Cytogenetic culture failure and its causes in hematological disorders; a single centre perspective.

OBJECTIVE: To highlight the reasons of culture failure in bone marrow aspirate samples sent for Cytogenetic analysis and to identify the associated parameters causing this impact. METHODOLOGY: This is a retrospective cross-sectional study conducted in the Clinical and Molecular Cytogenetics Laboratory of NIBD Hospital, Karachi, Pakistan. The rates of culture failure are assessed from the year 2017-2020 along with their reasons. Bone Marrow aspirate samples of patients with hematological malignancies were cultured for chromosomal analysis, both at the time of diagnosis or relapse. Statistical analysis was performed using SPSS version 25. RESULTS: A total of 1061 bone marrow aspirate samples were assessed for cytogenetic culture failures from the duration of 2017 to 2020. Ratio of males was predominantly higher i.e. 62.7% than female 37.3% with Mean ± SD age was 36.78 ± 18.94. Frequency of culture failure in the year 2020 was relatively high 20% as compared to the preceding years i.e. 8% in 2017, 6% in 2018, 7% in 2019. However, the patients were diagnosed with the following hematological malignancies; ALL 23%, CML 17.1%, AML 16.5% and AA 12.5%. Among the reasons of culture failure, cytogenetic analysis of patients with on-going chemo resulted in significant culture failures with p-value < 0.001 and the hematological malignancy, Acute Promyelocytic Leukemia, significantly impacted the growth of bone marrow aspirate cultures, with p-value < 0.001. CONCLUSION: Significant findings were associated with causative factors of culture failure including on-going treatment and sample issues of clotted bone marrow as well as with the clinical diagnosis. These evaluations facilitated in overcoming the rise in culture failures. As per our knowledge, no such data, discussing the effects of various parameters such as sample quality, diagnosis, effects of treatment etc., has been documented previously.

Fludarabine/Cyclophosphamide Conditioning Regimen in Aplastic Anemia Patients Receiving Matched-Sibling Donor Transplant Is Non-inferior to ATG/Cyclophosphamide: A Single-Center Experience from Pakistan.

The successful outcome of allogeneic hematopoietic stem cell transplant (HSCT) in aplastic anemia patients is driven by suitable donor selection, appropriate conditioning regimen, early intervention, and optimal supportive care after transplant. Pakistan, being a developing country, faces grave economic challenges due to meager health care budget; therefore, cost constraints remain the foremost impediment in optimizing transplant facilities for socioeconomically deprived patients. We conducted a single-center retrospective analysis of aplastic anemia patients (N = 130), who received matched sibling donor transplants from 2011 to 2019, treated with either fludarabine/cyclophosphamide (Flu/Cy) or antithymocyte globulin/cyclophosphamide (ATG/CY) conditioning regimen. Median age was 16 years (IQR, 11-20), and it ranged from 3 to 48 years. The median time from diagnosis to transplant was 3 months (IQR, 2 to 4), and it ranged from 1 to 8 months. The estimated overall survival (OS), relapse-free survival (RFS), and GvHD-free survival (GFS) were found to be 69.0%, 66.7%, and 64.3% in the ATG/Cy group while 76.1%, 72.7%, and 62.5% in the Flu/Cy group, respectively, after a median follow-up of 30 months (IQR, 8 to 55), and it ranged from 0 to 98 months for the study groups. The Flu/Cy regimen was well tolerated and was not associated with increased risk of GvHD. Hence, it may be an appropriate alternative conditioning regimen for developing countries with limited health care resources.

P-Loop mutations-Negative prognosticators in tyrosine kinase inhibitors resistant chronic myeloid leukemia patients.

INTRODUCTION: P-Loop mutations in CML patients prevent the conformational change in BCR-ABL1 necessary for drug binding. The present study aimed to evaluate the impact of mutations in this domain on the prognosis of the disease and also to associate the baseline Sokal relative risk score with the overall survival in non-responding CML patients. METHODS: Blood samples were analyzed using ARMS-PCR and then an association was assessed between presence/absence of mutations, hematological and molecular response, disease progression, overall survival, and Sokal score. RESULTS: Of the total 250 CML patients, 102 were found to be treatment-resistant. Fifty-three patients harbored P-Loop mutations with G250E (12.7%) being most frequent. Complete hematological response and major molecular response were achieved by only 27.7% and 5.7 patients, respectively. Worst survival (57.1%) was observed in Y253H positive patients while according to Sokal score in high-risk patients harboring Y253F (50%) and E255V (50%). CONCLUSION: The presence of P-Loop domain mutations negatively impacted the prognosis of the disease in terms of disease advancement and overall survival. So, the timely performance of the BCR-ABL1 mutational analysis and the modifications in the treatment plan based on the mutation identified would help in a better outcome of the disease.

Beyond the In-Practice CBC: The Research CBC Parameters-Driven Machine Learning Predictive Modeling for Early Differentiation among Leukemias.

A targeted and timely treatment can be a beneficial tool for patients with hematological emergencies (particularly acute leukemias). The key challenges in the early diagnosis of leukemias and related hematological disorders are their symptom-sharing nature and prolonged turnaround time as well as the expertise needed in reporting confirmatory tests. The present study made use of the potential morphological and immature fraction-related parameters (research items or cell population data) generated during complete blood cell count (CBC), through artificial intelligence (AI)/machine learning (ML) predictive modeling for early (at the pre-microscopic level) differentiation of various types of leukemias: acute from chronic as well as myeloid from lymphoid. The routine CBC parameters along with research CBC items from a hematology analyzer in the diagnosis of 1577 study subjects with hematological neoplasms were collected. The statistical and data visualization tools, including heat-map and principal component analysis (PCA,) helped in the evaluation of the predictive capacity of research CBC items. Next, research CBC parameter-driven artificial neural network (ANN) predictive modeling was developed to use the hidden trend (disease's signature) by increasing the auguring accuracy of these potential morphometric parameters in differentiation of leukemias. The classical statistics for routine and research CBC parameters showed that as a whole, all study items are significantly deviated among various types of leukemias (study groups). The CPD parameter-driven heat-map gave clustering (separation) of myeloid from lymphoid leukemias, followed by the segregation (nodding) of the acute from the chronic class of that particular lineage. Furthermore, acute promyelocytic leukemia (APML) was also well individuated from other types of acute myeloid leukemia (AML). The PCA plot guided by research CBC items at notable variance vindicated the aforementioned findings of the CPD-driven heat-map. Through training of ANN predictive modeling, the CPD parameters successfully differentiate the chronic myeloid leukemia (CML), AML, APML, acute lymphoid leukemia (ALL), chronic lymphoid leukemia (CLL), and other related hematological neoplasms with AUC values of 0.937, 0.905, 0.805, 0.829, 0.870, and 0.789, respectively, at an agreeably significant (10.6%) false prediction rate. Overall practical results of using our ANN model were found quite satisfactory with values of 83.1% and 89.4.7% for training and testing datasets, respectively. We proposed that research CBC parameters could potentially be used for early differentiation of leukemias in the hematology-oncology unit. The CPD-driven ANN modeling is a novel practice that substantially strengthens the predictive potential of CPD items, allowing the clinicians to be confident about the typical trend of the "disease fingerprint" shown by these automated potential morphometric items.

Diagnosis and Management of Diffuse Large B-Cell Lymphoma: Society of Medical Oncology, Pakistan Society of Hematology, and Pakistan Society of Clinical Oncology Joint Clinical Practice Guideline.

Diffuse large B-cell lymphoma (DLBCL) is the commonest non-Hodgkin lymphoma encountered by hematopathologists and oncologists. Management guidelines for DLBCL are developed and published by countries with high income and do not cater for practical challenges faced in resource-constrained settings. This report by a multidisciplinary panel of experts from Pakistan is on behalf of three major national cancer societies: Society of Medical Oncology Pakistan, Pakistan Society of Hematology, and Pakistan Society of Clinical Oncology. The aim is to develop a practical and standardized guideline for managing DLBCL in Pakistan, keeping in view local challenges, which are similar across most of the low- and middle-income countries across the globe. Modified Delphi methodology was used to develop consensus guidelines. Guidelines questions were drafted, and meetings were convened by a steering committee to develop initial recommendations on the basis of local challenges and review of the literature. A consensus panel reviewed the initial draft recommendations and rated the guidelines on a five-point Likert scale; recommendations achieving more than 75% consensus were accepted. Resource grouping initially suggested by Breast Health Global Initiative was applied for resource stratification into basic, limited, and enhanced resource settings. The panel generated consensus ratings for 35 questions of interest and concluded that diagnosis and treatment recommendations in resource-constrained settings need to be based on available resources and management expertise.

Sequencing of SARS-CoV-2 in local transmission cases through Oxford Nanopore MinION platform from Karachi Pakistan.

INTRODUCTION: The first case of severe acute respiratory syndrome 2 (SARS-CoV-2) was imported to Pakistan in February 2020, since then 8,260 deaths have been witnessed. The virus has been constantly mutating and local transmission cases from different countries vary due to host dependent viral adaptation. Many distinct clusters of variant SARS-CoV-2 have been defined globally. In this study, the epidemiology of SARS-CoV-2 was studied and locally transmitted SARS-CoV-2 isolates from Karachi were sequenced to compared and identify any possible variants. METHODOLOGY: The real time PCR was performed on nasopharyngeal specimen to confirm SARS-CoV-2 with Orf 1ab and E gene as targets. The virus isolates were sequenced through oxford nanopore technology MinION platform. Isolates from the first and second wave of COVID-19 outbreak in Karachi were compared. RESULTS: The overall positivity rate for PCR was 26.24% with the highest number of positive cases in June. Approximately, 37.45% PCR positive subjects aged between 19-40 years. All the isolates belonged to GH clade and shared missense mutation D614G in spike protein linked to increased transmission rate worldwide. Another spike protein mutation A222V coexisted with D614G in the virus from the second wave of COVID-19. CONCLUSIONS: Based on the present findings it is suggested that the locally transmitted virus from Karachi varies from those reported from other parts of Pakistan. Slight variability was also observed between viruses from the first and second wave. Variability in any potential vaccine target may result in failed trials, therefore information on any local viral variants is always useful for effective vaccine design and/or selection.

Usefulness of convalescent plasma transfusion for the treatment of severely ill COVID-19 patients in Pakistan.

BACKGROUND: Convalescent plasma(CP) was utilized as potential therapy during COVID-19 pandemic in Pakistan. The study aimed at appraisal of CP transfusion safety and usefulness in COVID pneumonia. METHODS: Single arm, MEURI study design of non-randomized open label trial was conducted in five centers. Patients werecategorized as moderately severe, severe, and critical. The primary endpoint was a) improvement in clinical status and change in category of disease severity; secondary endpoint was b) CP ability to halt disease progression to invasive ventilation. CP transfused to hospitalized patients. Statistical tests including median (interquartile ranges), Mann-Whitney U test, Fisher's exact test using SPSS ver. 23, ANOVA and Chi-square test were applied for the analysis of results parameters before and after CP treatment. SOFA score was applied for multiorgan failure in severe and critical cases. RESULTS: A total of 50 adult patients; median age 58.5 years (range: 29-92 years) received CP with infusion titers; median 1:320 U/mL (Interquartile range 1:80-1:320) between April 4 to May 5, 2020. The median time from onset of symptoms to enrollment in trial was 3 to 7 days with shortness of breath and lung infiltration as severity criterion. In 35 (70%) recipients, oxygen saturation improved from 80 to 95% within 72h, with resolution of lung infiltrates. Primary endpoint was achieved in 44 (88%) recipients whereas secondary endpoint was achieved in 42 (84%). No patient experienced severe adverse events. A high SOFA score (> 7) correlated with deaths in severe and critical patients. Eight (16%) patients expired due to comorbidities; cardiac arrest in 2 (4%), multiorgan failure secondary to cytokine storm in 5 (10%) and ventilator associated complications in 1 (2%). CONCLUSION: CP transfusion can be used as a safe and useful treatment in moderately severe and severe patients. TRIAL REGISTRATION: The trial registration number is NCT04352751  ( https://www.irct.ir/search/result?query=IRCT20200414047072N1 ). Trial Registration date is 28th April 2020.

Mature and Immature/Activated Cells Fractionation: Time for a Paradigm Shift in Differential Leucocyte Count Reporting?

Leucocytes, especially neutrophils featuring pro- and anti-cancerous characteristics, are involved in nearly every stage of tumorigenesis. Phenotypic and functional differences among mature and immature neutrophil fractions are well reported, and their correlation with tumor progression and therapy has emerging implications in modern oncology practices. Technological advancements enabled modern hematology analyzers to generate extended information (research parameters) during complete blood cell count (CBC) analysis. We hypothesized that neutrophil and lymphocyte fractions-related extended differential leucocytes count (DLC) parameters hold superior diagnostic utility over routine modalities. The present study was carried out over a four-and-a-half-year period wherein extended neutrophil (immature granulocyte [IG] and mature neutrophil [NEUT#&]), and lymphocyte (activated/high fluorescence lymphocyte count [HFLC] and resting lymphocyte [LYMP#&]) parameters were challenged over routine neutrophil [NEUT#] and lymphocyte [LYMP#] items in a study population of 1067 hematological neoplasm patients. Extending the classical statistical approaches, machine-learning-backed data visualization was used to explore trends in the study parameters. As a whole, extended neutrophil and lymphocyte count outperformed and was diagnostically more relevant than routine neutrophil and lymphocyte parameters by showing the least difference from their respective (gold-standard) manual DLC counts. The mature neutrophil count was compared to IG, and resting lymphocyte count was compared to HFLC by calling the function 'correlation' as a 'clustering function' for heatmap based visualization. The aforementioned study parameters displayed close clustering (rearrangement) for their respective study items by presenting distinct trends of equally valuable weights (deviated values), advocating fractions-based extended DLC reporting. Importantly, using a Bland and Altman analysis analogously to a manual neutrophil count, the mature neutrophil count [NEUT#&] remained unbiased since a routine neutrophil count [NEUT#] was found to be a negatively biased. The extended DLC-parameter-driven fractions-based reporting has superior diagnostic utility over classical routine approaches; this finding can largely minimize labor-intensive manual DLC practices, especially in hematology-oncology departments.

CLINICAL RELEVANCE OF EXTENDED PLATELET INDICES IN THE DIAGNOSIS OF IMMUNE THROMBOCYTOPENIA.

Immune thrombocytopenia (ITP) is an autoimmune disorder. Besides platelet count, immature platelet fraction (IPF) can be used as a tool to predict megakaryocytic activity in ITP patients. The aim of the study was to evaluate the utility of extended platelet indices in ITP diagnosis and their association with disease persistence and severity. This case-control study (1:1), conducted from January 2015 to December 2017, included 111 ITP patients and 111 healthy controls. ITP patients were grouped as newly diagnosed ITP, persistent ITP, chronic ITP, and refractory ITP patients. Peripheral blood was collected and complete blood profile parameters were recorded using Sysmex XN 1000. Significant (p≤0.05) difference between the groups of ITP patients and healthy control subjects was determined by Fisher exact test, while Pearson correlation was used to evaluate platelet count correlation with IPF using SPSS ver. 23. Low hemoglobin and platelet counts with high total leukocyte count and IPF were detected in ITP patients as compared to healthy subjects (p≤0.001). Among all groups of ITP patients, very low platelet count (6.9±6.02.x109/L) with highest mean IPF (27.1±19.2%) was observed in newly diagnosed ITP group. Other platelet parameters including mean platelet volume (MPV), plateletcrit, platelet large cell ratio (P-LCR) and platelet distribution width values were also altered in patient groups. Pearson correlation revealed negative relationship between platelet count and IPF in all patient groups. With the advent of new, sophisticated hematologic analyzers, the IPF and other platelet parameters provide simple, reliable and easier tools for predicting platelet disorders such as ITP, and to some extent the disease severity. Besides IPF, the MPV and P-LCR seemed to predict disease severity, treatment responsiveness, and duration of the disease to some extent.

Incorporating a clinical oncology pharmacist into an ambulatory care pharmacy in pediatric hematology-oncology and transplant clinic: Assessment and significance.

BACKGROUND: Beta thalassemia patients, post-bone marrow transplant, and leukemia patients require long term therapy with an intense care follow-up especially for pediatric hematology-oncology origin. Emergence of side effects and noncompliance to therapy lead to reduced efficacy of medicines resulting in relapse of diseases. There is an increasing fact to support the incorporation of a pharmacist into clinical team due to their distinctive skills. Clinical oncology pharmacist with experience and specialized training in hematological cancers and bone marrow transplantation (BMT) patient care has in-depth knowledge and skills of chemotherapy regimens including drug information, monitoring parameters of cancer treatment, dose adjustment, drug-drug interactions, adverse effects, and patient counseling skills. AIM AND OBJECTIVES: The main objective of our study was to assess the significance of incorporation of clinical oncology pharmacist in ambulatory care in pediatric hematology-oncology and transplant clinic. MATERIAL AND METHOD: This study was conducted at National Institute of Blood Diseases and Bone Marrow Transplantation hospital with duration of five months from 17 March 2019 to 16 July 2019. In this study the clinical oncology pharmacist was made available at ambulatory clinic of hematology-oncology and transplantation. The activities performed by a clinical oncology pharmacist were observed by resident BMT clinical pharmacist during the visits of patients and their families in a clinic. The BMT pharmacist is a clinical oncology pharmacist with experience and specialized training in hematological cancers and BMT patient care. Only pediatrics patients with beta thalassemia major and those who were on chemotherapy treatment and post-transplant patient were included in this study. RESULTS: During the five months' tenure, there were 1820 pediatric patients' visits in total. The clinical oncology pharmacist performed 980 direct patient interviews and documented 1665 pharmacist interventions. The majority of the documented clinical oncology pharmacist interventions were review of medication histories (n: 404, 24%) and "deferiprone" dose adjustments (n:400, 24%). Genomic profiling interventions were also among the commonly reported activities by the clinical oncology pharmacist. For beta thalassemia patients undergoing hydroxyurea therapy, the genomic profiling was performed to assess whether the hydroxyurea treatment is clinically effective or not (n:396, 23%). CONCLUSION: The involvement of clinical oncology pharmacist into a specialized outpatient clinic of hematology-oncology and transplant clinic plays an integral role in minimizing the adverse effect and reduction in readmission into the hospital. This is new expansion of pharmacist's role especially in underdeveloped country, considering the relevant clinical participation of clinical oncology pharmacist into specialized clinic revealing through optimized therapy and future prospect of clinical oncology pharmacist in pediatric hematology.

Peripheral Blood Stem Cell Harvest HPC Count Is an Effective Surrogate Marker for CD34+ Cell Count in Allogeneic Stem Cell Transplant Setting.

OBJECTIVE: We assessed the predictive potential of XN-HPC for CD34+ cell count as obtained through Sysmex automated hematology analyzers (XN-1000). METHODS: This study was conducted at the National Institute of Blood Diseases and Bone Marrow Transplantation in 84 donors between December 2012 and December 2017 in the first phase and later validated in 112 donors between December 2017 and December 2018. Sysmex XN-1000 and BD FACS Calibur estimated XN-HPC and CD34+ cells of peripheral blood apheresis product, respectively. Spearman's correlation was assessed between XN-HPC and CD34+ cell count followed by receiver operating characteristic curve calculation to determine the XN-HPC cutoff for a CD34+ count of ≥2 million cells/kg of recipient's body weight RESULTS: There is a moderately positive correlation (P value = .003) between XN-HPC and CD34+ count. Receiver operating characteristic curve analyses demonstrated that a cutoff value for XN-HPC of ≥1·845×106cells/kg of recipient's body weight has a specificity and sensitivity of 100% and 78·2%, respectively, for predicting the CD34+ count of ≥2 million cells/kg of recipient's body weight. This cutoff value of XN-HPC was prospectively validated in 112 donors. The positive predictive value was found to be 100%, while negative predictive value was 17%. CONCLUSION: XN-HPC has a highly promising potential to serve as a cost-effective and time-saving surrogate for CD34+ cell count.

A distinct molecular mutational profile and its clinical impact in essential thrombocythemia and primary myelofibrosis patients.

BACKGROUND: Classical MPNs including ET and PMF have a chronic course and potential for leukaemic transformation. Timely diagnosis is obligatory to ensure appropriate management and positive outcomes. The aim of this study was to determine the mutational profile, clinical characteristics and outcome of ET and PMF patients in Pakistani population. METHODS: This was a prospective observational study conducted between 2012 and 2017 at NIBD. Patients were diagnosed and risk stratified according to international recommendations. Response to treatment was assessed by IWG criteria. RESULTS: Of the total 137 patients analysed, 75 were ET and 62 were PMF. JAK2 positivity was seen in 51 cases (37.2%), CALR in 41 cases (29.9%), while triple-negative in 17 (12.4%) cases. None of the patients in the present study were MPL positive. Overall survival for patients with ET and PMF was 92.5 and 86.0% respectively and leukaemia free survival was 100 and 91.6% respectively, at a median follow-up of 12 months. Leukaemic transformation occurred in 6.5% of MF patients; among them, JAK2 mutation was frequently found. Molecular mutations did not influence the OS in ET whereas in PMF, OS was shortest in the triple-negative PMF group as compared to the JAK2 and CALR positive patient groups. CONCLUSION: This study shows a different spectrum of molecular mutations in ET and PMF patients in Pakistani population as compared to other Asian countries. Similarly, the risk of leukaemic transformation in ET and PMF is relatively lower in our population of patients. The factors responsible for these phenotypic and genotypic differences need to be analysed in large scale studies with longer follow-up of patients.

Case report of one month and 15 days old baby with type V hyperlipoproteinemia (HLP).

BACKGROUND: Most of the patients with type 1 and V hyperlipoproteinemia (HLP) present with symptoms and signs of acute pancreatitis due to marked elevation of triglycerides, but this baby presented with a chest infection, which was later diagnosed as type V HLP on laboratory workup. CASE PRESENTATION: We report a case of a 1 month and 15 days old baby boy, product of 2-nd degree consanguinity admitted to a nearby hospital with complaints of refusal to feed, cough and excessive crying. On examination his heart rate was 102 beats/min, respiratory rate was 55 breaths/min and temperature was within the normal range, provisional diagnosis of Pneumonia was made. His samples were tested at our laboratory, the lipid Profile at age of 1 month 15 days showed total cholesterol (TC) of 1400 mg/dl reference range (RR < 200 mg/dl), triglycerides (TG) of > 885 mg/dl after dilution it was 31,400 mg/dl (RR < 150 mg/dl), High density Cholesterol (HDL) of 35 mg/dl (RR > 40 mg/dl) and low density cholesterol (LDL) of 200 mg/dl (RR < 100 mg/dl). The patient's blood sample was grossly milky and lipemic in appearance. A "Refrigerator test" was performed after overnight storage of the sample in refrigerator at 4 °C, which gave a creamy layer at the top and clear infranatant due to caking of the Chylomicrons. Lipoprotein electrophoresis performed 1 month later showed Chylomicrons of 4.7% (RR 0-2%), Pre-beta lipoproteins of 51.5% (RR 5-22%), beta lipoproteins of 16.5% (RR 39-70%) and alpha of 27.3% (RR 23-53%). Initially he was diagnosed as type 1 HLP, but later on he was correctly diagnosed as type V HLP. Cholestyramine (Questran sachet) powder was started at a dose of 100 mg/kg on t.i.d basis with NAN-1 formula Milk at the age of 1 month and 15 days. On follow up, detailed advices regarding the weaning food was given to the mother (using olive oil in cooking, giving proteins and avoiding heavy fatty meals). His lipid profile was repeated at age of 3 months, which showed some improvement, his TGs were 1986 mg/dl and TC 105 mg/dl. CONCLUSION: There is no universal diagnostic criterion for diagnosing Type V HLP, most likely, due to a scanty literature on this disorder. It stimulated us to report this case so that our findings may help for a timely diagnosis of the affected patients.

Characterization of the mutation spectrum in a Pakistani cohort of type 3 von Willebrand disease.

INTRODUCTION: Type 3 von Willebrand disease (VWD), a severe autosomal recessive hereditary bleeding disorder, is described by the virtual absence of von Willebrand factor (VWF). In consanguineous populations, for example Pakistan, the disease is reported with a higher incidence rate than the worldwide prevalence. AIMS: This study aims to characterize molecular pathology and clinical profile of type 3 VWD cohort of Pakistani origin. METHODS: In total, 48 patients were enrolled in the current study. Initially, the index patients (IPs) were evaluated by a standardized questionnaire for recording bleeding manifestations and by performing conventional coagulation tests. The diagnosis of VWD type 3 was confirmed by VWF antigens less than 5 IU/dL. Direct sequencing of VWF gene (VWF) was carried out to identify causative gene variations. We evaluated the potential consequence of novel splice site and missense variations by predictive computational programs and in silico structural analysis. RESULTS: VWF mutations were detected in 46 out of 48 IPs (95.8%), predominantly as homozygous variants. In total, twenty-nine different gene defects were characterized in this cohort from which 10 (34.5%) are novel. The majority of the mutations were null alleles (66%; including gene conversions, nonsense, splice site variations, small deletions and insertions), and 34% of them were missense substitutions. CONCLUSION: Herein, we reported for the first time, the pattern of gene defects in Pakistani type 3 VWD cohort. We identified a wide heterogeneous mutation spectrum along with variability in the type of bleeding episodes.

Early and sustained deep molecular response achieved with nilotinib in high Sokal risk chronic myeloid leukemia patients.

BACKGROUND: Nilotinib (Tasigna®) is a second-generation tyrosine kinase inhibitor that shows faster and deeper molecular responses (MR) in comparison to Imatinib as initial therapy in chronic phase chronic myeloid leukemia (CML). Efficacy and safety data for nilotinib in the Asian population is scarce, particularly in Pakistan. We aimed to determine the MR to nilotinib and its safety profile in patients with chronic phase CML. PATIENTS AND METHODS: This observational study was conducted among 173 patients with newly diagnosed CML presenting in the chronic phase. Most patients (50.1%) had a high Sokal score at diagnosis. All patients received nilotinib 600 mg/day. The hematological and molecular responses were assessed at 3 and 6 months respectively and thereafter at 6-monthly intervals. Long-term event free survival (EFS), transformation free survival (TFS), overall survival (OS) and adverse events were observed. RESULTS: Cumulative incidence of major MR (MMR) was 86% and deep MR (DMR ie MR 4.0 and MR4.5) was 39%. Early MMR and DMR after 6 months of therapy were achieved by 74.9% and 37% of patients, respectively. Two-year EFS, TFS and OS rates for all patients were 91.9%, 92% and 92.3%, respectively. At median follow-up of 24 months, 81% and 49% of patients sustained MMR and DMR, respectively. The main adverse events were weight gain (4.6%) and abdominal pain (4%). CONCLUSION: This study showed promising results in terms of achievement of early and sustained DMR in chronic phase CML, therefore, we recommend nilotinib as frontline treatment in Pakistani population.

Lung resistance-related protein (LRP) predicts favorable therapeutic outcome in Acute Myeloid Leukemia.

There is conflicting evidence that MDR1, MRP2 and LRP expression is responsible for chemotherapy resistance. We conducted this study to explore their role in AML therapy outcomes. Bone marrow and peripheral blood samples of 90 AML patients, receiving chemotherapy, were analyzed by real time PCR. Gene expression was calculated by the 2-ΔΔCt method. The patients who had a persistent remission were labelled 'Good Responder' (GRes) whereas, those with relapse or drug resistance were labelled 'Poor Responders' (PRes). Higher LRP expression in bone marrow, but not in peripheral blood, was positively associated with persistent remission (p = 0.001), GRes (p = 0.002), 1-year overall as well as disease-free survival (p = 0.02 and p = 0.007, respectively). Marrow and blood MDR1 and MRP2 expression did not differ significantly between the above groups. Logistic regression analysis showed that only a diagnosis of acute promyelocytic leukemia (APL; M3) or high marrow LRP expression significantly predicted a favorable therapeutic outcome. This is the first report showing that high bone marrow LRP expression predicts significant favorable therapeutic outcome. Peripheral blood LRP expression as well as marrow and blood MDR1 and MRP2 expression have no predictive value in AML patients treated with standard dose cytarabine and daunorubicin 3+7 regimen.

Gene expression of hENT1, dCK, CDA, dCMPD and topoisomerase IIα as an indicator of chemotherapy response in AML treated with cytarabine and daunorubicin.

PURPOSE: Acute myeloid leukemia patients are commonly treated with cytarabine (Ara-C) and anthracyclines but the sustained remission rate is not very promising. We explored the role of drug-metabolizing enzymes and transporters in the therapeutic response. PATIENTS AND METHODS: Bone marrow and peripheral blood samples of 90 newly diagnosed acute myeloid leukemia patients treated with standard 3+7 regimen were analyzed through real-time PCR for expression of human equilibrative nucleoside transporter 1, deoxycytidine kinase, cytidine deaminase (CDA), deoxycytidine monophosphate deaminase (dCMPD) and topoisomerase IIα (Topo-IIa). The expression of these markers was studied in relationship with good (persistent remission) and poor therapeutic response (relapse/resistance). RESULTS: High Topo-IIa expression in peripheral blood was associated with good response (P=0.006). Relapse was higher among low expressors of Topo-IIa in peripheral blood (OR: 26.25). Bone marrow Topo-IIa expression followed a similar trend but did not reach statistical significance. In contrast, patients with high bone marrow dCMPD expression had poor response (OR: 3; P=0.043). One-year disease-free survival (DFS) was better among those with high bone marrow Topo-IIa (P=0.04) or CDA (P=0.03) expression. High bone marrow Topo-IIa expression also had better DFS at 6 months (P=0.04) and at 12 months (P=0.04). CONCLUSION: High expression of Topo-IIa in peripheral blood is a favorable indicator of persistent remission, good therapeutic response and DFS. High dCMPD and low CDA expression in bone marrow is associated with poor therapeutic outcome.

Evaluation of efficacy of serological methods for detection of HCV infection in blood donors: A single centre experience.

BACKGROUND AND OBJECTIVE: Blood transfusion is an essential and life-saving medical intervention. Despite multiple preventive measures transfusion-transmitted hepatitis C virus (HCV) infection continues to be a major healthcare issue in Pakistan. This study was conducted at National Institute of Blood Diseases & Bone Marrow Transplantation to evaluate the frequency of active HCV infection with or without co-infection in blood donors and also to determine comparative efficacy of Multisure HCV antibody assay (MHAA); a new serological device. METHODS: A total of 14652 blood donors visiting National Institute of Blood Diseases & Bone Marrow Transplantation (NIBD) Blood Bank from January 2013 to July 2014 were enrolled and screened for a range of blood borne infections such as HBV, HCV, HIV, malaria and syphilis. The HCV was screened simultaneously by Abbot Architect anti-HCV assay (CLIA) and MHAA. The active HCV infection was confirmed by nucleic acid testing (NAT) in reactive donors. Later; for determination of comparative efficacy of MHAA; all NAT positive samples were further tested using Monolisa™, HCV blot 3.0, Anti-HCV plus V2 and Anti-HCV-MPBIO-EIA. RESULTS: The HCV reactive sera were observed in 1.563% (226) donors. The NAT confirmed active HCV infection in 138 donors. Overall 27.84% of HCV positive donors exhibited co-infection either with HBV (2.57%), syphilis (22.78%). Triple infection was not observed in any donor. The efficacy of MHAA is comparable to all the serological tests with a sensitivity of about 96.89%. CONCLUSION: Active HCV infection was present in 0.94% donors. With a sensitivity of 96.89% (95% CI: 95.66-98.12) the multi-parametric device MHAA can effectively detect HCV infection in donors. Thus, it can be used in limited health care settings for HCV screening.

Detection of the Incidence of HBV, HCV Infection and Febrile Neutropenia Associated With CHOP With or Without Rituximab in Diffuse Large B-Cell Lymphoma-Treated Patients.

Background: Reactivation of hepatitis B virus (HBV) and hepatitis C virus (HCV) and febrile neutropenia (FN) are common in diffuse large B-cell lymphoma (DLBCL) patients undergoing cyclophosphamide, hydroxyrubicin, Oncovin, and prednisolone (CHOP) or cyclophosphamide, hydroxyrubicin, Oncovin, prednisolone - rituximab containing (R-CHOP) chemotherapy. This ultimately leads to delaying the therapy, increasing hospital stay, and raising the pharmacoeconomic burden on patients. Aim and Objective: The aim of this study was to determine the incidence of HBV and HCV infection and febrile neutropenia in DLBCL patients treated with R-CHOP and CHOP. Methodology: This was an institutional approved study in which patient records from a private hospital, specialized in hematology and oncology (Karachi, Pakistan), were reviewed retrospectively from 2014 to 2016. Patients aged above 18 years with known diagnosis of DLBCL who underwent CHOP-21 or R-CHOP-21 chemotherapy regimen were included. Baseline blood chemistry and liver function tests along with the data regarding HBV (hepatitis B surface antigen [HBsAg], hepatitis B surface antibody [anti-HBs]), HCV (antibody anti-HCV), and febrile neutropenia were collected from patient records. Results: In total, 35 cases of DLBCL were treated during a 3-year period (ie, from 2014 to 2016), of which 16 were on CHOP-21 regimen whereas 19 were treated with R-CHOP-21. Of the 19 patients who underwent R-CHOP chemotherapy, only 2 (10%) patients were HBsAg reactive. Before commencing the second cycle, 2 (10%) patients reported to hospital with fever and had hematological (low neutrophil count) and microbiological (Escherichia coli) proven febrile neutropenia. The incidence of HBV infection post treatment was lower in group treated with CHOP (1 patient showed HBsAg reactivity).

Bax, Bcl-2, and Bax/Bcl-2 as prognostic markers in acute myeloid leukemia: are we ready for Bcl-2-directed therapy?

PURPOSE: Many anticancer drugs induce apoptosis in malignant cells, and resistance to apoptosis could lead to suboptimal or no therapeutic benefit. Two cytoplasmic proteins, B-cell lymphoma protein 2 (Bcl-2)-associated X (Bax) and Bcl-2, act as a promoter and an inhibitor of apoptosis, respectively. Both Bax and Bcl-2 as well as their ratio have been regarded as prognostic markers in various cancers. However, conflicting results have been reported. A clear understanding of apoptosis has also become crucial due to reports about anti-Bcl-2 chemotherapy. We explored the relationship of Bax and Bcl-2 gene expression and their ratio with the therapeutic response in acute myeloid leukemia (AML) patients. PATIENTS AND METHODS: Bone marrow and/or blood samples from 90 AML patients treated with cytarabine and daunorubicin were included. Expression of Bax and Bcl-2 was determined through real-time polymerase chain reaction by using ΔΔCt method of relative expression. RESULTS: Bax and Bcl-2 expression among marrow and blood samples correlated with each other (rs=0.5, p<0.01). Although bone marrow expression of Bax and Bcl-2 tended to remain higher among responders (median 1.01 and 0.29, respectively) as compared to non-responders (median 0.66 and 0.24, respectively), the difference failed to reach statistical significance (U=784.5 and 733; p=0.68 and 0.28, respectively). Conversely, Bax/Bcl-2 ratio was higher among poor responders (median 3.07 vs 1.78), though again failed to reach statistical significance (U=698.5, p=0.07). CONCLUSION: Expression of Bax and Bcl-2 does not differ significantly among AML patients treated with cytarabine and daunorubicin in terms of remission, relapse, resistance, overall survival, and disease-free survival, thus questioning the utility of emerging anti-Bcl-2 therapy.