ALBERT

Description: Single-stranded DNA binding (SSB) proteins are essential for a variety of DNA metabolic processes and the maintenance of genomic stability. SSB1 and its homolog SSB2, share greater sequence and domain homology to the archaeal and bacterial SSBs than eukaryotic RPA. They form complexes with two other proteins, C9Orf80 and INTS3, and play roles in mediating transcription and DNA repair. SSB1 (also known as OBFC2B or NABP2) is recurrently mutated in various cancers, however the precise function in normal development is incompletely understood. We have previously shown that Ssb1 is required for skeletogenesis, telomeric homeostasis and genomic stability in vivo while Ssb2 knockout mice are viable and grow normally without any detectable phenotype. Interestingly, we observed pronounced upregulation of Ssb2 in response to Ssb1 deletion and modest up-regulation of Ssb1 in response to Ssb2 deletion, suggesting that Ssb1 and Ssb2 may have some overlapping functions. To investigate the specific roles of both Ssb1 and Ssb2 in adult tissue homeostasis, we generated conditional double-knockout (DKO) mouse models of both genes. DKO in adult mice was achieved by using a tamoxifen-inducible Cre (Ssb1fl/fl Ssb2fl/fl R26-CreERT2), in which Ssb1 and Ssb2 are conditionally deleted by the administration of tamoxifen. Induced DKO mice become moribund within seven days featured with pancytopenia and dramatic loss of hematopoietic stem and progenitor cells (HSPCs), suggesting that Ssb1 and Ssb2 are required for the maintenance of haematopoietic stem and progenitors cells (HSPCs). DKO bone marrow was markedly hypocellular with reduction in all lineages of haematopoietic development. Functionally, HSPCs in DKO mice show decreased quiescence at the early stage followed by decreased proliferation and increased cell loss due to apoptotic cell death at the later stage, suggesting the imbalanced bone marrow homeostasis upon DKO may eventually result in exhaustion of the stem cell pool in DKO mice. Furthermore, bona fide HSPC intrinsic functional deficiency caused by DKO was confirmed by competitive bone marrow transplant, where DKO bone marrows showed abolished differentiation capacity and failed to repopulate the bone marrows of recipient mice after induction of DKO in the established engraftments from the Ssb1fl/fl Ssb2fl/fl R26-CreERT2 donors. Gene expression of DKO HSPCs demonstrated an exacerbated p53/p21 DNA damage response and pronounced interferon response. Validating these findings, stabilization of p53 and increased apoptotic cell death were observed in DKO bone marrows and HSPCs and induction of cell cycle and expression of interferon target genes was confirmed by QPCR. DKO HSPCs have increased expression of IFN induced surface markers such as Sca1. The IFN response was intrinsic to HSPCs. Mechanistically, DKO HSPCs manifest a profile of stalled replication forks on DNA combing analysis, unrepaired double strand breaks (increased gammaH2Ax foci and alkaline comet tail moment) and telomeric loss resulting in widespread chromosomal instability. DKO HSPC showed aberrant cytoplasmic accumulation of single stranded DNAs, with R-loop formation (DNA:RNA hybrid), driving this genetic instability and cell-intrinsic interferon response. Altogether, these data provide strong evidence that Ssb1 and Ssb2 have essential functions in regulating haematopoiesis through repairing replication associated DNA damage as well as resolution of R-loop generated during transcription, to maintain genomic stability during normal HSPC homeostasis. Disclosures No relevant conflicts of interest to declare.

Description: Patients with the human genetic disorder ataxia-telangiectasia (A-T) are characterized by immunodeficiency and a predisposition to develop lymphoid malignancies. The gene mutated in A-T patients, ATM, codes for a high molecular weight protein that is implicated in DNA damage recognition and cell cycle control. The ATM protein does not change in amount or cellular distribution throughout the cell cycle or in response to DNA damaging agents. Because peripheral blood mononuclear cells (PBMCs) are largely in a state of quiescence and can be readily stimulated to enter a proliferative phase and because A-T cells exhibit growth abnormalities and senescence, indicative of a general intracellular defect in signalling, we chose PBMCs to examine the relationship of ATM to the proliferative status of the cell. We show here that ATM protein is present at low levels in freshly isolated PBMCs and increases approximately 6-fold to 10-fold in response to a mitogenic stimulus, reaching a maximum after 3 to 4 days. A similar, but delayed response, was evident in the presence of serum only. This increase in ATM protein was accompanied by an increase in ATM kinase activity. While expression of ATM protein increased during proliferation, ATM mRNA expression was unchanged in stimulated and unstimulated cells and there was no evidence for increased ATM protein stability in the phytohemagglutinin (PHA)-treated cells. In keeping with the reduced levels of ATM in quiescent cells, the extent of radiation-induction of the p53 pathway was significantly lower than in mitogen-stimulated cells. Basal levels of p21 were elevated in quiescent cells, and the response to radiation was negligible or reduced compared with proliferating cells over a 2-hour period. Overall, the data suggest that the increase in ATM protein in proliferating cells is due to posttranscriptional regulation and points to a role for ATM in more general signalling.

Description: Patients with the human genetic disorder ataxia-telangiectasia (A-T) are characterized by immunodeficiency and a predisposition to develop lymphoid malignancies. The gene mutated in A-T patients, ATM, codes for a high molecular weight protein that is implicated in DNA damage recognition and cell cycle control. The ATM protein does not change in amount or cellular distribution throughout the cell cycle or in response to DNA damaging agents. Because peripheral blood mononuclear cells (PBMCs) are largely in a state of quiescence and can be readily stimulated to enter a proliferative phase and because A-T cells exhibit growth abnormalities and senescence, indicative of a general intracellular defect in signalling, we chose PBMCs to examine the relationship of ATM to the proliferative status of the cell. We show here that ATM protein is present at low levels in freshly isolated PBMCs and increases approximately 6-fold to 10-fold in response to a mitogenic stimulus, reaching a maximum after 3 to 4 days. A similar, but delayed response, was evident in the presence of serum only. This increase in ATM protein was accompanied by an increase in ATM kinase activity. While expression of ATM protein increased during proliferation, ATM mRNA expression was unchanged in stimulated and unstimulated cells and there was no evidence for increased ATM protein stability in the phytohemagglutinin (PHA)-treated cells. In keeping with the reduced levels of ATM in quiescent cells, the extent of radiation-induction of the p53 pathway was significantly lower than in mitogen-stimulated cells. Basal levels of p21 were elevated in quiescent cells, and the response to radiation was negligible or reduced compared with proliferating cells over a 2-hour period. Overall, the data suggest that the increase in ATM protein in proliferating cells is due to posttranscriptional regulation and points to a role for ATM in more general signalling.

Description: Key Points Combined loss of Ssb1/Ssb2 induces rapid lethality due to replication stress–associated loss of hematopoietic stem and progenitor cells. Functionally, loss of Ssb1/Ssb2 activates p53 and IFN pathways, causing enforced cell cycling in quiescent HSPCs and apoptotic cell loss.

Description: Introduction: Haploidentical hematopoietic stem cell transplantation (HSCT) is a potentially curative intervention for various malignant and non-malignant hematological conditions. Its use has led to the near universal availability of donors, but major challenges compared to HLA-matched related donor (MRD) and HLA-matched unrelated donor (MUD) transplants still exist. Although the most frequently discussed complication of haploidentical HSCT is graft rejection or severe/fatal graft-versus-host disease (GVHD), infection remains a significant cause of morbidity and mortality compared to other forms of transplant. Methods: This was a retrospective, single institution study that analyzed the outcomes of 187 patients with various hematological diseases who received allogeneic HSCT with haplo donor transplants, MRD transplants, or MUD transplants from 2011-2018. Conditioning regimens included combinations of fludarabine, busulfan, cyclophosphamide, melphalan, antithymocyte globulin, and total body irradiation. GVHD prophylaxis included either the combination of cyclosporine, tacrolimus, and mycophenolate mofetil (MMF) or tacrolimus and methotrexate (MTX). All patients received anti-viral prophylaxis with acyclovir, anti-fungal prophylaxis with either an azole or echinocandin, and anti-bacterial prophylaxis with trimethoprim-sulfamethoxazole or levofloxacin if the absolute neutrophil count (ANC) was 0.05). Rates of chronic GVHD were also similar between the two groups at 45% in haplo patients and 48% in non-haplo patients (p 〉0.05). The 100-day infection-related mortality rate following transplant was significantly higher in the haploidentical group at 9% compared to 1% in the non-haploidentical group (p = 0.03, Figure 1). In addition, the 1-year rate of infection-related mortality after transplant was also significantly increased at 16% vs. 4% in the haplo vs. non-haplo groups, respectively (p = 0.01, Figure 2). The proportion of patients who experienced bacterial and fungal infections over this time period was comparable between the two groups (41% vs. 42% for bacterial infections and 18% vs. 12% for fungal infections in haploidentical patients vs. non-haploidentical patients, p 〉0.05 for both) while the incidence rate of viral infections was significantly higher in the haplo group at 72% vs. 38% in the non-haplo group (p 〈 0.001). However, all cases of infection-related mortality were due to bacterial and fungal infections rather than viral with 27% of bacterial or fungal infections leading to patient death in haploidentical patients compared to 7% in non-haploidentical patients (p = 0.02, Figure 3). Conclusion: The results of our study showed that compared to non-haplo transplant recipients, patients who underwent haplo transplants had significantly increased risks of 100-day and 1-year mortality secondary to infections. This finding is supported by prior data demonstrating that there is a longer time to immune reconstitution, specifically of the T-cell subset and dendritic cell subgroup, in the haploidentical population (Chang et. al. Journal of Clinical Immunology 2011). Furthermore, our results showed that the increased risk of infection-related mortality is not due to higher rates of acute or chronic GVHD, implying that the risk may be due to haploidentical transplantation itself as opposed to increased amounts of immunosuppressive therapy. While there was a greater incidence rate of viral infections amongst haploidentical patients, no viral infections led to mortality in either transplant group. In contrast, despite similar rates of bacterial and fungal infections between haploidentical and non-haploidentical patients, these infections led to death more often in the haplo population. These findings are important in considering future approaches to infection prophylaxis and treatment in haploidentical transplant recipients. Disclosures No relevant conflicts of interest to declare.

Description: Background: High dose chemotherapy (HDT) followed by autologous hematopoietic stem cell transplantation (auto-HSCT) is an effective choice of treatment for relapsed/refractory non-Hodgkin lymphoma (NHL) and Hodgkin lymphoma (HL). Historically, carmustine, etoposide, cytarabine, and melphalan (BEAM) has been the most widely used conditioning regimen, shown to significantly improve progression free survival (PFS) and overall survival (OS) with relatively low toxicity. However, high doses of carmustine and etoposide combined with melphalan (BEM), despite increased risks of pulmonary toxicity and severe mucositis, has also demonstrated efficacy as a more intensive preparative regimen with acceptable tolerability in younger populations. We performed a single institution descriptive study in order to compare the outcomes of patients with both NHL and HL treated with either consolidative BEAM or BEM preparative regimens and auto-HSCT. Methods: We identified 65 patients who were treated at USC Norris Comprehensive Cancer Center for relapsed/refractory NHL or HL with HDT and auto-HSCT between 2013 and 2018. 14 patients were excluded from statistical analysis due to insufficient data. The primary outcome was 1-year OS and the secondary outcomes were 100-day mortality and 1-year relapse rate (RR). BEAM consisted of carmustine 150mg/m2, etoposide 200mg/m2, cytarabine 200mg/m2, and melphalan 140mg/m2, while BEM was carmustine 12mg/kg, etoposide 60mg/kg, and melphalan 140mg/m2. Patient baseline characteristics, including age, sex, and diagnosis, were identified to describe the population. The T-test was used to describe groups with continuous outcomes and the Fisher's exact test was used to describe groups with categorical outcomes. A p-value of 〈 0.05 was considered statistically significant. Results: 22 patients who received BEM and 29 patients who received BEAM were included for statistical analysis. The patient's baseline characteristics are shown in Table 1. There was an insignificant trend towards increased 1-year OS (100% vs. 93.10%; p = 0.4996) but a higher 1-year RR (35.29% vs. 33.33%; p = 0.77) in BEM than BEAM. All patients were alive at 100 days; so, no difference was observed between BEAM and BEM with regards to 100-day mortality. Notable differences in demographics between the patients treated with BEAM versus BEM were observed primarily in age and diagnosis. Compared to BEAM, BEM was utilized significantly more in younger patients (mean age at transplant 41.7 vs. 58.6 years old; p 〈 0.0001) with a significantly larger percentage of HL diagnosis (48.28% vs. 5.56%; p = 0.0001). There was no significant difference in the 1-year OS, 100-day mortality or 1-year RR between HL and NHL (p = 0.5333, p = 1, and p = 0.7287, respectively) or between age 〈 50 and age ≥ 50 (p = 0.5271, p = 1, and p = 1, respectively). Conclusions: Both BEAM and BEM HDT for auto-HSCT to treat relapsed/refractory NHL or HL resulted in similar outcomes with no difference observed in 100-day mortality and only minor differences in 1-year OS and 1-year RR. However, significant differences in age and diagnoses of patients treated with these conditioning regimens serve as obstacles for more direct and applicable comparisons. Further analyses including tighter-matched patient cohorts, induction and salvage therapy prior to auto-HSCT, maintenance therapy, as well as treatment-related toxicities are necessary to better differentiate these two preparative regimens and provide additional utility in the clinical setting. Nonetheless, despite the much wider usage of BEAM as a preparative regimen for auto-HSCT in patients with relapsed/refractory lymphomas, BEM appears to be an efficacious alternative, and, therefore, warrants further consideration for future studies. Disclosures No relevant conflicts of interest to declare.

Description: Biphenotypic Acute Leukemia (BAL) is a rare, newly defined disease in the WHO classification of hematological neoplasms. There is little information about the laboratory presentation, and considerable difficulties exist in reaching a definite diagnosis. In this study, designed to examine the laboratory presentation of BAL, we report a total of 23 (3.4%) out of 676 adult and pediatric leukemia cases referred to our center during the last 4 years. The mean age at presentation was (21.57 ± 15.59 years; range 1 year – 66 years). There were 14 males. The mean hematological and biochemical parameters were as follows: WBC 68.76 ± 65.18 x 10 9/L, Hemoglobin 94.13 ± 15.13 g/L, Platelets 105.7 ± 104.23 x 10 9/L, and LDH 1351.44 ± 1118.29 u/L. Fourteen patients (60.8%) had mixed morphology of small and large blast cells and seven were M1 and M2 according to FAB criteria. Using the European Group for Immunological Classification of Leukemia (EGIL) scoring system, all patients had positive Myeloid markers with EGIL scores that ranged from 2.0 – 5.5. Eighteen patients were positive for B-cell markers with scores ranging from 2.0 – 8.5 but only 8 patients were positive for T-cell markers with scores of 0.5 – 4.0. Three patients were positive for markers of the three cell lines. In 7 patients myeloid lineage commitment was also demonstrable by ultrastructural myeloperoxidase. Five patients (22%) had normal cytogenetics. Philadelphia chromosome was positive by karyotype in 3 patients (13%) and MLL gene in 2 (8.7%). Three patients (13%) demonstrated trisomies involving chromosomes 19, 22 and 4, respectively. Out of the 23 patients, 11 (48%) are alive without disease, 4 had died with leukemia, 3 are alive with disease and 5 had lost follow up.

Description: Introduction: The presence of the Philadelphia chromosome has historically been associated with a poor prognosis in patients with acute lymphoblastic leukemia (ALL). Prior studies had demonstrated that the 5-year overall survival (OS) rate among adult patients with Philadelphia-chromosome positive (Ph+) ALL was 25% compared to 41% in Philadelphia-chromosome negative (Ph-) ALL (Moorman et. al. Blood 2007). While hematopoietic stem cell transplantation (HSCT) has long been regarded as the standard of care for adult Ph+ ALL patients despite its significant transplant-related morbidity and mortality, its benefit is less clear in the era of newer-generation tyrosine kinase inhibitors (TKI) such as dasatinib. Methods: We conducted a retrospective study at a single urban transplant center with academic affiliation to analyze the outcomes of adult patients diagnosed with Ph+ ALL between 2005-2018. Patients were treated with either combination chemotherapy plus dasatinib or combination chemotherapy plus dasatinib and allogeneic HSCT. Chemotherapy regimens included hyper-CVAD, Berlin-Frankfurt-Munster-like (BFM-like) protocol, and a regimen developed at the University of Southern California (USC ALL regimen) using pegaspargase (Douer et. al. Journal of Clinical Oncology 2014). All patients in both the transplant group and the non-transplant group received TKI therapy with dasatinib throughout their treatment courses; in the former, patients received dasatinib both prior to and following their transplants. Statistical analysis was performed using the Fisher Exact Probability Test with p-values 30 x 109/L at the time of diagnosis. When comparing rates of OS and RFS between transplant vs. non-transplant patients in this group, there was also no significant difference. Conclusion: This study showed that in the management of adult Ph+ ALL patients, there was no significant difference in OS or RFS between patients who underwent allogeneic HSCT compared to those who received only combination chemotherapy with dasatinib. In addition, prior studies of Ph+ ALL patients treated with either chemotherapy alone or chemotherapy followed by HSCT concluded that the OS rates were approximately 50%, 35%, and 30% at the 1-year, 2-year, and 3-year marks, respectively (Rowe et. al. Blood 2005). Our analysis, however, demonstrated improved rates of survival at all time points for this patient population with the addition of dasatinib. Subgroup analysis of patients with a WBC 〉30 x 109/L at the time of diagnosis also showed no significant difference in OS between transplant and non-transplant patients despite previous reports showing a survival benefit from HSCT (Huang et. al. Blood 2016). This may be attributed to the fact that our patients received the newer and more potent agent dasatinib compared to imatinib in these prior studies. Since allogeneic HSCT demonstrated no survival benefit in our study and can also introduce risks of serious infectious complications, venoocculsive disease (VOD), and graft-versus-host disease (GVHD) that contribute to patient morbidity and mortality, it may serve a less beneficial role for the Ph+ ALL population in the era of newer-generation TKIs. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Thrombotic thrombocytopenic purpura (TTP) is a rare, life-threatening disorder characterized by microangiopathic hemolytic anemia, thrombocytopenia, neurological symptoms, renal dysfunction and other end organ failure. It is associated with inherited or acquired antibody-mediated deficiency of ADAMTS13 resulting in an inability to cleave von Willebrand factor. It occurs in approximately 3 in 1 million adults and 1 in 10 million children annually. First line therapy is plasmapheresis, typically coupled with steroids. Patients recovering from TTP may demonstrate persistent or intermittent ADAMTS13 deficiency without symptoms or thrombocytopenia. Most relapses of acquired TTP occur in the first week after stopping plasma exchange. There are multiple factors contributing to relapses, which include age 〉 60, severe neurological symptoms at presentation and a persistently elevated lactate dehydrogenase. Thus young patients with low ADAMTS13 activity are appropriate candidates for prophylactic treatment. The early initiation of immune-modulatory therapy targeting the antibody inhibitor of ADAMTS13 could potentially reduce the number of plasma exchange procedures required to achieve remission, increase the response rate and decrease the incidence of relapses in patients with TTP. Since TTP is a rare disease, the aim of our study was to outline the trends and outcomes of hospitalizations due to TTP using the nationally representative database for future use in risk stratification and treatment protocols. Methods: We performed retrospective study using the National Inpatient Sample database, a part of the Healthcare Cost and Utilization Project (HCUP) of the agency for Healthcare Research and Quality (AHRQ). We extracted the study cohort of adult admissions with TTP from 2007-2017 by using International Classification of Diseases (9th/10th editions) Clinical Modification diagnosis codes. In the final study cohort we only included patients who received plasmapheresis to restrict patient population with active TTP disease. This approach has been validated in prior publications from administrative databases. Other diagnosis of interests and other comorbidities were identified by ICD-9/10-CM codes and Elixhauser comorbidity software. We utilized Cochran Armitage trend test and survey logistic multivariable regression modeling to analyze trends and predictors of outcomes with weighted analysis. Statistical analyses were performed using SAS software, version 9.4. Results: There were a total 22,054 hospitalizations due to TTP, which increased from 1,620 in 2007 to 1,870 in 2017. The median age was 47-years (IQR:33-60) with 25% hospitalizations among the age group of 18-34 years, 66.1% were females, 46.7% were Caucasians followed by 39.2% African American race. The overall length of stay was 16-days which declined from 17-days in 2007 to 15-days is 2017 (pTrend

Description: Background: A community of practice (COP) is a group of people who share a passion for something, and learn how to perform better as they interact regularly. COPs have been shown to be effective models for achieving quality outcomes in healthcare. We report the development and evaluation of a COP in stem cell donor recruitment in Canada. Methodology: In 09/2017, we launched a COP in stem cell donor recruitment in Canada. Stakeholders in donor recruitment were invited via email and Facebook posts to participate in regular e-meetings and a Facebook group. E-meeting topics included running larger stem cell drives, recruiting the most needed donors, redirecting non-optimal donors, reviews of drive outcomes and strategies to improve, using patient stories to support donor recruitment, and reducing donor attrition. Each e-meeting included speakers and roundtable discussion relevant to the theme. The Facebook group facilitated discussion and sharing of resources between e-meetings (see Fig. A for examples of posts). COP participants were also invited to join subcommittees which focused on developing needed resources or achieving specific objectives identified by the COP. A survey was sent to COP participants in 01/2020 to evaluate the perceived impact of the COP to donor recruitment practice. Recruitment outcomes by COP participants of the Canadian donor recruitment organization Stem Cell Club were compared before and after the launch of the COP. Results: As of 07/2020, the COP Facebook group included 333 stakeholders in donor recruitment (312 donor recruiters from Stem Cell Club; 15 patients/donors; 6 donor registry staff). 51 unique attendees participated in 7 e-meetings, 21 of whom attended 2 or more meetings. COP participants collaboratively set the following goals for the COP: 1) to foster teamwork and collaboration in donor recruitment efforts; 2) to improve knowledge and practice related to donor recruitment; 3) to improve recruitment of the most-needed donors; and 4) to improve donor recruiters' ability to run high quality stem cell drives. 141 posts were published to the Facebook group about patient/donor stories (41%), resources in stem cell donation (23%), stem cell drive outcomes and campaigns (15%), updates related to donor recruitment (14%), and questions posed to the community by COP participants (5%). 44 COP participants completed the COP evaluation survey. The majority agreed/strongly agreed that the Facebook group (86%) and e-meetings (59%) supported the development of a community. 64-84% agreed/strongly agreed that participating in the COP fostered collaboration; improved their knowledge and practice in donor recruitment; and improved their ability to run higher quality drives and recruit most-needed donors (Fig. B). Stem Cell Club's donor recruitment outcomes improved following the launch of the COP: in 2016-2017, Stem Cell Club recruited 2918 donors (46% male; 55.9% of males non-Caucasian) compared to 3418 donors in 2017-2018 (52.7% male; 57.8% of males non-Caucasian), and 4531 donors in 2018-2019 (52.9% male; 62.7% of males non-Caucasian) (Fig C). Finally, a number of outputs were generated as a result of collaboration through the COP, including development of resources such as an infographic (stemcellclub.ca/promo.html), a whiteboard video series (youtu.be/V4fVBtxnWfM), and a stem cell donation story library (#WhyWeSwab; facebook.com/WhyWeSwab). COP participants collaborated on national donor recruitment campaigns, securing coverage in major media outlets across Canada (including Toronto Star: thestar.com/life/2019/11/15/stem-cell-donors-wanted-get-swabbed-campaign-coming-to-university-campuses.html; Toronto Sun: torontosun.com/news/local-news/working-to-build-canadas-network-of-stem-cell-donors; London Free Press: lfpress.com/news/local-news/toddlers-case-proves-patients-must-harness-social-media-in-quest-for-stem-cell-donors-advocates; and Victoria News: vicnews.com/news/stem-cell-drive-at-uvic-aims-to-find-lifesaving-donors-for-patients-in-need) and recruiting thousands of needed donors (Fig. D). Conclusion: We describe the first COP in stem cell donor recruitment to our knowledge. The COP was valued by participants and supported efforts to improve donor recruitment. The COP model can be adapted by donor recruitment organizations around the world to improve recruitment outcomes. Figure Disclosures No relevant conflicts of interest to declare.