Hematologic Complications with Age in Shwachman-Diamond Syndrome

Shwachman-Diamond Syndrome (SDS) is an inherited bone marrow failure syndrome with leukemia predisposition. An understanding of the hematologic complications of SDS with age could guide clinical management, but data are limited for this rare disease. We conducted a cohort study of 153 subjects from 143 families with confirmed biallelic SBDS mutations enrolled on the North American Shwachman Diamond Registry or Bone Marrow Failure Registry. The SBDS c.258+2T>C variant was present in all but one patient. To evaluate association of blood counts with age, a total of 2146 blood counts were analyzed for 119 subjects. Absolute neutrophil counts were positively associated with age (P<0.0001). Hemoglobin was also positively associated with age up to 18 years (P<0.0001) but thereafter the association was negative (P=0.0079). Platelet counts and marrow cellularity were negatively associated with age (P<0.0001). Marrow cellularity did not correlate with blood counts. Severe marrow failure necessitating transplant developed in 8 subjects at a median age of 1.7 years (range 0.4-39.5), with 7 of 8 requiring transplant prior to age 8 years. Twenty-six subjects (17%) developed a myeloid malignancy (16 MDS and 10 AML) at a median age of 12.3 years (range 0.5-45.0) for MDS, and 28.4 years (range 14.4-47.3) for AML. A lymphoid malignancy developed in one patient at the age of 16.9 years. Hematologic complications were the major cause of mortality (17/20 deaths, 85%). These data inform surveillance of hematologic complications in SDS.

Analysis of Hematopoietic Niche Components Post Allogeneic Transplantation

Timely and effective reconstitution of the hematopoietic niche is imperative post-allogeneic stem cell transplantation (alloHCT) for prevention of life-threatening opportunistic infections and relapses. Efficient engraftment is dependent on the ability of donor hematopoietic stem cells (HSCs) to expand and provide long-term multi-lineage progenitors in the recipient. Mesenchymal stem cells (MSCs) are essential in providing a conducive environment for HSC reconstitution and maintenance. Conditioning chemotherapy with or without total body irradiation is required to confer a competitive advantage to donor HSCs. However, myeloablative conditioning (MA) regimens also destroy the specialized stromal niches which support HSC self-renewal and maintenance (Acar et al., 2015), providing a plausible explanation for the long term cytopenias observed post allogeneic transplantation (Domenech et al., 1995). Previous studies in humans showed CD146-expressing stromal cells support the establishment of the hematopoietic cell microenvironment (Sacchetti et al., 2007). Expression of markers of stromal/progenitor cells (CD146, CD31 and CD34) was evaluated pre and post alloHCT at day 30 in bone marrow biopsies obtained from patients treated with either MA or reduced intensity conditioning (RIC) approaches. CD146 and CD31 expression was evaluated by immunohistochemistry performed on formalin-fixed, paraffin-embedded bone marrow core biopsies. Whole slide imaging (Aperio) was used to digitize the histology and positive cells were scored using image analysis software (strong positive pixel count; ST PPC). In bone marrow, CD146 is expressed on vascular endothelial cells, pericytes on sinusoids, adipose and bone marrow stromal cells. CD31 positive cells include vascular endothelial cells, sinusoidal cells, megakaryocytes, and myeloid cells (Fig. 1). The percent of CD34 positive stem cells was determined by flow cytometry in concurrent bone marrow aspirate cells. The indications for an alloHCT were AML (5/10), ALL (2/10) or MDS (3/10). Median age was 62 (59-69) years in the RIC group and 34 (25-46) years in the MA group. Average HCT CI score was higher in the RIC group (2.5 versus 1.2) (Table 1). 3/10 patients received total body irradiation (TBI) based conditioning. All patients received peripheral blood stem cell alloHCT and 7/10 were MUDs. Chronic GVHD was present (grade 2 or 3) in 3/10 patients in the RIC groups and 1 patient (grade 1) in the MA cohort. Chimerism studies showed optimum engraftment of donor cells in 2 patients in the RIC group and 1 patient in the MA (Table 1). On average, pretransplant bone marrow cellularity was lower in the RIC group. The average overall cellularity on D30 was similar in the two groups (Fig.2). The average percent of CD34 positive cells was similar pretransplant and lower at D30 in the RIC cohort compared to the MA group (0.96% CD34 pre alloHCT versus 0.30% D30 post alloHCT RIC; pre alloHCT 0.96% CD34 versus D30 post alloHCT 0.92% MA). The mean number of CD31 positive cells was higher in the RIC group compared to the MA cohort both pre-transplant and at D30 (Fig 3). On average, an increase in CD146 positive cells on D30 was noted in the alloHCT RIC group compared to the MA cohort (Fig. 4). In conclusion, the RIC cohort showed a pre-post-transplant increase in average overall bone marrow cellularity, more numerous CD31 and CD146 positive cells on average and an increase in CD146 positive cells at D30 post alloHCT compared to the MA group. Despite the modest number of patients, this is the first study to date comparing pre and post alloHCT niche components which may provide important insights in the reconstitution process and impact of conditioning. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Bone Marrow Lymphoid Infiltrates and Aplasia after CD19-Directed CAR T Cell Therapy in Pediatric B-Lymphoblastic Leukemia

Introduction Chimeric Antigen Receptor (CAR) T cell therapy is used to treated relapsed/refractory B-Acute Lymphoblastic Leukemia (B-ALL) patients. Long term side effects include B cell aplasia and cytopenia that are treated with supportive therapy. Prolonged cytopenia is seen in 16% of CAR T-cell treated B-ALL patients. The etiology of cytopenia is not clear, and could be attributed to the impact of chemotherapy, CAR T cells and disease or patient-specific factors. The impact of CAR T cell infusion on bone marrow cellularity, lymphocyte compartment and its correlation with cytopenia has not been investigated. Methods We analyzed pre- and post-CAR bone marrow biopsies in 178 B-ALL patients who received a CD19-directed CAR T-cell product between 2012-2017 and followed for at least 12 months. Responses were categorized into sustained responders, non-responders, CD19-positive relapses and CD19-negative relapses as previously described. Bone Marrow biopsy (BMB) overall cellularity, complete blood counts (CBC) in a 12-month post infusion period were analyzed. BMB were considered mildly, moderately and severely hypocellular based on cellularity of 50, 25 and 5% respectively. Hypocellularity was further stratified by CBC per aplastic anemia (AA) classification guidelines: Non-Severe Aplastic Anemia (NSAA) and Very Severe Aplastic Anemia (VSAA). Immunohistochemistry (IHC) for CD3, CD4, CD8, CD163, granzyme and perforin were performed on pre- and post-CAR BMB. CD3 IHC was digitally scanned and analyzed quantitatively (Leica Aperio ImageScope) and qualitatively. Targeted RNA sequencing-based gene expression profiling (EdgeSeq Immuno-Oncology Panel, HTG Diagnostics) was performed on pre-and post-treatment biopsies, with differential expression assessed by DESeq2 within HTG Reveal software. RNAseq gene expression was deconvoluted to impute relative expression of immune cell subsets. Results 31% of patients were mildly hypocellular but none were severely hypocellular at baseline pre-CAR timepoints. The highest proportion of hypocellularity was at the 1-month time point. 81% of patients were mildly hypocellular (≤50%), 42% were markedly hypocellular (≤25%), and 13% were severely hypocellular (≤5%) at the 1-month time point. By month 12, the proportion of mildly hypocellular patients was 74%, markedly hypocellular patients was 26%, and severely hypocellular patients was 7% (Figure 1). The proportion of NSAA and VSAA was 57% and 10% in moderately hypocellular BMB. The proportion of NSAA and VSAA was 64% and 18% in severely hypocellular BMB. Severely hypocellular patients had a higher average day minus 1 disease burden (33% involvement) compared to their moderate (24%) and non-hypocellular patients (16%). VSAA patients had a lower baseline BMB cellularity (47%) compared to NSAA patients (71%) and those without AA (60%) Increased CD3+ T cells were noted in the post-CAR BMB compared to the pre-CAR BMB (Figure 1B). Lymphocyte were singly scattered or formed loose aggregates and tight lymphohistiocytic clusters. IHC and RNAseq analysis showed increased CD8+ granzyme+ cells in the post-CAR BMB compared to pre-CAR BMB. Sustained responders showed higher T cell infiltration compared to other categories of patients (Figure 1C and D). Lymphoid infiltrates did not correlate with hypocellularity or cytopenia. Conclusion We describe for the first time the changes in bone marrow cellularity and lymphocyte compartment after CD19-directed CAR T-cell infusion in B-ALL. A subset of patients were moderately or severely hypocellular and met criteria for aplastic anemia. However, most of them recovered bone marrow cellularity and CBC. Hypocellularity and AA were correlated with pre-CAR disease burden and baseline cellularity rather than post-CAR lymphocyte infiltration. We also show for the first time that sustained responders to CD19-CAR showed increased CD3+ CD8+ T cell infiltrates compared to CD19-positive relapses and non-responders. Figure 1. A. Bone marrow hypocellularity post-CAR T-cell infusion. Proportion of patients who were mildly hypocellular, markedly hypocellular, and severely hypocellular in the 12month follow up period. B. Increased CD3+ T cells and aggregates after CAR T cell infusion. C and D. Sustained responders show significantly greater CD3+ T cells after CAR T cell infusion compared to other categories. *P<0.05. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Targeting USP7 Suppresses Tumor Development By Promoting Ubiquitination of PHF8 and Suppressing SNAI1/Wnt Signaling in T-Cell Acute Lymphoblastic Leukemia

Introduction T-cell acute lymphoblastic leukemia (T-ALL) is a common hematological malignancy with a high unfavorable prognosis. Ubiquitin-specific-processing protease 7 (USP7) is one of the deubiquitinating enzymes attracting concentrated attention in current studies for cancers; it is also involved in regulation of oncogenic transcriptional program in T-ALL, and serves as a potential target to treat T-ALL. USP7 is interacted with PHD Finger protein 8 (PHF8), a histone lysine demethylase in T-ALL. However, the impact of the interaction in T-ALL development is undetermined. In this study, we explored the anti-tumor effect of targeting USP7 and its downstream substrate in T-ALL and underlying mechanism. Methods In vitro GST pull-down assay, co-immunoprecipitation assay, protein deubiquitination and stability assays were used to detect the interaction of USP7 with its direct substrate and the ubiquitination level. Gain-of-function(overexpression) and loss-of-function (lentiviral shRNA knockdown) approaches for USP7 were conducted for gene expression, CCK-8 cell proliferation assay, and apoptosis assay with annexin V + PI staining. ChIP-qPCR assay was used to explore the enrichment of histone markers in promoter region of target gene. In vivo human T-ALL xenograft mouse model was developed in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mouse by tail vein injection of 2.5 × 10 6 /mouse and the leukemia engraftment was evaluated with human CD45+ cells by flow cytometry. The qPCR and western blot were carried out for detection mRNA level and protein level of related genes. The mRNA level of USP7 and its substrate PHF8 was examined by qPCR in 43 newly-diagnosed T-ALL patients with an approval of the Ethics Committee. Microarray datasets of T-ALL patients from GEO database were used to determine the USP7 and its substrate -related genes with web-based platform MEM and Limma. Results Results showed that USP7 directly interacted with PHF8; and overexpressed USP7 deubiquitinated and stabilized PHF8, and enhanced cell proliferation of JURKAT T-ALL cells. These data indicates targeting USP7 promotes cell proliferation of T-ALL cells through its substrate PHF8. Moreover, SNAI1 was identified as one of key co-expressed genes with PHF8 in microarray datasets from T-ALL patients, and knock-down of PHF8 suppressed the SNAL1 expression and increased the recruitment of repressive histone marker, H3K9me2 but decreased the enrichment of H3K4me3 in SNAI1 promoter. SNAI1 knockdown significantly induced the cell proliferation arrest and apoptosis of T-ALL cells (Fig.1A & 1B). With in vivo human leukemia xenograft mouse model, we further demonstrated that SNAI1 knockdown significantly reduce the spleen size and weight, the ratio of human CD45(+) cells, bone marrow cellularity and also the inflammatory cell infiltration compared to the scramble shRNA control (Fig.1C-1F). Moreover, Wnt signaling was identified as SNAI1 interaction partners by the pathway analysis in the SNAI1 interaction genes in the microarray data from T-ALL patients. SNAI1 knockdown significantly suppressed the expression of the Axin2 and Survivn, the key components of Wnt signaling. These data suggested oncogenic role of USP7/PHF8/SNAI1/Wnt signaling in T-ALL. Next, we explored the effect of SNAI1 on USP7 knockdown-induced anti-tumor effect. We found that USP7 knockdown induced cell proliferation arrest and apoptosis, and overexpression of SNAI1 could block the effect in vitro. Furthermore, the in vivo data showed that USP7 knockdown significantly suppressed spleen size and weight, the ratio of human CD45(+) cells, bone marrow cellularity, inflammatory cell infiltration, and the protein expression of PHF8, SNAI1, Axin2 and Survivin; and SNAI1 overexpression completely rescue the USP7 knockdown-mediated antitumor effect and restored the expression of Axin2 and Survivin in vivo (Fig.2). Conclusion: Our results demonstrated that targeting USP7 by shRNA induces the cell proliferation arrest and apoptosis by promoting ubiquitination of PHF8 and suppressing SNAI1/Wnt signaling in T-ALL. Our data also revealed the oncogenic roles of USP7/PHF8/SNAI1/Wnt signaling in T-ALL and suggested targeting the signaling pathway as potential therapy in T-ALL. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Prospective Phase I/II Study of Eltrombopag for the Treatment of Bone Marrow Failure in Fanconi Anemia

Fanconi anemia (FA) is an inherited disorder associated with loss of function mutations in gene members of the FA-BRCA pathway involved in interstrand cross-link DNA damage repair during cell division. Progressive bone marrow failure (BMF) is a primary cause of morbidity and mortality in patients with FA. Allogeneic hematopoietic stem and progenitor cell (HSPC) transplantation is the only curative treatment for FA-associated BMF. However, donor availability, graft failure, and FA-specific transplant toxicities remain significant hurdles. Attempts at genetic correction of FA are underway but collection of sufficient numbers of autologous HSPCs is challenging in subjects with advanced BMF. Androgens have been successfully used but side effects often prevent prolonged therapy. The orally bioavailable small molecule mimetic of thrombopoietin, eltrombopag (EPAG), was recently shown to stimulate multipotent long-term repopulating HSPCs in patients with acquired BMF, resulting in persistent trilineage hematopoiesis. EPAG promotes DNA repair (Guenther, Exp Hematol 2019) and maintains HSPCs under inflammatory conditions (Alvarado, Blood 2019), indicating potential relevance in FA. In this study, we investigated whether EPAG may offer a novel therapeutic modality for FA-associated BMF. We have enrolled 10 patients (FANCA, n=9 and FANCC, n=1) to date in a prospective phase I/II study of EPAG in Fanconi anemia (NCT03204188). All subjects received EPAG at an oral daily dose adjusted for age and ethnicity. The primary efficacy endpoint was the proportion of subjects with at least 2-fold increase in marrow cellularity or CD34+ HSPC frequency (marrow response), or clinically significant improvement in peripheral blood (PB) counts at 6 months (blood response). The primary safety endpoint was the global toxicity profile assessed at 6 months using CTCAE criteria. Responders were invited to continue on the extension phase of the study for an additional 3 years. Four of 10 patients have reached the 6-month assessment endpoint, and a pre-specified 3-month interim analysis is available on two additional subjects. A marrow response was observed in all 4 subjects at 6 months. Mean marrow cellularity increased by a factor of 4 (Fig. A/B), and CD34+ HSPC frequency improved 2.5-fold at 6 months relative to pre-treatment values (Fig. C/D). Marrow response criteria were also met in the other two subjects at the 3-month interim assessment. Primary PB response was observed in 2 of 4 patients in one (hemoglobin) or two lineages (hemoglobin and platelets) at 6 months of treatment. Responders continue to receive EPAG at 10 or 25 months with sustained improvements in blood counts and transfusion independence. The subject treated for 25 months recently met response criteria in a third lineage (neutrophils) (Fig. E). Both patients who had a marrow but no PB response are clinically well; one subject (FANCA) underwent an unrelated allogeneic HSPC transplantation one year ago, and the other subject (FANCC) recently entered the extension phase of this study. Unilineage PB response criteria were also met in one of two subjects at the 3-month interim assessment. No drug-related serious adverse events have occurred during EPAG treatment. All patients tolerated the maximum dose of EPAG and no interruption or dose reduction was required for adverse events attributable to EPAG. Patients without transfusional iron overload at study entry developed progressive depletion of iron stores attributable to the known potent iron chelating and mobilizing properties of EPAG. At a median of 3 months after initiating EPAG (range, 1 to 6 months), affected patients initiated daily oral iron supplementation with gradual amelioration of iron store levels. To date, there has been no occurrence of marrow fibrosis, solid malignancies or clonal evolution, defined as abnormalities on standard metaphase analysis of bone marrow or overt clinical transformation to MDS/AML. In sum, treatment with EPAG was associated with increased marrow HSPCs in all subjects and PB responses in a subset of patients with Fanconi anemia. The overall safety profile was favorable. Further follow-up and expansion of the ongoing patient cohort will confirm EPAG's potential as a safe and efficient long-term therapeutic modality for FA-associated BMF. Alternatively, EPAG could also be used safely to boost marrow CD34+ cell numbers prior to autologous gene therapy applications. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Clinicopathologic characteristics of myeloid neoplasms that harbor both a DDX41 germline and an acquired DDX41 genetic alteration

Introduction/Objective Myeloid neoplasms associated with DDX41 germline genetic alterations are becoming increasingly recognized as a unique subset of hematologic cancer predisposition syndromes. As such, we sought to review their clinicopathologic characteristics. Methods/Case Report We searched our next-generation sequencing database for cases with two DDX41 variants; one variant at heterozygous variant allele fraction (VAF) indicating known or putative germline status, and second variant at low VAF consistent with an acquired subclone. We reviewed the clinicopathologic features. Results (if a Case Study enter NA) We identified 18 cases - male:female ratio 2.6:1, average age at presentation, 69 years (range 57-89). The diagnoses included no diagnostic abnormality (3), MDS-MLD (1), MDS-EB1 (1), increased blasts only(5-19%) (7), and AML (6). All had anemia [average hemoglobin (10.2) (range 6.5-12.9 g/dL)], 17/18 with thrombocytopenia [average 87 (range 22-222 K)], 17/18 with neutropenia [average absolute neutrophil count (ANC) (819) (range 90-1800)] and 13/18 with macrocytosis [average (101.5) (range 83.7-114 fL)]. Bone marrow cellularity (corrected for increased blasts >20%) was predominantly hypocellular (11/19) followed by normocellular (5/19). 3/19 cases showed erythroid dysplasia; no cases demonstrated granulocytic dysplasia; 7/19 cases showed megakaryocytic dysplasia. Of non-AML cases, 8/12 cases showed increased blasts [average 10% (range 5-19%)]. Of 4 cases without an increase in blasts, 1 showed MDS, 2 no dysplasia [1 -ANC of 666 and preserved platelets (222); 2- thrombocytopenia with preserved ANC (1800)] and 1 had slight megakaryocytic atypia. 16/17 cases were karyotypically normal. 14/18 of the patients are alive (median follow-up, 48) (8-125 months). Conclusion This cohort finds that myeloid neoplasms arising from DDX41 germline predisposition syndrome tend to present in older individuals, have infrequent dysplasia and are associated with a prolonged clinical course despite elevated blast counts at diagnosis. Recognition of these disorders is challenging and DDX41 testing should be included as part of genetic profiling. The presence of a suspected DDX41 germline variant may prompt confirmatory and familial testing, particularly in the event a hematopoietic transplant is a treatment option.

JAK inhibition for murine HLH requires complete blockade of IFNg signaling and is limited by toxicity of JAK2 inhibition

Hemophagocytic lymphohistiocytosis (HLH) is an inflammatory disorder in which numerous cytokines are elevated, though interferon gamma (IFN-g) is central to disease pathogenesis and a key therapeutic target. Experimental and early clinical reports have shown that ruxolitinib, a small molecule inhibitor of Janus kinases (JAKs) which are essential for cytokine signaling, may be therapeutic in HLH. In contrast, we found that intermittently administered ruxolitinib at various dose levels failed to prevent HLH development or treat established murine HLH. High doses of ruxolitinib blocked IFN-g signaling only transiently after administration, consistent with human pharmacokinetics, and only continuously administered drug could prevent HLH development or treat established HLH. Continuously administered ruxolitinib was therapeutic in only a narrow dose range and intermittently dosed ruxolitinib worsened survival and decreased bone marrow cellularity of animals concurrently treated with anti-IFN-g antibody, indicating a narrow therapeutic window and potential toxicity. As JAK2 is essential for hematopoietic cytokine signaling, we also tested a JAK1-selective inhibitor and observed therapeutic benefit without apparent toxicity, though it did not improve survival when combined with anti-IFN-g. We conclude that continuous blockade of IFN-g signaling is necessary for optimal control of HLH and that JAK2 inhibition may be toxic in this disorder.

Clinicopathological Evaluation of Metastatic Carcinomas of Bone Marrow Presenting as Cytopenia

Background – Metastatic carcinomas can involve bone marrow and may lead to subsequent marrow fibrosis and failure. The Bone marrow examination is important in patients diagnosed or patients on chemotherapy for cancer, who presented with peripheral cytopenia. The metastasis of bone marrow by these tumors is a sign of advanced stage of disease with poor prognosis. Methods – Our study is a retrospective study, in which we reviewed a total of 702 bone marrow procedures, out of which 118 bone marrow procedures done in patients with a diagnosis of cancer or patients on chemotherapy presented as cytopenia in Great Eastern Medical College and Hospital during a period of 10 years. Result – In our study of 118 patients 74 males and 44 females. Peripheral smear examination of these cases - 24 out of 32 (75%) presented with anemia, which was the commonest clinical presentation. Others were, thrombocytopenia in 18 (50%), bleeding manifestations in 10 (31.2%), Pancytopenia in 9 (28.1%), bi-cytopenia in 4 (12.5%). During this study period, among 118 malignancies reported in histopathology, 32 cases show bone marrow metastasis. 17.1% were carcinoma breast, 25% were carcinoma stomach 33.3% were carcinoma prostate and carcinoma urinary bladder, 23.5% were SCC carcinoma lung, 0% carcinoma Cervix, all cases of Ewing’s sarcoma, neuroblastoma and poorly differentiated carcinoma show bone marrow metastasis. Conclusion - Bone marrow examination is valuable tool in the diagnosis and staging of hematologic and nonhematological disease, as well as in the assessment of overall bone marrow cellularity, pattern of marrow involvement in metastatic carcinomas.

Influence of the Herbal Preparation "Licorice Oil" on the State of Hematopoiesis in Rats Under Ionizing Irradiation

The radioprotective effect of the herbal preparation "Licorice oil" on the hematopoietic system and oxidative stress was studied. The experiment was carried out on 30 male Wistar rats, divided into 3 groups. The first group is control group, the second is irradiation with 6Gy, third group - a week before irradiation and two weeks after, received "Licorice oil" intragastrically at a dose of 2.5 ml/kg of body weight. Gamma irradiation significantly reduced the number of erythrocytes, hemoglobin, hematocrit, leukocytes, thrombocytes in peripheral blood and bone marrow cellularity. The frequency of micronuclei in polychromatophilic erythrocytes of the bone marrow has significantly increased. The level of lipid peroxidation in the blood increased against the background of a significant decrease in the activity of antioxidant enzymes. The introduction of "Licorice oil" for 21 days provided a protective effect. In application of "Licorice oil", there was an increase in the number of cellular elements in the peripheral blood and against the background of a decrease in the frequency of micronuclei in the bone marrow. The activity of antioxidant enzymes in blood plasma increases against the background of a decrease in the amount of peroxidation products. The herbal preparation "Licorice oil" exhibits antioxidant activity, reduces genotoxicity and cytotoxicity under gamma irradiation. "Licorice oil" can be used to prevent radiation damage.