Hypocellular Bone Marrow Research Articles

Platelet counts on peripheral blood and Mean Platelet Volume as markers of clinical severity in Sickle Cell Disease

Paroxysmal nocturnal hemoglobinuria (PNH) can occur as a hemolytic form or small PNH clone found in a patient with bone marrow failure.Describe Brazilian retrospective PNH cohort and identify the impact of disease burden on long-term follow-up.167 patients, mean age at diagnosis 28.4 (7.1–71.2 years), four years mean interval between onset of cytopenia/aplasia diagnosis and PNH clone detection. Patients were divided into 15 Classic PNH, 55 Hemolytic PNH with bone marrow hypoplasia (PNH/AA), and 97 Subclinical PNH (sc-PNH). Hypocellular bone marrow was found in 89.2%; 55 had hemoglobinuria and 22 thrombosis during monitoring. WBC PNH clone correlated with RBC PNH clone, LDH and cytopenia. Subclinical patients had lower median lower RBC clone (2.0% vs 24.0% vs 57.8%) and WBC clone (11.7% vs 58.8% vs 81.2%) than PNH/AA and Classic PNH, respectively. PNH granulocyte clone was 89.1% in thrombotic patients. Ten-year overall survival 80.4% and mortality in transplanted patients 9.6%. Sepsis was mortality cause in subclinical PNH (16/18, 88.8%), and thrombosis in hemolytic PNH (11/13, 84.6%).Large PNH clones and LDH burden were associated with increased hemolysis and thrombosis risks, while young patients were associated with small PNH clones and subclinical form of the disease. Knowledge of the patient profile, the low risk associated with HSCT, and the use of long-term IST may be instrumental in the clinical management of PNH in restricted-resources countries.

Allogeneic Hematopoietic Stem-Cell Transplantation for GATA2 Deficiency Using a Busulfan-Based Regimen

Background: GATA2 deficiency is an increasingly recognized bone marrow failure syndrome responsible for: MonoMAC, monocytopenia with atypical mycobacterial infection (MAC); DCML, dendritic cell, monocyte and lymphoid cell deficiency; Emberger syndrome with lymphedema and monosomy 7; and familial myelodysplastic syndrome (MDS)/acute myelogenous leukemia (AML). GATA2 deficiency can be reversed by hematopoietic stem cell transplantation (HSCT). However, the intensity of conditioning required to ensure engraftment and reverse the myelodysplastic syndrome (MDS) characteristic of the disease remains controversial.Methods: We carried out a prospective study of allogeneic HSCT in 22 patients (mean age 26 years; range 17 to 45 years) with GATA2 deficiency, or the MonoMac syndrome, using a myeloablative, busulfan-based conditioning regimen. All 22 patients had MDS and two had AML. Eleven patients had clonal cytogenetic changes in the bone marrow. There were two matched related donor (MRD) recipients, 13 matched unrelated donor (URD) recipients, and 7 haploidentical related donor recipients. MRD and URD recipients received conditioning with 4 days of busulfan (days -6 to -3) to target an AUC between 3600 and 4800 min*microm/L and 4 days of fludarabine 40 mg/m2/day (days -6 to -3). Haploidentical related donor recipients received low-dose cyclophosphamide for two days (days -6 and -5), fludarabine 30 mg/m2/day for 5 days (days -6 to -2), 2-3 days of busulfan depending upon cytogenetics, along with 200 cGY total body irradiation (TBI) on day -1. MRD and URD recipients received tacrolimus and short course methotrexate for post-transplant graft-versus-host disease (GVHD) prophylaxis, whereas haploidentical recipients received high-dose post-transplant cyclophosphamide (PT/CY) followed by tacrolimus and mycophenolate mofetil for GVHD prophylaxis.Results: Nineteen of the 22 patients are alive with resolution of the clinical phenotype and correction of the bone marrow MDS with eradication of the cytogenetic changes at a median follow-up of 23 months (range, 6 to 48 months) giving an overall disease-free survival of 86 percent. The three deaths all occurred in the URD group. Of the three deaths, one resulted from refractory acute myelogenous leukemia (AML), and two from complications related to treatment for GVHD. There was a 26 percent incidence of grade III-IV acute GVHD (aGVHD) in the MRD and URD groups, and no grade III-IV aGVHD in the haploidentical related donor cohort. Similarly, there was a 46 percent incidence of chronic GVHD (cGVHD) in the MRD and URD cohort, whereas two of the 7 (28 percent) of the haploidentical related donor recipients had cGVHD.Conclusions: Despite overall disease-free survival of 86 percent with eradication of the clonal cytogenetic abnormalities in the bone marrow, GVHD remains a limitation in the MRD and URD cohorts treated with standard post-transplant tacrolimus/methotrexate prophylaxis for GVHD. We are currently de-escalating the busulfan dosing based on the AUC in all cohorts and incorporating PT/CY in the MRD and URD cohorts to decrease toxicity and reduce the incidence of aGVHD and cGVHD. Together with our previous studies using a nonmyeloablative regimen, it appears that when GATA2 deficiency patients are transplanted when they have a hypocellular bone marrow with MDS and without cytogenetic changes, a nonmyeloablative regimen results in reliable engraftment in GATA2 deficiency since the GATA2 deficient marrow has a proliferative disadvantage. However, with clonal progression and unfavorable cytogenetic changes and/or a hypercellular marrow, where the malignant clone has a proliferative advantage, a higher dose regimen results in more reliable engraftment and eradication of the clone. Studies to identify the precise level of conditioning for the stages of the disease are ongoing. DisclosuresNo relevant conflicts of interest to declare.

Bone Marrow Cellularity and Recovery of Blood Counts after Venetoclax and Azacitidine in Elderly Acute Myeloid Leukemia Patients Unfit for Induction Chemotherapy

Introduction: Cytotoxic chemotherapy is frequently employed in the treatment of aggressive malignancies such as acute myeloid leukemia (AML). Patients (pts) who respond to this therapy universally experience significant cytopenias after a period of bone marrow hypocellularity that is the result of toxicity to normal hematopoietic cells. Older pts with AML poorly tolerate cytotoxic chemotherapy, largely due to these cytopenias, and novel therapies that spare this toxicity are needed. In the setting of a multi-center phase 1b dose escalation/expansion study (NCT02203773), we explored the combination of the BCL-2 inhibitor venetoclax (ven) with azacitidine (aza) for elderly AML pts unfit for induction chemotherapy. We noted distinctive post treatment morphologic appearances of some pts' bone marrow biopsies that could be grouped based on cellularity. This observation, coupled with a high overall response rate that included some pts who did not achieve full count recovery, led to a hypothesis that bone marrow cellularity, either at baseline or after 1 treatment cycle, might predict full vs. incomplete count recovery in the context of this novel therapy.Methods: We enrolled 33 pts at our institution into this dose escalation/expansion study involving the sequential administration of aza at the standard dose and schedule with daily ven; 23 received 400 mg venetoclax, 9 received 800 mg and 1 received 1200 mg. Cycles lasted 28 days and were repeated indefinitely in the absence of progression or limiting toxicity. Baseline and post cycle 1 bone marrow biopsies were assessed as hypercellular (>80% cellularity) or hypocellular (<20% cellularity) (Figure 1). Univariate logistic regression analysis was used to determine whether these features predicted the ability to achieve a complete remission (CR) versus CR with incomplete count recovery (CRi) or morphological leukemia free state (MLFS), and McNemar's test for paired samples was used to predict whether pre-treatment cellularity predicted post-treatment cellularity.Results: 17 pts at baseline met pre-defined criteria for hypercellularity and 2 were hypocellular. 10/33 pts had AML with myelodysplasia related changes; of these, 6 were hypercellular and none were hypocellular at baseline. After 1 cycle, 2 pts met criteria for hypercellularity and 9 were hypocellular. Hypercellularity at baseline predicted post treatment hypercellularity (P=0.0001) and hypocellularity at baseline predicted post treatment hypocellularity (P=0.0196) (Table 1). Median time to platelet and neutrophil recovery was 25 and 42 days, respectively. Hypercellularity at baseline was significantly associated with a reduced ability to recover blood counts (OR to achieve CR vs. CRi/MLFS was 0.15, 95% CI 0.02-0.89, P=0.0369). Hypocellularity at baseline was not associated with failure to recover blood counts (P=0.9618). Hyper or hypocellularity after cycle 1 did not predict ability to recover counts (p=0.9403 and 0.1593, respectively) (Table 2).Conclusion: Novel therapies that spare elderly pts long periods of cytopenias are necessary. Ven with aza is an active combination in this population. Although nearly all pts in our cohort achieved an International Working Group defined overall response, several had a CRi or MLFS. After cycle 1 of treatment, some pts achieved a remission without an apparent period of hypocellularity. Surprisingly, hypercellularity at baseline was associated with a lower likelihood of achieving full count recovery after treatment. Patients that were hypercellular after cycle 1 were more likely to have been hypercellular at baseline, however, hypercellularity after cycle 1 was not associated with delayed count recovery. Neither baseline nor post treatment hypocellularity had a higher association with poor count recovery. These observations will need to be tested in a larger sample set, but they suggest that marrow recovery may not be linked to morphologic features like baseline cellularity. In the setting of non-intensive therapies other features may predict inferior count recovery, such as the presence or duration of an antecedent hematologic malignancy like MDS. [Display omitted] DisclosuresPollyea:Takeda, Ariad, Alexion, Celgene, Pfizer, Pharmacyclics, Gilead, Jazz, Servier, Curis: Membership on an entity's Board of Directors or advisory committees; Agios, Pfizer: Research Funding.

Marrow Hypocellularity, but Not Residual Blast Count, Is Prognostic on Day-14 Assessment in Acute Myeloid Leukemia

Background: Standard induction chemotherapy for acute myeloid leukemia (AML) with “7+3” regimen consisting of cytarabine and daunorubicin (DNR) yields a complete remission (CR) rate of 40-80%. Current practice, endorsed by the National Comprehensive Cancer Network, is to perform a repeat bone marrow assessment on day 14 (D14) of initial induction. A blast count greater than 5-10% with a marrow cellularity of 10-20% at that time suggests residual leukemia and recommendation is for a second round of induction chemotherapy. Most studies only note blast count as the key value in decision making. Large studies have confirmed a prognostic value of D14 bone marrow biopsy based on blast percentage, yet controversy lies in its use as treatment decision point. In this single center retrospective study, we evaluated the role of cellularity on clinical outcomes in patients with residual morphologic blasts on day-14 bone marrow biopsy. Methods: All consecutive patients 18 years or older who received induction chemotherapy for newly diagnosed AML from 1/1/06 to 5/1/13 at the Lifespan Cancer Institute were identified. Outcomes were followed until 5/1/17. On D14 marrow assessment, patients were determined to have residual leukemia if the morphologic blast count was ≥5%, deemed to have an intermediate response (IR) if blasts counts were 5-19%, and considered hypocellular if the cellularity was Results: Of 113 patients identified, 68 underwent D14 bone marrow biopsy and 35 had residual disease on D14 determined by blast count. Sixteen of these patients received reinduction. Nineteen had hypocellular bone marrows and 16 were non hypocellular. Between the hypocellular and non hypocellular groups no demographic or leukemia specific variables were significantly different (Table 1). In terms of outcome data, more patients with hypocellular D14 bone marrows attained an IR (12 of 19, 63%) compared to a non hypocellular marrow (3 of 16, 19%, p=.008). Furthermore, hypocellular D14 marrows showed improved post induction complete response (74% v. 31%, p=0.12) and improved median overall survival (17 v. 8.5 months, p=0.03, Figure 1) compared to non hypocellular D14 marrows. Based on logistic regression, there was no benefit for reinduction based on cellularity for either CR (OR 0.056, 95% CI 0.002-1.47) or OS (HR 1.96, 95% CI 0.4-9.6). Using a blast count of ≥5% on the day-14 marrow assessment as a positive screening test for residual leukemia demonstrated PPV of 42%, NPV of 79%, sensitivity of 53% and specificity of 70% for achieving CR. Using the alternative cutoff of Conclusions: In our single center retrospective review, we found that D14 bone marrow hypocellularity significantly predicted CR and OS in patients with residual morphologic blasts. We found no benefit to additional chemotherapy based on D14 marrow assessment in terms of attaining CR or improvement in OS. Furthermore, there was no benefit for reinduction based on D14 marrow cellularity using logistic regression. There is improved specificity for D14 bone marrow biopsy to detect residual disease when a morphologic blast cutoff of 20% or greater and bone marrow cellularity of 20% or greater is utilized. For those patients with residual blast counts over 20% and non hypocellular marrow, strong consideration should be made for clinical trial or novel therapeutic agent. Although these are relatively small numbers, this data underscores the continued value of D14 bone marrow morphologic assessment for prognostication. Download : Download high-res image (142KB) Download : Download full-size image Figure 1 . Disclosures Reagan: Teva: Membership on an entity's Board of Directors or advisory committees.

Characteristics and Outcomes of Pediatric Patients with t(16;16)-AML: Results of a Case Series By the International BFM Study Group

IntroductionCore binding factor (CBF) acute myeloid leukemia (AML) inv(16)(p13.1q22)/t(16;16)(p13.1;q22)/CBFβ-MYH11 [inv(16)/t(16;16)] is associated with a favorable outcome in children. Aberrations inv(16) and t(16;16) result in the same fusion of the CBFβ subunit at 16q22 and the myosin-11 heavy chain gene at 16p13, and are thought to share common biological features and outcomes. However, CBFβ-MYH11 fusion transcripts are heterogeneous and may vary in terms of biological activity. Therefore, in this cohort study by the International Berlin-Frankfurt-Munster (I-BFM) study group, we evaluated clinical characteristics, prognostic features and outcomes in pediatric patients with t(16;16)-AML.MethodsPatients (age 0‒19 years) with cytogenetically or molecularly confirmed t(16;16)-AML, diagnosed by members of the I-BFM study group between 1990 and 2015, were included in the study. Patients with previous malignancies, chemotherapy, or radiotherapy were excluded. Data on baseline characteristics, including cytogenetic and molecular information, fusion type, treatment details and follow up were collected using standardized case report forms. If data on the CBFβ fusion type were missing, real-time quantitative PCR for fusion types A, D, and E was performed on available biobank material. The molecular characterization part of the study is ongoing. Herein, we present descriptive statistics and estimated outcomes related to achieving complete remission (CR) and probabilities of event-free survival (pEFS) and overall survival (pOS).ResultsThe study included 46 patients with t(16;16)-AML. Median age was 9.8 [interquartile range (IQR) 1.7‒11.8] years, median white blood cell (WBC) count at diagnosis was 74 [IQR 35‒80] ×109/L and the predominant FAB-type was M4Eo (49%). Eleven patients (24%) had central nervous system involvement at diagnosis. Information on common additional molecular aberrations (e.g. cKIT and RAS mutations) was unavailable for more than 50% of patients and therefore not considered. Information on fusion types was available for 13 patients (28%). Biobank material was used to determine CBFβ fusion type in 2 patients for whom it had previously been unknown. Another 9 samples from international groups are available and pending analysis. Most patients for whom information was available on CBFβ fusion type had type A (n=7, 54%), followed by type D (n=3, 23%) and type E (n=3, 23%). No other fusion types were identified. Patients with type non-A (n=6, 46%) were predominantly male (67%) and had FAB-type M4Eo (83%). They were older than patients with type A (11.9 vs 8.8 years, respectively) and had a higher WBC count (69 vs 48 ×109/L, respectively), but these differences were not significant. Additional cytogenetic aberrations occurred in 38% of patients. Gain of chromosome 8, 21 or 22 were the most common additional chromosomal aberrations. Distribution of additional cytogenetic aberrations was comparable between patient with fusion type A and other types.Patient outcome was excellent with a 100% CR rate and a 5-year pEFS and pOS of 80% [standard error (SE), 6.4%] and 87% [SE, 5.6%], respectively (Figure 1). Five patients suffered a relapse. Of them, 4 had WBCs >50 ×109/L at diagnosis. None of the 5 patients had additional cytogenetic aberrations. Two patients had fusion type A and type was unknown for the other 3 patients. Of 46 patients, 2 died after relapse and 3 died from toxic complications in first CR. These complications included failure to recover bone marrow hypocellularity and subsequent multi-organ failure (n=1), and sepsis after stem cell transplantation in CR (n=2).ConclusionThis is the first report of a relatively large cohort of pediatric t(16;16)-AML patients. Patient outcomes were very good, especially compared with those reported for patients with non-CBF leukemias. However, these patients were at risk of toxic death in CR. Clinical characteristics and outcomes were comparable with those reported previously for patients with inv(16). Although this case series was large, patient numbers remained too small to identify significant prognostic factors associated with outcome. Prospective trials are needed to determine other biological features that may explain differences in outcomes among patients with either t(16;16) or inv(16)-AML. [Display omitted] DisclosuresKaspers:Janssen-Cilag: Research Funding.

Evaluation of Nudt15 -Guided Thiopurine Dose Individualization In Vivo Using CRISPR/Cas9-Edited Mouse Model

Prolonged exposure to thiopurines (e.g., 6-mercaptopurine [MP]) is an essential component of curative therapy of acute lymphoblastic leukemia (ALL) in children and adults. However, this class of drugs are also associated with frequent dose-limiting hematopoietic and liver toxicities, at least in part explained by genetic polymorphisms in metabolizing enzymes such as TPMT . In fact, pre-emptive TPMT- genotype guided thiopurine dose reduction has been implemented in clinical practice and shown to effectively mitigate toxicity without compromising therapeutic efficacy. More recently, we and others identified NUDT15 as a novel genetic determinant of thiopurine toxicity (Nature Genetics , 2014 and 2016), particularly in Asian and Hispanic populations. While results from correlative studies in patients point to potential effectiveness of NUDT15 -genotype based thiopurine individualization, the exact dosing algorithm remains unknown and there have been no laboratory models to test such strategies. To this end, we established a Nudt15 knockout mouse model on FVB/N background by CRISPR-Cas9 genome editing, with which we systematically evaluated NUDT15 -genotype directed thiopurine dose reduction and its effects on hematopoietic toxicity in vivo .Nudt15+/+ and Nudt15-/- mice received daily intra-peritoneal MP injection at the dosage of 1, 5 or 20 mg/kg. Consistently across dosage levels, Nudt15-/- mice experienced more severe and precipitous myelosuppression (especially reduction in neutrophil count), more rapid weight loss, and earlier toxic death, compared to wildtype mice (P < 0.001). For example at the MP dosage of 20 mg/kg/day, the median time to toxic death was 12 days in Nudt15-/- mice whereas wildtype mice survived a median of 25 days (P = 6.9 × 10-15). Myelosuppression (white blood cell count < 2.0 × 106 /ml) occurred as early as 9 vs 22 days following the start of MP treatment in Nudt15-/- vs wildtype mice, respectively (P < 0.01). Severe bone marrow hypocellularity at the time of death was confirmed by necropsy, and was particularly pronounced in deficient mice. Notably, MP treatment induced damage to the mucosal epithelial cells of the digestive tract (e.g., esophagus and small intestine) in Nudt15-/- mice, whereas there were no mucosal lesions in wildtype animals. In Nudt15-/- mice, toxicities were significantly reduced with decreasing MP dosage. In fact, at MP dosage of 1mg/kg/day, Nudt15-/- mice showed a toxicity profile (bone marrow suppression, weight loss, and toxic death) that was comparable to that of Nudt15 wildtype mice receiving 20 mg/kg/day of MP, suggesting that a 95% dose reduction might be needed to completely mitigate MP toxicity in individuals with homozygous NUDT15 deficiency.To assess the impacts of Nudt15 genotypes on thiopurine activation, we also characterized the acute accumulation of MP metabolite (i.e., DNA incorporated thioguanine, DNA-TG) following daily MP injection at increasing dosages (1, 5, 20 and 40 mg/kg/day). Although the level of DNA-TG in hematopoietic cells was positively correlated with MP dosage in both genotype groups, this active metabolite accumulated much more rapidly in Nudt15-/- mice with increasing MP dosages than in wildtype mice (p < 0.001). Interestingly, DNA-TG level in Nudt15-/- mice exposed to 1 mg/kg/day of MP was comparable to that in Nudt15+/+ mice dosed at 20 mg/kg/day, consistent with our observation that a 20mg-to-1mg dosage reduction effectively eliminated the excessive MP toxicity associated with Nudt15 deficiency. Finally, we also examined intra-patient variations of leukocyte DNA-TG levels during MP dose titration in 46 children with ALL enrolled on the Japanese Pediatric Leukemia/Lymphoma Study Group ALL B-12 trial. The increase in DNA-TG when MP dose was titrated up was significantly higher in patients homozygous for NUDT15 risk alleles than those with heterozygous genotype and lowest in wildtype patients (P = 0.05), in a fashion similar to results from the Nudt15-/- mouse model. Taken together, these data suggested that DNA-TG may be directly related to hematopoietic toxicity of MP and this metabolite may be used to guide MP dosage titration to avoid excessive toxicity.In conclusion, we established a Nudt15 knockout mouse model, with which we comprehensively evaluated NUDT15 -genotype guided thiopurine dose individualization in vivo . DisclosuresNo relevant conflicts of interest to declare.

The WASP P460S Mutation Causes a New Phenotype of WASP Mutations Related Disorder: X-Linked Pancytopenia

Mutations of the Wiskott-Aldrich Syndrome Protein (WASP) are responsible for a rare and severe disease: Wiskott-Aldrich Syndrome (WAS), including classic Wiskott-Aldrich Syndrome (WAS), X-linked thrombocytopenia (XLT), and congenital X-linked neutropenia (XLN). Classic WAS patients present typical WAS manifestation: eczema, thrombocytopenia, small platelet and recurrent infection, while XLT patients present thrombocytopenia, and XLN patients present congenital neutropenia. Bone marrow failure (BMF) is a series diseases characterized by pancytopenia in peripheral-blood(PB), different degree of bone marrow hypocellularity. In this study, we present two unrelated boys with pancytopenia who were admitted to our hospital on account of declining blood cells and different degree of hematopoietic failure. We identified a new WAS gene mutation C1378T inducing the WASP P460S mutation in both penitents by the second generation sequencing. Furthermore, one case developed T cell acute lymphoblastic leukemia (T-ALL) after severe pancytopenia recovered for 4 years and relapsed in mediastinum transplanted with cord blood for 1 year, another case recovered after haploidentical transplantation and kept stable for 1 year. To date, the role of WAS gene mutation in aplastic anemia-like hematopoietic failure remains unknown. We further verified WASP P460S mutation in these two patients by Sanger sequencing. In patient's PB cells, normal expression level of total WASP proteins was detected. This notion was different from general WASP deficiency classic WAS or XLT patients. To test the function of WASP P460S mutation in pancytopenia, we examined the effect of WASP P460S mutation on cell proliferation. Human myelogenous leukemia K562 cell line, which lacks endogenous WASP, was transfected with lentivirus vectors carrying mutant or wild type of WAS gene. We found that the cells expressing WASP P460S grew significantly slower than the cells expressing the wild type. Moreover, actin polymerization assay showed that increased actin polymerization function in WASP P460S mutation compared with wild type. In addition, P460S mutation had no obvious effect on Daunorubicin-induced apoptosis response. Our findings revealed that the WASP P460S mutation played a role in the inhibition of cell proliferation and actin polymerization which may lead to a new phenotype for WASP mutation related X-linked pancytopenia. DisclosuresNo relevant conflicts of interest to declare.

Oral Squamous Cell Carcinomas in Patient with Graft Versus-Host Disease Following Allogenic Bone Marrow Transplantation: A Case Report during Mutant COVID-19 Pandemic

Aplastic anaemia is a severe haematological disorder characterized by an inadequate number of hematopoietic stem cells, resulting in pancytopenia, formed by a hypocellular bone marrow. Disorders of this nature are widely treated with haematopoietic stem cell transplantation (HSCT). A potential chronic complication following (HSCT) is the growth of secondary malignancies. Notably, patients suffering from chronic graft versus host disease (cGvHD) secondary to HSCT have been shown to be more susceptible to oral squamous cell carcinoma (OSCC). Here, we present a rare case of a 30-year-old Libyan woman treated with HSCT for aplastic anaemia, with subsequent complications of cGvHD and OSCC after few months of HSCT. These carcinomatous lesions were detected in the buccal gingiva and retromolar pad area at the age of 31. The present case report emphasizes the connection between oral cGvHD and OSCC, and the potential appearance of OSCC after HSCT at any time of patient life. Thus, closer follow-up is mandatory for all patients treated with HSCT who developed cGvHD, and efficient cGvHD prevention and therapeutic approaches are needed.

Pretransplant bone marrow cellularity and blood count recovery are not associated with relapse or survival risk following allogeneic stem cell transplant for AML in CR.

Allogeneic hematopoietic cell transplantation (HCT) can be curative for acute myeloid leukemia (AML). Novel therapies may render patients' bone marrow hypocellularity and lead to prolonged post-therapy pancytopenia. Patients' bone marrow cellularity (BMC) at pretransplant assessment and post-treatment pancytopenia (classification CR-incomplete [CRi]) may manifest AML persistence. We retrospectively examined the impact of BMC and ELN response (ELNr) on a single-center cohort of 337 patients who underwent allogeneic HCT for AML in CR1. Median follow-up was 33months. Overall survival (OS) for the whole cohort was 55.8% at 2years, while cumulative incidence of relapse (CIR) was 20.8%, and non-relapse mortality was 27.5%. OS and CIR were not significantly different between BMC groups; and neither was ELNr. ELNr CRi was associated with BMC aplastic and hypocellular marrow states (P<2.6e-8). Multivariate analysis confirmed neither BMC nor attainment of ELNr CR vs CRi affected OS or relapse. Significant factors for survival included age at transplant, cytogenetic risk, development of acute Gr II-IV GvHD, and moderate-severe chronic GvHD, while cytogenetic risk and chronic GvHD affected relapse. Neither ELNr status nor pretransplant BMC influenced relapse post-HCT or OS. Hypocellularity and CRi are not negative prognostic factors for post-HCT outcomes of AML.

Manifestations of Bone Marrow Abnormalities of HIV/AIDS Patients

Background: Human immunodeficiency virus can involve almost any organ system. Anemia is themost common hematological manifestation in HIV/AIDS patients. Bone marrow changes includevarying degrees of dysplasia in one or more cell lines, plasmacytosis, opportunistic infections andhematological malignancies. There are only a few studies where hematological manifestations ofHIV/AIDS patients had been described. Materials and Methods: 100 HIV positive patients, agedbetween 12-65 years were enrolled in this hospital-based cross-sectional study. The study wasconducted from March 2016 to March 2018. A complete blood count, CD4 counts were done, besidesa thorough history and clinical examination. HIV positive patients were classified as those havingAIDS and Non-AIDS, according to NACO criteria. Written informed consent was taken from patientsand bone marrow aspiration was done. Results: Total number of patients included in the study was100. We were able to do a CD4 count of 91 patients. As per criteria, out of 91 patients, 37 cases hadAIDS. The most common hematological abnormality was anemia, seen in 95.45%of patients. Bonemarrow was normocellular in 86.48% of AIDS and 85.18% of non-AIDS, hypocellular in 8.10% ofAIDS and 9.25%o f non- AIDS, hypercellular in 5.40% of AIDS and 5.55% of non-AIDS patients.Dysplasia was statistically and significantly associated with anemia. The commonest dysplasticfeatures are seen in the granulocytic and erythroid series. L.D. bodies were seen in 2 cases andHistoplasma was found in one case. Conclusion: Normocytic normochromic anemia was the mostcommon peripheral smear finding. Hypocellular bone marrow was more common than hypercellularmarrow in an advanced stage of the disease. Dysplastic changes were more common in AIDS thanNon-AIDS. Granulocytic dysplasia was the most common type of dysplasia. There was evidence ofopportunistic infections and gelatinous transformation were detected in our study.

Cystic Lesions of Celiac Disease

Cystic Lesions of Celiac Disease

Febrile Illness in 3 Adolescent Patients.

Febrile Illness in 3 Adolescent Patients.

Stanozolol for the treatment of anemic lower-risk myelodysplastic syndromes without del(5q) after failure of epoetin alfa: findings from a retrospective study.

Options for anemic lower-risk myelodysplastic syndromes (MDS) without del(5q) after failure of erythropoiesis-stimulating agents (ESAs) are very limited. The effectiveness of second-line treatments is uncertain. We retrospectively reviewed the clinical effectiveness and overall survival (OS) of lower-risk MDS without del(5q) patients exclusively treated with stanozolol (STZ) after failure of epoetin alfa. The response was defined according to the 2006 International Working Group (IWG) criteria. Fifty-six patients were included. The median follow-up time was 55 months (range: 5-156 months). Twenty-seven patients (48.2%) achieved hematologic improvement-erythroid response (HI-E). Higher response rates were observed in patients with lower IPSS-R scores (≤3.5, P = 0.008) and hypocellular bone marrow (P = 0.002). In univariate Cox analysis, HI-E was the strongest factor associated with better OS (P = 0.0003). In multivariate Cox, HI-E, age ≤ 50, and transfusion independence (TI) at the onset of STZ were factors associated with better OS. The estimated 5-year OS was 88.6% (68.7-96.2%) and 33.8% (14.9-54.0%) in responders and non-responders (P < 0.01), respectively. The most common side effects included masculinization and liver damage, but they were manageable with supportive measures and dose adjustments. STZ may be considered an alternative treatment in lower-risk MDS after failure of epoetin alfa.

Pediatric MDS and bone marrow failure-associated germline mutations in SAMD9 and SAMD9L impair multiple pathways in primary hematopoietic cells

Pediatric myelodysplastic syndromes (MDS) are a heterogeneous disease group associated with impaired hematopoiesis, bone marrow hypocellularity, and frequently have deletions involving chromosome 7 (monosomy 7). We and others recently identified heterozygous germline mutations in SAMD9 and SAMD9L in children with monosomy 7 and MDS. We previously demonstrated an antiproliferative effect of these gene products in non-hematopoietic cells, which was exacerbated by their patient-associated mutations. Here, we used a lentiviral overexpression approach to assess the functional impact and underlying cellular processes of wild-type and mutant SAMD9 or SAMD9L in primary mouse or human hematopoietic stem and progenitor cells (HSPC). Using a combination of protein interactome analyses, transcriptional profiling, and functional validation, we show that SAMD9 and SAMD9L are multifunctional proteins that cause profound alterations in cell cycle, cell proliferation, and protein translation in HSPCs. Importantly, our molecular and functional studies also demonstrated that expression of these genes and their mutations leads to a cellular environment that promotes DNA damage repair defects and ultimately apoptosis in hematopoietic cells. This study provides novel functional insights into SAMD9 and SAMD9L and how their mutations can potentially alter hematopoietic function and lead to bone marrow hypocellularity, a hallmark of pediatric MDS.

Minimal role of interleukin 6 and toll-like receptor 2 and 4 in murine models of immune-mediated bone marrow failure.

Immune aplastic anemia (AA) results from T cell attack on hematopoietic cells, resulting in bone marrow hypocellularity and pancytopenia. Animal models have been successfully developed to study pathophysiological mechanisms in AA. While we have systemically defined the critical components of the adaptive immune response in the pathogenesis of immune marrow failure using this model, the role of innate immunity has not been fully investigated. Here, we demonstrate that lymph node (LN) cells from B6-based donor mice carrying IL-6, TLR2, or TLR4 gene deletions were fully functional in inducing severe pancytopenia and bone marrow failure (BMF) when infused into MHC-mismatched CByB6F1 recipients. Conversely, B6-based recipient mice with IL-6, TLR2, and TLR4 deletion backgrounds were all susceptible to immune-mediated BMF relative to wild-type B6 recipients following infusion of MHC-mismatched LN cells from FVB donors, but the disease appeared more severe in IL-6 deficient mice. We conclude that IL-6, TLR2, and TLR4, molecular elements important in maintenance of normal innate immunity, have limited roles in a murine model of immune-mediated BMF. Rather, adaptive immunity appears to be the major contributor to the animal disease.

Implication of therapeutic outcomes associated with molecular characterization of paediatric aplastic anaemia

ObjectivesSevere aplastic anemia is characterized by a hypocellular bone marrow and peripheral cytopenia. Mesenchymal stem cells (MSCs) play a crucial role in haematopoietic stem cells (HSCs) development and the development of microenvironment suitable for hematopoiesis. Molecular characterization of telomere maintenance pathway and gene expression profiling of MSCs can be important for the therapeutic interventions among paediatric aplastic anaemia patients. MethodsThe study involved paediatric aplastic anaemia patients (n = 10) and age matched paediatric healthy donors (n = 8). Peripheral blood samples were collected from the individuals. Average leucocyte telomere length and gene expression of the telomere maintenance genes were determined by quantitative real time PCR. Microarray based gene expression profiles (GSE33812) of MSCs for five paediatric aplastic anaemia patients were analyzed compared to five healthy controls and the data was downloaded from the GEO database. ResultsThe telomere length was significantly shorter among paediatric AA patients compared to age matched healthy donors. Interestingly, one subgroup (n = 2) of paediatric AA patients has moderate telomere length comparable to age matched healthy donors. Based on the gene expression analysis of telomere maintenance pathway, TERF2 was significantly downregulated among paediatric patients with shorter telomere length but not among paediatric patients with moderate telomere length. Gene expression profiling of MSCs revealed three differentially expressed genes (GAS2L3, MK167 and TMSB15A) among the patients and was associated with therapeutic outcome. ConclusionTelomere length estimation and gene expression patterns of the MSCs and telomere length maintenance pathway may serve as a potential biomarker and could be associated with therapeutic choice of paediatric aplastic anaemia patients.

Hypoplastic Myelodysplastic Syndromes: Just an Overlap Syndrome?

Simple SummaryHypoplastic myelodysplastic syndromes (hMDS) represent a diagnostic conundrum. They share morphologic and clinical features of both MDS (dysplasia, genetic lesions and cytopenias) and aplastic anemia (AA; i.e., hypocellularity and autoimmunity) and are not comprised in the last WHO classification. In this review we recapitulate the main clinical, pathogenic and therapeutic aspects of hypo-MDS and discuss why they deserve to be distinguished from normo/hypercellular MDS and AA. We conclude that hMDS may present in two phenotypes: one more proinflammatory and autoimmune, more similar to AA, responding to immunosuppression; and one MDS-like dominated by genetic lesions, suppression of immune surveillance, and tumor escape, more prone to leukemic evolution.Myelodysplasias with hypocellular bone marrow (hMDS) represent about 10–15% of MDS and are defined by reduced bone marrow cellularity (i.e., <25% or an inappropriately reduced cellularity for their age in young patients). Their diagnosis is still an object of debate and has not been clearly established in the recent WHO classification. Clinical and morphological overlaps with both normo/hypercellular MDS and aplastic anemia include cytopenias, the presence of marrow hypocellularity and dysplasia, and cytogenetic and molecular alterations. Activation of the immune system against the hematopoietic precursors, typical of aplastic anemia, is reckoned even in hMDS and may account for the response to immunosuppressive treatment. Finally, the hMDS outcome seems more favorable than that of normo/hypercellular MDS patients. In this review, we analyze the available literature on hMDS, focusing on clinical, immunological, and molecular features. We show that hMDS pathogenesis and clinical presentation are peculiar, albeit in-between aplastic anemia (AA) and normo/hypercellular MDS. Two different hMDS phenotypes may be encountered: one featured by inflammation and immune activation, with increased cytotoxic T cells, increased T and B regulatory cells, and better response to immunosuppression; and the other, resembling MDS, where T and B regulatory/suppressor cells prevail, leading to genetic clonal selection and an increased risk of leukemic evolution. The identification of the prevailing hMDS phenotype might assist treatment choice, inform prognosis, and suggest personalized monitoring.

Are Peripheral Blood Counts Predictor of Bone Marrow Infiltration and Hypocellularity in Malignant Neoplasms?

To determine the association of peripheral blood counts (cytopenia and normal counts) with bone marrow findings in patients with malignant neoplasms. Descriptive study. Place and Duration of the Study: King Edward Medical University from 2017 to 2019. Patients having malignant diseases, of both gender and all ages, were included. Clinical data and results of bilateral bone marrow aspiration and biopsy were noted. Results were categorised as groups with bone marrow infiltration, normal morphology with normal cellularity and normal morphology with decreased cellularity. Results were analysed using SPSS V21 applying Chi-square test, keeping the confidence interval 95% and p-value of <0.05 as significant. A total of 157 patients were included (107 men, 50 women). The main categories of neoplasm included non-Hodgkin's lymphoma [93 (59.2%)] Hodgkin's lymphoma [31 (19.7%)], and solid organ malignancy [33 (21.0%]. Bone marrow infiltration was seen in 50 (31.8%) patients, normal morphology with decreased cellularity in 51 (32.5%) and normal morphology with normal cellularity in 56 (35.7%) patients. Cytopenias were seen in 88 (56.1%) patients (out of these 28 (31.8%) had bone marrow infiltrarion, 32 (36.4%) had decreased cellularity and 28 (31.8%) with normal cellularity). In 69 (43.9%) patients with normal CBC, 22 (31.9%) had infiltration, 19 (27.5%) had decreased cellularity and 28 (40.6%) had normal cellularity. No association of cytopenia with bone marrow results was obtained (p = 0.086). Bone marrow infiltration and hypocellularity are not reflected by peripheral blood counts. Therefore, bone marrow biopsy remains decisive in patients with malignant neoplasm for disease staging and selection of treatment options. Key Words: Peripheral blood counts, Bone marrow infiltration, malignant neoplasms, bone marrow hypocellularity.

A Unique Constitutional Robertsonian Translocation t(13;14) Associated with Severe Aplastic Anaemia

Aplastic anaemia is characterised by cytopenias and hypocellular bone marrow without any evidence of marrow fibrosis or marrow infiltration. There is no specific cytogenetic abnormality associated with aplastic anaemia. Most common abnormalities are trisomies of chromosome 6, 8 and loss of 7. A 17-year-old female, presented with generalised weakness, exertional breathlessness and menorrhagia for last six months. She also gave a history of 12 units Packed Red Blood Cells (PRBC) transfusion, at the rate of 2-3 units per month in the last five months. Routine haematology showed severe pancytopenia with reticulocytopenia. Bone marrow evaluation revealed hypoplastic marrow with 15% bone marrow cellularity suggesting aplastic anaemia. Karyotyping using Giemsa (GTG) banding of unstimulated culture showed a very unique constitutional Robertsonian Translocation (RT) karyotype 45+XX, der(13;14)(q10;q10). Patient responded partially to treatment with cyclosporine and anabolic steroids. The final diagnosis was severe aplastic anaemia associated with constitutional RT t(13;14) karyotype. Although cytogenetic abnormalities are neither common nor specific in aplastic anaemia, some of them can have diagnostic and therapeutic implications.

Bicytopenia/pancytopenia in children - clinico etiological profile and importance of bone marrow evaluation in a tertiary care centre

Background: Peripheral cytopenia is a common hematological problem in our day-to-day clinical practice resulting from various diseases ranging from transient infection induced bone marrow suppression to life threatening hematological malignancies. The objective of this study was to know the clinicoetiological profile,hematological analysis and importance of bone marrow evaluation of bicytopenia/pancytopenia in children in a tertiary care teaching hospital.Methods: This was a retrospective, observational study, conducted in pediatric department, NRIIMS, Visakhapatnam, Andhra Pradesh from May 2019 to May 2020. All children in the age group of 1-18yrs of age admitted with new onset bicytopenia/pancytopenia who underwent hematological analysis and bone marrow examination in our hospital were included in this study. Children with bicytopenia/ pancytopenia evaluated outside/treated in other hospitals were excluded from our study. Data was obtained from bone marrow biopsy register from pathology department, admission register of pediatric ward and hospital medical records.Results: Out of the 31 children studied, males were (54.8%), females were (45.2%).11-18 years of age were (51.6%), 1-5 years of age were (38.7%). Fever (77.4%), pallor (74.1%), hepatosplenomegaly (41%) was the most common clinical feature noted in these patients. ALL (52.3%), followed by ITP (9.52%) was the most common etiology for bicytopenia. Aplastic anaemia (40%), megaloblastic anaemia (20%) were most common etiology noted for pancytopenia. Bicytopenia (67.7%) was more common than pancytopenia (32.2%).Peripheral smear examination picked up only 11 out of 14 cases of haematological malignancy, identified by bone marrow examination. Most common bone marrow finding noted was ALL (38.7%), followed by hypocellular bone marrow (22.5%).Conclusions: When there is high index of clinical suspicion and peripheral smear is unable to pick up these cases, it is advisable to do bone marrow examination at the earliest for early confirmation.