Armed Forces Bone Marrow Transplant Research Articles

Acute Graft versus Host Disease in Beta Thalassemia Patients Following Allogeneic Haematopoietic Stem Cell Transplantation.

To analyse the frequency, risk factors, and clinical symptoms of acute graft-versus-host disease (aGvHD) in patients with beta-thalassemia major after allogeneic haematopoietic stem cell transplantation (HSCT). Descriptive study. Place and Duration of the Study: Department of Clinical Haematology, Armed Forces Bone Marrow Transplant Centre, Rawalpindi, Pakistan, from January 2017 to December 2021. Data were obtained from patients diagnosed with bone and tissue malignancies (BTM) who had undergone haematopoietic stem cell transplantation (HSCT) and experienced aGVHD. Patients who experienced initial graft failure and individuals who underwent subsequent bone marrow transplantation were excluded. Total of 117 patients diagnosed with BTM underwent fully matched HSCT, including 76 (65%) males, and 41 (35%) females. The median age of the patients undergoing transplantation was 7.34±7.32 years and the donors' median age was 7.6±9.85 years. Among the donors, 53 (45.3%) were males and 64 (54.7%) were females. Gender disparity was observed in 46 (39.3%) instances as a female donor matched with a male recipient. A total of 106 individuals underwent bone marrow harvest (BMH); with 5 (4.3%) patients receiving peripheral blood stem cells (PBSC) and 6 (5.2%) patients receiving both BMH and PBSC. Acute GvHD was observed in 50 (42.7%) patients, including 30 (60%) males and 20 (40%) females. Grade I GvHD occurred in 32 (27.3%) individuals, Grade II GvHD in 16 (13.7%) patients, and Grade III GvHD in one (0.8%) patient. It had no statistically significant association with recipient/donor age, gender disparity, the source of the graft source, the dose of stem cells, or the presence of thymoglobulin (TG). Acute GvHD was observed in high frequency in Beta-thalassemia patients receiving morrow harvesting proportional to their gender distribution. Associated factors were GvHD prophylaxis measure, mucositis and, CMV reactivation. Beta thalassemia major patients, Acute graft versus host disease, Allogeneic haematopoietic stem cell.

Densitometry of STR-PAGE for donor chimerism in acute leukemia’s: A simple method for routine use

Objective: To evaluate a PCR based method of polyacrylamide gel electrophoresis of short tandem repeats and its quantification for detecting donor chimerism after haematopoietic stem cell transplantation in acute leukaemias.
 Methods: The descriptive study was conducted at Genetic Resource Centre (GRC) Lab Rawalpindi from Feb 2018 - Nov 2020. A total of twenty patients with acute leukaemias having undergone HSCT were selected and assessed for the analysis of chimerism status. DNA extraction from the whole blood was done by chelex method and short tandem repeats were amplified by using conventional STR- PCR assay. Electrophoresis was carried out and 6% polyacrylamide gels were used for the resultant amplified DNA products and then followed by their densitometry. These patients had undergone HSCT from Pakistan Institute of Medical Science and Armed Forces Bone Marrow Transplant Centre.
 Results: The peaks in the PAGE densitometry represented the donor chimerism in all post transplant samples of the patients.
 Conclusion: Our study showed that densitometry of STR PCR PAGE is a useful and cheaper method for demonstration of donor chimerism in acute leukaemia patients having undergone HSCT. Hence this method can be a valuable option in the monitoring of chimerism status in these patients and therefore helps in preventing graft failure by fast and early treatment strategies for these patients.
 doi: https://doi.org/10.12669/pjms.40.5.9216
 How to cite this: Nayyar A, Ahmed S. Densitometry of STR-PAGE for donor chimerism in acute leukemia’s: A simple method for routine use. Pak J Med Sci. 2024;40(5):---------. doi: https://doi.org/10.12669/pjms.40.5.9216
 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Cytomegalovirus Reactivation in Allogenic Haematopoietic Stem Cell Transplant Patients with Haploidentical Versus Fully Human Leukocyte Antigen Matched Donor

Objective: To determine CMV reactivation in hematopoietic stem cell transplant patients with haploidentical versus fully HLA matched donors Methodology: Retrospective Observational study was conducted at Armed Forces Bone Marrow Transplant Centre (AFBMTC) between March 2019 and October 2022. We enrolled a total of 60 consecutive patients.CMV DNA PCR analysis was carried out every week from day +14 to day +100 in order to monitor CMV reactivation during the 100-day post-transplantation period. The SaMag viral nucleic acid extraction kit (Sacace Biotechnologies, Como, Italy) was used to extract the DNA from two millilitres of blood collected in EDTA tubes. The kit's lower limit of detection for blood and serum was 5 copies/mL, while for other body fluids it was 500 copies/mL. An Applied Biosystems 7500 real-time PCR machine (Thermo Fisher Scientific, Waltham, MA) was used to conduct the PCR analysis. Result: The age range from 0.6 to 53 years ago was a median of 15±10.6. 39 patients had CMV reactivation (65%). With a range of 13 to 66 days, the median time to CMV DNAemia was 19.25 days.. Only two (3.3%) of the 39 patients received ganciclovir, while 26 (43.3%) received valganciclovir. Patients with aplastic anaemia had the highest rate of CMV infection (41%), followed by those with ALL (20.5%), AML (12.8%), and primary immunodeficiency (10.2%). A statistically significant connection between the underlying diagnosis and CMV infection. CMV infection was higher in haplo identical transplant recipients (66.6%) than in completely matched recipients (33.3%) and was statistically significantly. Sadly, 14 people passed away, yielding a mortality rate of 23.3%. Primary graft failure was the most frequent cause of mortality, resulting in neutropenic sepsis in 6 patients (42.85%), but 3 of these patients also had CMV reactivation. The rate of overall survival (OS) was found to be 76.7%. The rate of disease-free survival (DFS) was estimated at 71.7%. Notably, neither OS nor DFS in our analysis showed a statistically significant connection with CMV infection. Conclusion: The findings demonstrated a statistically significant association between haploidentical transplantation and CMV reactivation, with higher rates observed in the haploidentical group compared to fully matched recipients. Various factors, including underlying diagnosis, conditioning regimen, and GVHD prophylaxis, were also found to be correlated with CMV reactivation. However, while CMV reactivation was associated with specific factors, it did not impact overall survival or disease-free survival in the study population.

Immune thrombocytopenia (ITP) with corticosteroids: “Evaluation of Response”

Objective: to evaluate the response of corticosteroids in newly persistent ITP patients. Methodology: Descriptive Case series conducted from 30th October 20202 to 29th April 2021 at Armed Forces Bone Marrow Transplant Centre Rawalpindi. A total of 74 individuals with ITP who were both males and females of various ages. Prednisolone was administered orally to patients in maximal dose of 1mg/kg. The initial reaction of platelets count was assessed on day 5 and subsequently every two weeks until the full response was attained. An informed consent was obtained. Results: Age was 32.0±19.1 years on average. Twenty-four (32.4%) of the patients were over the age of 40 while the majority (n=50, 67.6%) of the patients were under the age of 40 years. There were 1:1.1 patients that were male and female with 36 (48.6%) men and 38(51.4%) women. Twenty (27.0%) individuals had persistent ITP compared to 54 (73.0%) patients who had just been diagnosed. 34 (45.9%) patients had an initial response whereas 43 patients (58.1%) showed a full response. The average initial response time was 5.47±2.39 days and 11.79±6.73 days for full response. The frequency of first didn’t differ statistically significantly by patient age, gender or other subgroups. Conclusion: Regardless of age, gender, or ITP type, steroids were associated with an initial and complete response in a significant majority of patients with immune thrombocytopenia. Keeping in view with their affordability accessibility favors the use of steroids as first line in these patients.

Incidence, management and outcome of Hepatic Veno-Occlusive disease /Sinusoidal Obstruction Syndrome after hematopoietic stem cell transplant in Thalassemia major patients: A prospective study of Pakistani BMT.

Hepatic Veno occlusive disease (VOD), also known as sinusoidal obstruction syndrome (VOD/SOS), is a post-transplant life threatening complication. In this study, we aimed to discuss the incidence, management and outcome of VOD in post allogenic transplant patients of beta thalassemia major (BTM). A prospective study was conducted in Armed Forces Bone Marrow Transplant Center, between 2001-2022. A total of 385 fully Human Leucocyte Antigen (HLA) matched BTM patients, with Ursodeoxycholic acid for prophylaxis, were included in the study. Incidence of VOD was calculated through cumulative incidence estimates. Chi square test and Mann Whitney test were used to compare discrete and continuous variables respectively. VOD was diagnosed and graded according to European Society for Blood and Marrow Transplantation EBMT Pediatric diagnostic criteria. Risk factors for VOD were grouped as recipient, transplant and donor related. Univariate analysis was performed by log-rank test. All patients who developed VOD were managed primarily with fluid restriction and strict input output monitoring. Statistical analyses were performed using SPSS v 25.0. Out of 385 transplant patients, forty developed VOD. Median time from date of transplant till onset of VOD was 14 days (range 6-30). Cumulative incidence of all grade VOD was 10.39% (95% CI, 7-14). Eleven out of 40 patients who developed VOD died. Cumulative incidence of Transplant related mortality (TRM) for patients with and VOD was 20.5% (95% CI, 16.6-25.1) vs 27.5% (95% CI, 16.1-42) (p value 0.318) respectively. Among risk factors, age of recipient and fibrosis (p value of 0.04 and 0.000 respectively) were found to be significantly associated with VOD. Careful selection of transplant candidates before transplant can help reduce the incidence of VOD.

Impact of donor marrow cellularity on outcome of allogeneic stem cell transplantation

BackgroundOutcomes of allogeneic hematopoietic stem cell transplant (HSCT) are affected by a number of donor and patient related factors like extent of human leucocyte antigen mismatch, age, source of stem cells, female donor to male recipient and stem cell dose. All of these factors have been extensively investigated; however, effect of donor bone marrow cellularity on HSCT outcomes has not been evaluated. MethodsThis was a prospective study carried out at Armed forces bone marrow transplant center (AFBMTC/NIBMT) from January 2018 to December 2022. Bone marrow cellularity of donors was determined by separate assessment by two experienced Hematopathologists and classified as normocellular, hypocellular or hypercellular according to donor age. Total nucleated cells (TNC) were assessed by automated hematology analyzer and stem cell quantification was done by flowcytometric assay based on CD34, CD45 and 7-AAD immunophenotyping markers. Primary outcome measures were time to achieve neutrophil and platelet engraftment. Secondary outcome measures assessed were overall survival (OS), disease free survival (DFS) and graft versus host disease (GVHD). Frequency and percentage were calculated for categorical variables while Chi-square test was used for quantitative variables. Multivariate Cox regression analysis was used to determine significance of different variables and effect of cellularity on them. Kaplan Meier estimates with group differences were calculated using log rank tests for OS and DFS. A p value of 0.05 or less was considered statistically significant. ResultsCellularity of 95 donors was assessed, 39 (41.1 %) had normocellular marrow while 56 (58.9 %) were hypocellular for age. Median time from diagnosis to transplant was 13 months. In 38 (40 %) the indication for donation was for patients with bone marrow failure syndromes, 23 (24.2 %) for hematological malignancies, 21(22.1 %) for beta thalassemia major and 13 (13.6 %) for miscellaneous disorders including immune deficiencies. Median stem cell / CD 34 dose was 6 × 106/kg and median TNC dose was 5.09 × 108/kg. Median time of neutrophil engraftment was 13.6 days while that of platelet engraftment was 27.1 days. Mean OS was 84.2 %. OS for normocellular donors was 84.6 % and that for hypocellular donors was 83.9 % (p 0.995). DFS for normocellular donors was 84.6 % and for hypocellular donors was 83.9 % (p 0.96). No statistically significant association between the disease group and transplant type with donor marrow cellularity (p value 0.32 and 0.358 respectively) was determined. Multivariate logistic regression model and Backwald test showed no significant association between donor marrow cellularity and CD 34 dose (p 0.65), TNC (p 0.78), neutrophil engraftment (p 0.23), platelet engraftment (p 0.27), Acute GVHD (p 0.83), and chronic GVHD (p 0.44). ConclusionMajority of the donors had hypocellular bone marrow. Bone marrow cellularity had no statistically significant impact on the CD34 and TNC doses obtained; neither did it affect post-transplant neutrophil and platelet engraftment, OS, and DFS. Further population-based studies are required to confirm normal marrow cellularity in Pakistani population and the effects of various genetic and environment factors that make it hypocellular.

Outcomes of Allogeneic Stem Cell Transplant in Chronic Myeloid Leukaemia - A Single-Centre Experience from Pakistan

Objective: To determine indications and outcomes of stem cell transplant in Chronic Myeloid Leukemia in a tertiary care centre in Pakistan. 
 Study Design: Retrospective longitudinal study
 Place and Duration of Study: Armed Forces Bone Marrow Transplant Center, Rawalpindi Pakistan, from Jan 2002 to Jan 2019.
 Methodology: Retrospective analysis of all the patients who had undergone transplants for chronic myeloid leukaemia at our centre from 2002 to 2019 was done. Cases enrolled included patients of both genders and any age for whom data related to disease status and follow-up post-transplant was available. Patients with incomplete records and follow-ups were excluded from the study.
 Results: Out of 73 patients, data from 53 patients was analyzed. At a median follow-up of 40 months, overall survival was 72%. Survival in patients <40 years was better (78%) compared to the >40 years (50%). Among the three conditioning regimens, Bu16Cy120 was the most commonly used (n=35, 66%) and had better overall survival (n=29, 83%) though statistical significance could not be established (p=0.172).
 Conclusion: In addition to the established indications of resistance or intolerance to tyrosine kinase inhibitors for bone marrow transplant in CML, the non-availability/ affordability, young age, and lack of access to novel drugs can be included in underdeveloped countries like Pakistan.

Oral Mucositis in Patients Undergoing Hematopoietic Stem Cell Transplantation.

To determine the factors affecting the frequency and severity of oral mucositis are following hematopoietic stem cell transplantation. Descriptive study. Place and Duration of the Study: Armed Forces Bone Marrow Transplant Centre Rawalpindi, from September 2020 to February 2022. Patients who underwent allogenic stem cell transplantation were enrolled. Patients were analysed based on history and examination for oral mucositis (OM) as per the WHO mucositis scale, from the start of conditioning chemotherapy till discharge, total duration of mucositis and type of medication were noted. Its association with risk factors like age, gender, conditioning chemotherapy, methotrexate (MTX) for GVHD prophylaxis, and prior history of irradiation was determined. Mean age of the 72 transplant recipients was 21.9 ± 14 years, with 48 males and 24 females. The common underlying diseases were beta-thalassemia major (30.6%, n=22), acute lymphoblastic leukaemia (n=15, 20.8%), aplastic anaemia (n=10, 13.9%), and multiple myeloma (n=8, 11.1%). The frequency of mucositis in those aged under 15 years was 79.3% (n=23) and in those older than 15 years was 74.4% (n=32). Frequency of mucositis was statistically significant in patients who received myeloablative conditioning regimen (85% vs. 20%, p <0.01), and who had prophylactic. MTX (91% vs. 48%, p<0.01) and who had prior craniospinal (CSI) radiation (100% vs. 70.2%, p=0.01). There was no statistical significance between stem cell dose (CD34/TNC) and mucositis. Severity of mucositis was significantly greater in Allogenic vs. auto HSCT (p=0.04). All the patients with mucositis required analgesics for pain management. Oral mucositis is a common but potentially debilitating complication of stem cell transplant, requiring opioid analgesia in a significant number of cases. Myeloablative conditioning, prophylactic MTX, and prior CSI are significantly associated with mucositis in transplant patients. Hematopoietic stem cell transplantation (HSCT), Oral Mucositis, Myeloablative conditioning, Methotrexate, Analgesia.

Cytomegalovirus Infection Post-Allogeneic Stem Cell Transplantation: Experience from a Country with High Seropositivity

Cytomegalovirus (CMV) infection is a major cause of morbidity and mortality after hematopoietic stem cell transplantation (HSCT). Limited data are available from developing countries regarding the frequency of CMV infection and treatment outcomes. We enrolled 230 consecutive patients undergoing allogeneic HSCT for various hematologic disorders at the Armed Forces Bone Marrow Transplant Center/National Institute of Blood And Marrow Transplant between February 2017 and December 202. CMV reactivation post-HSCT was monitored weekly starting at day +30 and continuing until day +100, and preemptive antiviral therapy was administered to prevent CMV disease in all HSCT recipients with ≥2000 CMV copies/mL. The median age of the study cohort was 9.5 years (range, .6 to 53 years), and the male:female ratio was 2.4:1. The most frequent indication for HSCT was beta thalassemia major (36.1%), followed by aplastic anemia (23.9%). Malignant disorders constituted 20% of all the patients. Pretransplantation CMV seropositivity was 99.1% for the recipients and 99.5% for the donors. CMV infection was seen in 66.1% of the patients, and the median time to CMV DNAemia was 36 days (range, 12 to 95 days). Preemptive antiviral therapy was administered to 140 patients with a CMV viral load ≥2000 copies/mL (61%). In multivariate analysis, patient age >12 years, steroid administration, and use of mycophenolate mofetil with or without post-transplantation cyclophosphamide was associated with the greatest probability of CMV reactivation. Overall survival was 97.4% in patients without CMV reactivation, compared to 80.3% in those with CMV reactivation (P=.001). Event-free survival was 78.7% in the total study cohort, including 89.7% for patients without CMV reactivation and 73% for patients with CMV reactivation (P=.003). Our study is the first from this region to explore the frequency of CMV seropositivity and CMV infection, risk factors for CMV reactivation, and outcomes of antiviral therapy in HSCT recipients.

Haematopoietic Stem Cell Transplantation (HSCT) for Primary Immune System Disorders in Children: A Single Centre Experience.

To determine the outcomes of allogeneic HSCT in children with primary immune system disorders (PID). Descriptive Cross-sectional study. Place and Duration of the Study: Armed Forces bone marrow transplant centre / National Institute of Bone Marrow Transplant (AFBMTC / NIBMT), Rawalpindi, Pakistan, from October 2012 to December 2021. Data of all cases undergoing HSCT for immune system disorders were analysed for variables affecting outcome and overall survival in the first 180 days after allogeneic HSCT. All patients presenting to AFBMTC / NIBMT with PID, age <12 years. Patients with organ dysfunction secondary to repeated infections were excluded from the study. Data of all patients and their donors undergoing HSCT for immune system disorders were analysed for variables affecting outcome and overall survival in the first 180 days after allogeneic bone marrow transplant. Neutrophil engraftment was defined as absolute neutrophil count ≥0.5 × 109/L for 3 consecutive days, while platelet engraftment as platelet count ≥20 × 109/L without platelet transfusion for one week. Overall survival (OS) was taken as time from the date of HSCT till day + 180 post-transplant. A total of 42children including 29 boys and 13 girls underwent HSCT for PID. The mean age was 2.1±2.8 years. Underlying diagnosis was haemophagocytic lymphohistiocytosis (HLH), severe immune deficiency (SCID), leukocyte adhesion defect (LAD), X-linked agammaglobulinemia, chronic granulomatous disease (CGD) and Job's syndrome in 18 (42.9%), 16 (38.1%), 3(7.1%), 2 (4.8%), 2 (4.8%) and 1 (2.4%) patients respectively. Thirty-one (73.8%) children had fully HLA-matched donors while 11 (26.2%) had haplo-matched donors. Major immediate post-transplant complications were febrile neutropenia, mucositis and SOS/VOD in 31 (73.8%), 9 (21.4%) and 4 (10.0%) cases, respectively. Eight (19.0%) had CMV reactivation, acute GVHD was seen in 17 (40.4%) cases, while 1 (2.3%) case had chronic GVHD. Twelve (28.6%) patients died, out of which 5 had graft failure, 3 had VOD, 2 had pneumonia, 1 had severe GVHD, and 1 died due to seizures. Overall survival (OS) in this study was 71.4% with survival reaching up to 80.6% in fully matched HSCT. HLH and SCID were the commonest immune disorders requiring HSCT. Graft failure leading to neutropenic sepsis was the commonest cause of mortality. OS was better in fully matched HSCT as compared to haplo-identical HSCT. Immune deficiency, Severe combined immunodeficiency, Haematopoietic stem cell transplantation.

To determine the frequency of aldehyde dehydrogenase type 2 (aldh2) deficiency in aplastic anaemia: A single center experience from pakistan.

Aplastic anaemia is a rare bone marrow failure syndrome and is defined by pancytopenia associated with a hypo-cellular bone marrow with no increase in reticulin and the absence of any abnormal infiltrate. The objective of the study was to determine the frequency of Aldehyde Dehydrogenase type 2 (ALDH2) deficiency in patients with Aplastic Anaemia and investigate its correlation with patient and disease characteristics. It was a descriptive cross-sectional study conducted at Armed Forces Bone Marrow Transplant Centre Rawalpindi from 01-08-2022-01-02-2023, over 6 months. A total of 56 patients who were diagnosed with aplastic anaemia during this period, fulfilling inclusion criteria were enrolled. Patients were genotyped as GG (homozygous) and GA (heterozygous). GG had normal ALDH2, while GA were patients with ALDH2 deficiency. Data was collected on the patient's demographics, type and severity of anaemia, type of hematopoietic stem cell transplant (HSCT) and frequency of ALDH2 deficiency. Results were analyzed for ALDH2 deficiency and its correlation with patient and disease characteristics was investigated. A total of 56 patients were included in the study. The median age of the patients was 28 years (20-39). According to the type of aplastic anaemia, 2 (3.6%) had Fanconi anaemia and 54 (96.4%) had acquired aplastic anaemia. In our study, 18 (32.1%) patients had undergone HSCT while the remaining 38 (67.9%) could not undergo HSCT. The frequency of the presence of ALDH2 deficiency was 2 (3.6%). There was no statistically significant correlation between the frequency of ALDH2 deficiency with variables like gender, age distribution, type of aplastic anaemia, the severity of aplastic anaemia and hematopoietic stem cell transplant. We concluded from our study the frequency of ALDH2 was rare in patients with aplastic anaemia. There was no statistically significant correlation between the frequency of ALDH2 deficiency with variables like gender, age distribution, type of aplastic anaemia, the severity of aplastic anaemia and hematopoietic stem cell transplant.

Response to Hypomethylating Agents Plus Venetoclax in Patients with Acute Myeloid Leukemia and Myelodysplastic Syndrome: Real World Data from a Developing Country

Background: Myelodysplastic syndrome (MDS) is a pre-malignant condition characterized by ineffective hematopoiesis, cytopenias and potential to transform into acute leukemia. Acute myeloid leukemia (AML) is a hematological malignancy deriving from myeloid progenitor cells. About one quarter of patients fail to respond to initial therapy (refractory AML). In addition, around 50% of initial responders relapse after a transient remission (relapsed AML). Commonly used salvage chemotherapy regimens include FLAG, FLAG-Ida, HIDAC and Hypomethylating agents (HMA) with or without Venetoclax. In patients who are ineligible for transplant or with poor functional status and in relapsed/refractory cases, treatment options are minimal. However recent data from Western countries has shown promising results with HMA plus Venetoclax in such patients. No such data is available from Pakistan. Objectives: To assess response of HMA plus Venetoclax in patients with AML and MDS treated at Armed Forces Bone Marrow Transplant Center, Rawalpindi, Pakistan. Methods: Study design: Single arm observational prospective study Research Setting: Armed Forces Bone Marrow Transplant Center, Rawalpindi, Pakistan. Study population: Cases included in study were patients of AML and MDS who received HMA plus Venetoclax. A total of 35 patients (25 AML, 10 MDS) were found eligible and included in the final analysis. The primary outcome measured was response to HMA plus Venetoclax. Results: Out of 25 AML patients 17 (68%) were male and 8 (32%) were female. Median age of patients was 44 (range 20-67). Six (24%) patients had AML MRC, 18 (72%) had AML NOS which included 2 (8%) AML-M0, 1 (4%) AML-M1, 9 (36%) AML-M2, 3 (12%) AML-M4 and 3 (12%) patients with AML-M5 while other 3 patients included 1 (4%) patient each of t-AML and AML transformed from Fanconi anemia and aplastic anemia. One (4%) AML-M2 patient had Monosomy 7 and 1 (4%) AML-M5 had trisomy 8. Three (12%), 16 (64%) and 6 (24%) patients were categorized into favorable, intermediate and high risk respectively. Nine (36%) had relapsed disease including 3 (12%) who had relapsed after allogenic HSCT, 8 (32%) had refractory disease while 7 (28%) were unfit for intensive chemotherapy due to advanced age or poor functional status. Median number of HMA+ Venetoclax cycles given were 3 (range 1-16). Overall response rate (ORR) was 44% (n=11), including 9 (36%) patients with CR and 1 (4%) with CRi. One (4%) patient had partial response (PR). Fourteen (56%) patients were non-responders. Of the 10 patients who responded, 6 (24%) achieved remission after 2 cycles while other 4 achieved remission, 1 (4%) patient each, after 1, 3, 5 and 6 cycles. Two (8%) patients underwent allogenic HSCT after achieving remission, including 1 (4%) who had relapsed after first HSCT and underwent second HSCT after achieving CR with HMA+Venetoclax. Out of 10 MDS patients 8 (80%) were male and 2 (20%) were female. Median age of patients was 51 (range 29-71). Two (25%) patients had MDS-MLD, 6 (60%) MDS-EB2 and 2 (20%) had fibrotic MDS. One (10%), 7 (70%) and 2 (20%) patients had intermediate, high and very high risk disease (as per R-IPSS score) respectively. One (10%) patient had relapsed after allogenic HSCT. Median no of cycles of HMA+Venetoclax given were 2 (range 1-6). Three (30%) patients achieved CR (after 2 cycles each), 2 (20%) had stable disease and 5 (50%) patients were non-responders. Two (20%) patients underwent allogenic HSCT after receiving HMA+Venetoclax (Table:1) Common side effects noted were prolonged cytopenias, febrile neutropenia and infections (cellulitis, perianal infections and fungal infections). No treatment related mortality was noted. Conclusion: HMA plus Venetoclax is effective treatment regimen in relapsed/ refractory AML and MDS, however our population shows inferior results as compared to Western population and further large randomized controlled trials are needed to validate this observation. Key Words: Relapsed AML, refractory AML, MDS, salvage chemotherapy, Hypomethylating agents, Venetoclax Research funding: None Word count: 691 Presentation type: Oral Category: Acute Leukemia/ Myelodysplastic syndrome Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Efficacy and Safety of Allogenic Hematopoietic Stem Cell Transplant in Paroxysmal Nocturnal Hemoglobinuria: A Single Centre Study from Pakistan

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired disorder of clonal hematopoiesis1 .The resulting abnormalities in complement regulatory proteins can manifest as hemolysis, thrombosis, and marrow failure2. The management and clinical outcomes of patients with PNH significantly changed with the development of complement inhibitor therapies3. Allogenic Hematopoietic stem cell transplant (HSCT) despite being the sole curative option for PNH is not recommended as first line therapy owing to its toxicities 4,5. Current indications for HSCT include a suboptimal response to eculizumab, severe AA/PNH, clonal evolution to myelodysplastic syndrome (MDS), or acute myelogenous leukemia2. However, in low and middle income countries (LMIC) where complement inhibitor therapies are not available, allogenic HSCT still remains the only treatment option for classical PNH as well as for PNH in the setting of another specified bone marrow disorder. Objective: To analyze the efficacy and safety of Allogenic HSCT in patients with paroxysmal nocturnal hemoglobinuria (PNH). Study design: Observational, retrospective study. Place and duration of study: Armed Forces Bone Marrow Transplant Centre/ National Institute of Blood and Marrow Transplant (AFBMTC/NIBMT) Rawalpindi, Pakistan, from June 2008 till February 2022. Methodology: From June 2008 till February 2022, a total of 65 patients of PNH were registered at AFBMTC/NIBMT. Out of these16 patients underwent allogenic HSCT and were included in the final retrospective analysis. PNH diagnosis was confirmed by flow cytometry by the demonstration of GPI-negative cells in at least two cell lineages i.e neutrophils, red blood cells (RBCs) in 62.5% (n=10) patients and by fluorescent aerolysin (FLAER)-based assay in 37.5% (n=6) patients. Median time from diagnosis to transplant was 380 days. All patients received HLA identical sibling donor HSCT. Stem cell source was Bone marrow harvest (BMH), BMH plus peripheral blood stem cell (PBSC) and PBSC alone in 81% (n=13), 12.5% (n=2) and 6% (n=1) patients respectively. Graft versus host disease (GVHD) prophylaxis was with Antithymocyte globulin (ATG) + Cyclosporin (CSA) in patients receiving NMA conditioning or ATG + CSA + short term Methotrexate (MTX) in those receiving MAC. Results: Median age of the study cohort was 24 years (range, 18 to 42 years) and all were male. 68.7% (n=11) patients received immunosuppressive therapy before HSCT with partial or no response. History of thrombosis prior to HSCT was present in only one patient (cPNH). Allogenic HSCT done for classical PNH (cPNH) in 75% (n=12) patients and for PNH in the setting of another specified bone marrow disorder (PNH/AA) in 25% (n=4) patients. Patients with classical PNH received Myeloablative conditioning (MAC) i.e Flu150Bu12.8IV (75%, n=9), Bu16Cy120 (8.3%, n=1), or Flu150Cy200 (8.3 %, n=1) except for one patient who received Flu150Cy160. Patients with PNH/AA, received non myeloablative conditioning (NMA) i.e Flu150Cy120, except for one who received Flu150Bu12.8IV. Out of 16 patients, 94% (n=15) patients achieved successful engraftment with full donor chimerism. One patient (cPNH) had Primary graft failure. Median time to neutrophil and platelet engraftment was day +12 and day +22 respectively. Disease relapse was monitored by donor chimerism till Day +180 post BMT and thereafter by peripheral blood counts. PNH clone post BMT was not tested. Incidence of acute GVHD grade I/II involving skin and gut, was 25% (n=4). No incidence of chronic GVHD was noted. One patient (PNH/AA) had secondary graft failure on Day +46. With a median post-transplant follow-up of 15 months (range, 4 to 157) overall survival (OS) was 93.7% and disease-free survival (DFS) was 87.5%. Conclusion: In resource constrained, LMIC, where complement inhibitor therapies are currently unavailable, allogenic HSCT remains the valid option for treatment of PNH, with good overall survival rate and acceptable treatment related toxicites. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Promise of Next-Generation Sequencing in Improving the Diagnosis of Rare Genetic Disorders in Developing Countries

Introduction Genetic testing is frequently required for aiding and confirming the diagnosis, selecting treatment and predicting prognosis in patients with hematological disorders (1). However, large-scale genetic studies based on whole exome or genetic panel sequencing have not been a primary focus of developing countries due to lack of awareness, a dearth of genetic testing laboratories in clinical facilities, and the limited financial resources of affected families. As a result, the underlying genetic patterns in these patients often go undetected. Nevertheless, these countries constitute a rich source of clinical data with atypical phenotypes and novel mutations due to large family sizes, inter-family marriages, and potential founder effects (2). These genotype and phenotype data are worth being presented in the international data repositories for better identification of pathogenic variants in this population (3). Armed Forces Bone Marrow Transplant Centre (AFBMTC) is the country's largest hospital providing clinical hematology and stem cell transplantation services to a catchment area of over 560,000 Km2. We sought to identify underlying genetic causes of various difficult-to-diagnose rare hematological disorders with heterogenous/overlapping symptoms and challenging clinical presentation using next-generation sequencing (NGS). Materials and Methods Peripheral blood samples from 33 individuals with diverse hematological diseases were sent to Invitae Corporation (USA) or Centogene GmbH (Germany) for panel sequencing or whole exome sequencing, respectively. These samples were from patients who were difficult-to-diagnose, required risk stratification or genetic testing for selecting appropriate treatment. Results Whole exome sequencing or panel sequencing was performed in a cohort of 33 patients with various hematological disorders (figure 1). A total 115 variants were identified in these patients including 15 pathogenic variants (13%), 7 likely pathogenic variants (6%), 82 variants of uncertain significance-VUS (71%), 9 benign variants (7.8%) and 1 increased risk allele variant (0.86%). A total of 42 variants were found in musculoskeletal disorders panel, 20 in primary immunodeficiency panel, 11 each in bone marrow failure syndrome panel and dyskeratosis congenita panel, 9 in comprehensive deafness panel and 9 in hereditary hemolytic anemia panel among others. The most frequent diagnosis in our cohort was inherited bone marrow failure syndromes (27.3%), followed by hemolytic anemias (15.2%), immunodeficiency disorders (12.2%), inherited platelet function disorders (12.2%), myelofibrosis disorders (12.2%), skeletal disorders (12.2%) and miscellaneous disorders (9.1%). Patients clinical and demographic characteristics are summarized in table 1. Conclusion Use of high-throughput NGS in our clinical hematology setting has proven to be useful in identifying genetic variants for accurate diagnosis, selecting appropriate therapies, and predicting prognosis. We intend to develop a genetic facility that will provide genetic testing and identify ways to make it more accessible for people of all socioeconomic backgrounds. A second objective is establishing the prevalence of most frequent genetic disorders in the country. The high number of variants of uncertain significance (VUS) is most likely driven by a lack of sufficient population-based genetic data and proof of functional impact, though the patients identified having VUS were phenotypically disease presenting. Thus, supporting the hypothesis that these VUS may eventually be classified as pathogenic after appropriate testing on larger scales and animal models. To achieve a dependable and precise categorization of VUS, we plan to include functional genomics in our research focus to support the clinical database of this region. 1. Haferlach TJh. The time has come for next-generation sequencing in routine diagnostic workup in hematology. 2021;106(3):659. 2. MALTESE P, Poplavskaia E, Malyutkina I, Sirocco F, Bonizzato A, Capodicasa N, et al. Genetic tests for low-and middle-income countries: a literature review. 2017;16(1). 3. Gutierrez-Rodrigues F, Calado RTJH, Transfusion, Therapy C. The interpretation of rare or novel variants: Damaging vs. disease-causing. 2018;40:3-4. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

BK virus associated haemorrhagic cystitis in related donor haematopoietic stem cell transplant recipients: A developing country experience.

A retrospective cross sectional study was conducted at the Virology Department, Armed Forces Institute of Pathology (AFIP) and Armed Forces Bone Marrow Transplant Centre (AFBMTC), Rawalpindi, from January 2016 to July 2018. Medical records of 193 patients were examined to determine the number of patients developing Haemorrhagic Cystitis associated with BK virus (BKV). BKV PCR testing was done on the patients' urine samples. Cytomegalovirus reactivation was also assessed weekly from day 30 to day 100, by CMV quantitative PCR testing on blood samples. Out of 193 patients, 11 (5.6%) developed haemorrhagic cystitis and all these patients were positive for BKV on urine samples. The maximum number of positive cases, i.e. 5 (2.6%) was in the age group three months to 10 years. Primary disease in seven out of 11 cases was Beta-Thalassemia Major.

Association of early donor chimerism status with survival outcomes in post allogeneic haematopoietic stem cell transplant patients of nonmalignant diseases.

To highlight the association of early donor chimerism status at 2nd month with various survival outcomes. The retrospective study was conducted at the Armed Forces Bone Marrow Transplant Centre, Rawalpindi, Pakistan, and comprised patient data from January 2011 to July 2016. Data related to participants who underwent human leukocyte antigen-matched transplants for bone marrow failure syndrome and beta thalassemia major. Short tandem repeat-based polymerase chain reaction was used to assess donor chimerism status. Overall survival, disease-free survival, relapse-free survival, and graft versus host disease-free survival rates were noted. Data was analysed using SPSS 23. Of the 106, 64(60.4%) had bone marrow failure syndrome and 42(39.6%) had beta thalassemia major. The overall median follow-up was 13.53 months (range: 1.81-62.73 months). Early donor chimerism status was associated with overall survival (p=0.02) and disease-free survival (p=0.01). Mixed donor chimerism was less hazardous in terms of overall survival (p=0.04) and disease-free survival (p=0.02). Early mixed donor chimerism contributed to optimal survival in nonmalignant disease.

COMPLICATIONS IN ABO-INCOMPATIBLE HEMATOPOIETIC STEM CELL TRANSPLANT IN PAKISTAN

Background: Hematopoietic stem cell transplantation (HSCT) is therapeutic option for many blood diseases. It has increased risk of complications with incompatible pair. The objective of this study was to determine the frequency of complications in ABO-incompatible HSCT in Pakistan.Materials Methods: This cross-sectional study was conducted at Armed Forces Bone Marrow Transplant Centre , Rawalpindi, Pakistan from 11th August 2018 till 15th March 2021. A sample of 73 ABO-incompatible HSCT patients was selected. Variables were sex, age groups, acute delayed hemolysis, pure red cell aplasia and acute GvHD. All variables being categorical were described by count and percentage with 80%CI. Complications in sample vs. population were compared through chi-square goodness of fit test.RESULTS: Seventy three patients with ABO-incompatible HSCT included 52 (71.23%) men 21 (28.77%) women, and 49 (67.12%) in age group ≤14 years 24 (32.88%) in ≥15 years. Out of 73 patients, eight (10.96%) had acute hemolysis, 26 (35.62%) had delayed hemolysis, four (5.84%) had pure red cell aplasia and 34 (46.58%) had acute GvHD. The observed prevalence in sample was similar to population for acute (p=.46893) and delayed hemolysis (p=.30759) and acute GvHD (p=.55841), while it was different for pure red cell aplasia (p=.00006).CONCLUSION: Most common complication in our study was acute GvHD, followed by delayed hemolysis, acute hemolysis pure red cell aplasia. The observed prevalence in sample was similar to population for acute delayed hemolysis and acute GvHD, while it was different for pure red cell aplasia.

MAGNITUDE OF VANCOMYCIN RESISTANT ENTEROCOCCUS FECAL COLONIZATION AND BACTEREMIA IN PATIENTS WITH HEMATOLOGICAL DISEASES AT TERTIARY BONE MARROW TRANSPLANT CENTRE RAWALPINDI

Objective: To discover the frequency of vancomycin resistant enterococci (VRE) fecal colonization and subsequent bacteremia in patients with hematological diseases in a bone marrow transplant center.&#x0D; Study Design: Cross-sectional study.&#x0D; Place and Duration of Study: Department of Microbiology, Armed Forces Institute of Pathology (AFIP), in collaboration with Armed Forces Bone Marrow Transplant Center, Rawalpindi, from Jan 2016 to Dec 2019.&#x0D; Methodology: Stool specimens/anal swabs from all enrolled patients were collected aseptically and transported to the laboratory without delay. Blood cultures were collected aseptically from only those enrolled patients who developed signs and symptoms of bacteremia. Stool and blood cultures were processed as per standard microbiological protocols. Enterococci were identified to species level by colony morphology and biochemical tests. Modified Kirby Bauer disc diffusion method and VITEK-2 system (Version-8.01 bio Merieux, France) were used to perform antimicrobial sensitivity of each isolate.&#x0D; Results: A total number of 246 patients were studied. Among them, 67 (27%) patients had fecal colonization by vancomycin resistant enterococci. We report a statistically significant association of recent hospitalization, prolonged exposure to antimicrobial therapy, chemotherapy exposure and use of indwelling devices during the hospital stay with vancomycin resistant enterococci colonization. Vancomycin resistant enterococci bacteremia was detected in 57 (23%) patients. Among these 57 patients, 53 (93%) were vancomycin resistant enterococci colonizers. Vancomycin resistant enterococci colonization was significantly associated with vancomycin resistant enterococci bacteremia.&#x0D; Conclusion: A considerable burden of vancomycin resistant enterococci fecal colonization exists among patients with hematological diseases. vancomycin resistant enterococci colonization poses a considerable risk of vancomycin........

ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION IN FANCONI ANEMIA; A SINGLE CENTRE EXPERIENCE

Objective: To determine the treatment outcome of Hematopoietic stem cell transplantation in Fanconi Anemia.&#x0D; Study Design: Case series.&#x0D; Place and Duration of Study: Armed Forces Bone Marrow Transplant Center, Rawalpindi, from Jan 2001 to Jun 2018.&#x0D; Methodology: Data of all Fanconi anemia patients who had fully HLA matched bone marrow transplant during this period was analysed for variables affecting the outcome in terms of overall survival. Those fanconi anemia patients who had myelodysplastic changes or acute myeloid leukemia were excluded.&#x0D; Results: Total 27 patients underwent fully HLA matched allogeneic bone marrow transplant for Fanconi Anemia. Mean age of patients at transplant was 12.12 ± 5.16 years. All patients at transplant were in aplastic phase. Conditioning was done with fludarabine 120mg/m2 , ATG 20 mg/kg and Cyclophosphamide at a dose of 20-40 mg/kg. Mean time to neutrophil engraftment was 12.3 ± 2.92days and for platelets 20 ± 10.3 days. Major posttransplant complications were neutropenic fever in 26 (96%), hypertension in 18 (66.6%), mucositis in 12 (44.4%), azotaemia in 8 (29.6%), gut toxicity in 7 (25.9%) and haemorrhagic cystitis in 5 (18.5%) patients. Four patients (14.8%) had acute graft versus host disease while 7 (26%) patients had chronic GVHD. Overall survival at 6 months, 1, 5 and 8 years was 67%, 63%, 59% and 55% respectively. While overall survival in patients transplanted at younger age (&lt;11 years) was 81.8% compared to 37.5% in older age group (&gt;11years) and was statistically significant (p-value = 0.03).&#x0D; Conclusion: Our study demonstrated...................

HIGH ALTITUDE AND VENOUS THROMBOSIS: FREQUENCY AND RISK FACTORS

Objective: To determine the effect of high altitude on frequency and risk factors of venous thrombosis inindividuals ascending to high altitude&#x0D; Study Design: Cross-sectional analytical study.&#x0D; Place and Duration of Study: Armed Forces Bone Marrow Transplant Centre, Combined Military HospitalSkardu, from Apr 2015 to Aug 2018.&#x0D; Methodology: High Altitude was taken as height equal to or more than 8000 feet. Polycythemia as hemoglobin&gt;16.5 g/dl OR hematocrit &gt;49%. Medical records of all patients evacuated from high altitude through CombinedMilitary Hospital Skardu were analyzed retrospectively. A proforma was designed to include necessary variables.&#x0D; Results: We evaluated medical records of 539 individuals and found frequency of 86 (15.9%) for venous thromboembolism (VTE) at high altitude. Cerebral venous sinus thrombosis (CVST) was most common thrombotic complication seen in 39 patients (46%). Majority of thrombotic episodes occurred at &gt;18000 feet (likelihood ratio (LR) 5.99, p-value 0.009). Majority of thrombotic episodes 34 (39%) occurred within first 45 days of ascent to high altitude. Thrombosis was linked to smoking 47 (55%) (likelihood ratio 21.3 and p-value &lt;0.001) and use of melted snow as a source of drinking water 56 (65%) (likelihood ratio 57.6 and p-value &lt;0.001).&#x0D; Conclusion: This study showed a very high frequency of thrombotic complications at high altitude. There isa need for a robust prospective study covering epidemiology, clinical information and diagnostics in order todevelop appropriate standard operating procedures and guidelines for prevention of thrombosis at HA