients of tinnitus were seen between age of 40 to 60 years and 76.6% patients were male. Hearing loss accompanied tinnitus in 90% patients and 53% patients had tinnitus of moderate intensity. Pitch match frequency of 4 kilohertz was seen in 25.3% while 29% patients had loudness match of tinnitus ranging from 55 to 70 decibel [dB]. In vast majority [24%] minimal masking level ranged from 41 to 55 dB. Seventy nine percent patients had pitch match frequency at or around frequency of maximum hearing loss. Tinnitus is seen predominantly among males and its frequency increases with age .It is usually of moderate intensity and is of high pitch which corresponds to frequency of maximum hearing loss. Pitch of tinnitus is not a good indicator of degree of annoyance while loudness match gives a good measure of annoyance. Minimal masking levels are at or around loudness match. Tinnitus in majority of patients is associated with mild to moderate degree of hearing loss

Cholecystectomy; is drainage necessary?

To compare the outcome of cholecystectomy with and without drainage. The study was carried out from Jun 2005 to Nov. 2006 at Unit III, Department of Surgery, Liaqat University of Medical and Health Sciences, Jamshoro. All the patients diagnosed as cholelithiasis were treated with open cholecystectomy. The patients were randomly divided in group A and B. Number 18 Nasogastric tube was inserted in subheptic space after cholecystectomy in Group A, and no drain tube was placed in group B patients. Postoperatively patients in both groups were given same antibiotics. Postoperative complications and hospital stay were monitored in both groups. Exclusion criteria were cardiopulmonary disease, cirrhosis liver and diabeties mellitus. Patients with acute cholecystitis and choledocholithiasis were also excluded from study. Z-test is used to test the difference between proportions of two groups are statistically insignificant. During the study period a total of 100 patients were operated for cholelithiasis, with 50 patients in each group. The mean age for group A and B were 46 and 45 years respectively. The female to male ratio in the group A and B were 45:5 and 43:7 respectively. Mortality rate in both groups was zero. Group A had two cases of infected collection in subhepatic space and five cases of wound infection. In group B one patient with bile collection, one infected collection and two cases had wound infection. Mean hospital stay was 3.7 in group A as compared to 2.26 in group B. Both groups are statistically insignificant with respect to complications. Routine drainage after cholecystectomy is unnecessary

Pneumocystis carinii and Trichosporon beigelii pneumonia following allogeneic haemopoeitic stem cell transplantation

Pneumocystis Carinii and Trichosporon beigelii are opportunistic infections in immunocompromised patients. We report a case of a young lady who underwent haemopoeitic stem cell transplantation for relapsed acute lymphoblastic leukemia. This 25 years old female developed fever, dry cough and rapidly progressive dyspnoea during post transplant neutropenia and was found to be suffering from Pneumocystis carinii pneumonia. She was successfully treated with Co-trimoxazole. The patient again presented with similar symptoms on day 55 post transplant. This time Trichosporon beigelii was isolated from bronchoalveolar lavage and she responded to prompt antifungal therapy. Other complications encountered during the subsequent course were extensive subcutaneous emphysema and spontaneous pneumothorax that required chest intubation and brief hospitalization. The patient is presently nine months post transplant and is asymptomatic

Frequency of tuberculosis in haematological malignancies and stem cell transplant recipients

To assess magnitude of tuberculosis [TB] in patients suffering from various haematological malignancies and stem cell transplant [SCT] recipients. Descriptive study. Oncology Department, Combined Military Hospital, Rawalpindi, and Armed Forces Bone Marrow Transplant Centre, Rawalpindi, from July 2001 to December 2002. Patients suffering from various haematological malignancies treated between July 2001 and December 2002 were included in the study. The hospital records and out-patient follow-up charts were reviewed for demographic information, diagnosis, clinical presentation, laboratory investigations, radiological and pathological examinations, sites involved in TB, methods of diagnosis, number and type of anti-tuberculosis drugs given and response to treatment. During the study period a total of 213 [including 25 allogeneic stem cell transplant [SCT] recipients] patients with different haematological disorders were treated. Out of these, 34, including 4 SCT recipients developed tuberculosis. Overall frequency of TB was 16%. Median age of TB patients was 33.5 years [range 8-80 years]. Median time between diagnosis of haematological disorders and tuberculosis was 21 weeks. Sites of involvement by TB were lung [18], disseminated [6], lymph node [5], pleura [2], spine [2] and pericardium [1]. Three of the patients died of TB; one undiagnosed, second with multi-drug resistant TB and the third soon after the start of anti-tuberculosis treatment while remaining 31 cases responded to anti-tuberculosis treatment. Tuberculosis is a major problem in immunocompromised patients and there is need to establish guidelines for TB chemoprophylaxis in our setup