Supplemental Table 3. Multivariate analysis showing impact of cGVHD characteristics on treatment related mortality, disease free survival and overall survival in patients with CML who developed cGVHD

Supplemental Table 1. Multivariate analysis of late relapse, transplant-related mortality, overall survival after HCT for each disease (AML, ALL, CML, MDS)

Supplemental Figure 1. Cumulative incidence of cGVHD, relapse and death in patients with AML, ALL, CML, MDS

Supplemental Table 2. Multivariate analysis showing impact of cGVHD characteristics in CML patients on relapse

Treatment for relapse of chronic myeloid leukemia (CML) following hematopoietic cell transplantation (HCT) includes tyrosine kinase inhibitors (TKIs) with or without donor lymphocyte infusions (DLIs), but the most effective treatment strategy is unknown. This study was performed through the Center for International Blood and Marrow Transplant Research (CIBMTR) database. We retrospectively reviewed all patients reported to the CIBMTR registry from 2002 to 2014 who underwent HCT for CML and were alive 30 days postrelapse. A total of 215 HCT recipients relapsed and were analyzed in the following groups: (1) TKI alone (n = 128), (2) TKI with DLI (n = 48), and (3) DLI without TKI (n = 39). In multivariate analysis, disease status prior to HCT had a significant effect on overall survival (OS). Patients who received a DLI alone compared with a TKI with a DLI had inferior survival (hazard ratio, 2.28; 95% confidence interval, 1.23 to 4.24; P= .009). Those who received a TKI alone had similar survival compared with those who received a TKI with a DLI (P = .81). These data support that despite use of TKIs pretransplantation, TKI salvage therapy continues to provide significant survival following relapse in patients with CML following HCT. These data do not suggest that adding a DLI to a TKI adds an improvement in OS. (C) 2020 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc.

Post-transplant cyclophosphamide (PTCy) has significantly increased the successful use of haploidentical donors with a relatively low incidence of graft-versus-host disease (GVHD). Given its increasing use, we sought to determine risk factors for GVHD after haploidentical hematopoietic cell transplantation (haplo-HCT) using PTCy. Data from the Center for International Blood and Marrow Transplant Research on adult patients with acute myeloid leukemia, acute lymphoblastic leukemia, myelodysplastic syndrome, or chronic myeloid leukemia who underwent PTCy-based haplo-HCT (2013 to 2016) were analyzed and categorized into 4 groups based on myeloablative (MA) or reduced-intensity conditioning (RIC) and bone marrow (BM) or peripheral blood (PB) graft source. In total, 646 patients were identified (MA-BM = 79, MA-PB = 183, RIC-BM = 192, RIC-PB = 192). The incidence of grade 2 to 4 acute GVHD at 6 months was highest in MA-PB (44%), followed by RIC-PB (36%), MA-BM (36%), and RIC-BM (30%) (P = .002). The incidence of chronic GVHD at 1 year was 40%, 34%, 24%, and 20%, respectively (P < .001). In multivariable analysis, there was no impact of stem cell source or conditioning regimen on grade 2 to 4 acute GVHD; however, older donor age (30 to 49 versus <29 years) was significantly associated with higher rates of grade 2 to 4 acute GVHD (hazard ratio [HR], 1.53; 95% confidence interval [CI], 1.11 to 2.12; P = .01). In contrast, PB compared to BM as a stem cell source was a significant risk factor for the development of chronic GVHD (HR, 1.70; 95% CI, 1.11 to 2.62; P = .01) in the RIC setting. There were no differences in relapse or overall survival between groups. Donor age and graft source are risk factors for acute and chronic GVHD, respectively, after PTCy-based haplo-HCT. Our results indicate that in RIC haplo-HCT, the risk of chronic GVHD is higher with PB stem cells, without any difference in relapse or overall survival.

Lenalidomide, bortezomib, and dexamethasone (RVD) is standard-of-care induction for fit multiple myeloma patients, with response rate (RR)>90% in first line. Standard RVD utilizes a 21-day cycle with bortezomib IV 1.3 mg/m2 days 1, 4, 8, and 11; lenalidomide 25 mg days 1-14; and dexamethasone 160-320 mg per cycle. Bortezomib-induced neuropathy may be treatment-limiting, occurring in up to 80% of patients. Weekly administration of bortezomib, subcutaneous (SC) dosing, and extending the cycle to 28 or 35 days may optimize tolerance. Those adjustments were studied primarily in transplant-ineligible or relapsed/refractory patients. We report the results of a retrospective analysis of patients who received RVD on a 21-day cycle with weekly subcutaneous bortezomib to improve tolerability or logistics.

Introduction Allogeneic hematopoietic stem cells transplant (HSCT) is a curative treatment for acute myeloid leukemia (AML). The persistence of disease-associated somatic mutations following HSCT for myelodysplastic syndrome has been shown to be associated with a high risk of disease progression. While a recent study in AML found that the presence of somatic mutations at day 21 post-HSCT is associated with higher risk of relapse, there is paucity of data at a later time point. We hypothesized that persistence of mutations at day 100 resulted in increased relapse rates and inferior outcomes than in patients who had cleared their mutations. Methods All adult patients with AML, diagnosed in 2016-2017, who underwent allogeneic HSCT, were included in the study. Somatic mutations in diagnostic and post-transplantation bone marrow specimens were detected by a 97-gene next generation sequencing (NGS) assay. Baseline characteristics including age, sex, risk stratification, type of transplant, conditioning regimen and presence of acute and chronic graft-versus-host disease (GHVD) were collected. We evaluated somatic mutations in bone marrow samples obtained 100 days after transplantation (T+100). Results We identified 32 patients, four had no detectable mutations in the diagnostic bone marrow NGS and were excluded from the analysis. Most patients were >60 years with male-predominance (68%). Fourteen (50%) patients fell into the poor-risk AML category. Twenty eight (87.5%) patients had at least one mutation in the diagnostic bone marrow; the mean number of mutations at diagnosis was 3 (1-11). FLT3, NPM1, RUNX1, TET2, RAS and ASXL-1 were the most common mutations. Eighteen patients (64%) underwent matched unrelated HSCT and 15 (54%) had reduced-intensity conditioning (RIC) chemotherapy. Twenty three (82%) out of 28 patients had no detectable mutations in the day 100 bone marrow. All five patients with persistent mutations at T+ 100 had evidence of disease relapse. The T+100 bone marrow demonstrated evidence of relapse in two patients while the other three relapsed after a median of 6.53 months from transplant. This compared to a relapse rate of 17% in patients with no detectable mutations at T+100 (p=0.001, Fisher-Exact test). Median progression-free survival (PFS) was longer in patients who had no mutations at day 100 compared to those with persistent mutations (15.03 months vs. 5.50 months). Conclusions Our data show, that the persistence of somatic mutations at T+100 after allogeneic HCT can predict relapse. While the number of patients in this study is limited, the signal observed is significant and expansion of this analysis to a larger study is warranted, as identification of this high-risk population for relapse potentially allows for the initiation of early post-transplantation intervention strategies.

Purpose: Acute myeloid leukemia (AML) is characterized by multiple somatically acquired mutations that affect genes of different functional categories. It has been well established in myelodysplastic syndrome (MDS) that the cumulative number of somatic mutations has an impact on overall survival. However, no such data exist for AML. In this study, we sought to determine the number of clinically significant somatic mutations for each cytogenetically defined risk group of AML and to determine whether this had an impact on overall survival (OS). Methods: In this retrospective, single-center study, all adult patients diagnosed with AML from August 2016–December 2017 were reviewed. Baseline characteristics, somatic mutations in the diagnostic bone marrow as detected by Next Generation Sequencing (NGS), and survival outcomes were analyzed. NGS panel was done in-house and could identify 94 genes. Patients were divided into favorable, intermediate, and poor risk groups based on cytogenetics, and molecular abnormalities using NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for AML, version 1.2018. Kaplan-Meier plots and Cox regression analyses were utilized. Results: A total of 105 AML patients were included; baseline characteristics and frequency of identified clinically significant (CS) mutations are described in the presentation. The FLT3 mutation occurred in the highest frequency (22%) followed by DNMT3A & ASXL1 (15%). 17 (16%) patients were favorable risk, 33 (31%) intermediate risk, and 55 (52%) were poor risk. 67.6% of patients were male, and the median age was 64 (20–79) years. There was a difference in the number of CS mutations between the intermediate risk group and favorable risk group (P=.007), but not between the favorable risk and poor risk groups (P=.221) or between the intermediate risk group and poor risk group (P=.093). Increased number of CS mutations (≥ 5) was seen with equal frequency across risk groups and predicted for shorter overall survival in both univariate (HR=2.80; P=.039) and by multivariate Cox regression analysis (P=.001) independently from assigned risk group. There were no differences in age, gender, smoke, geographic, and different risk groups by multivariate analyses. Conclusion: Our study shows that ≥ 5clinically significant somatic mutations were associated with adverse outcomes and decreased survival, independent of risk groups and induction regimen. Thus, it may be a useful prognostic factor. This finding needs to be validated using a larger sample size.

Anti-thymocyte globulin (ATG) is a polyclonal antiserum introduced into clinical medicine more than 30 years ago. It induces a broad non-specific immunosuppression. In haematology, standard indications are severe aplastic anaemia and prophylaxis and treatment of graft-versus-host disease (GVHD) (after allogeneic transplantation). For aplastic anaemia, ATG from horses has been found to be superior to ATG from rabbits. In the situation of allogeneic transplantation, ATG lessens the risk of chronic GVHD but may not improve survival. There is current controversy regarding which patients benefit most from ATG and what the ideal dosage is. It is likely that in the coming years a more specific immunosuppressive will be developed that will minimize GVHD while maintaining the graft-versus-malignancy effect.

BackgroundImmune reconstitution represented by lymphocytes recovery post autologous hematopoietic stem cell transplantation (HCT) has been shown to improve outcome in relapsed/refractory lymphoma. This in turn has been linked to the infused peripheral blood autograft absolute lymphocyte count (ALC). Shorter time from last chemotherapy to collection (CTC) has been found to negatively ALC at pre-pheresis and leads to poor outcome in relapsed diffuse large B cell lymphoma. We hypothesized that in relapsed/refractory classical Hodgkin lymphoma, CTC affects the ALC on the date of collection.MethodRetrospective review of all adult patients who underwent autologous HCT (2007-2017) for histologically confirmed classical Hodgkin lymphoma at the Markey Cancer Center- University of Kentucky was done. Exclusion criteria included lack of salvage chemotherapy data, and those undergoing allogeneic HCT. Descriptive statistics were done for baseline characteristics. Median ALC was compared using Mann-Whitney U test. Kaplan-Meier curve with log rank testing was used to compare progression free survival (PFS) as well as overall survival (OS). Statistical analysis was done on SPSS 24.ResultsA total of 44 patients met the inclusion criteria. The median age was 34 (range 18-63) yrs. Male to female ratio was 2:1. The most common histology was nodular sclerosis (60%). The first line chemotherapy was ABVD in 83%. The most commonly used salvage regimens were Ifosfamide, Gemcitabine, and Vinorelbine (IGEV) in (55%) and Ifosfamide, Carboplatin and Etoposide (ICE) in (30%). Stem cell harvest was done from peripheral blood only using Cytoxan+GCSF (45%) and GCSF (18%). GCF+Plerixafor was used in 36% of the patients. Most of the patients (93%) collected a median of (5.5 × 106 CD 34 cells/kg) in 3 days. The Median CTC was 43 (range 26-91) days. Patients were then divided into two cohorts, Short CTC (6 weeks or less) and long CTC (more than 6 weeks). Baseline characteristics were balanced between the two groups (see table 1). There was no statistical difference in the median ALC (2.35 vs 1.47, p=0.226) (figure a), or CD34 cell count (6.06 vs 4.5, p=0.184) (figure b). At a median follow up time of 17 months (range 0-116 months), the progression free survival between the two groups were comparable (46 mo vs not reached, p=0.423) (figure c). Overall survival did not differ significantly (not reached for both groups, p=0.894) (figure d).ConclusionIn our single institutional experience, time to collection of peripheral stem cell harvest did not affect ALC or outcome following salvage chemotherapy followed by autologous HCT in classical Hodgkin lymphoma. Other prognostic markers need exploration to improve outcome of relapse/refractory classical Hodgkin lymphoma. Immune reconstitution represented by lymphocytes recovery post autologous hematopoietic stem cell transplantation (HCT) has been shown to improve outcome in relapsed/refractory lymphoma. This in turn has been linked to the infused peripheral blood autograft absolute lymphocyte count (ALC). Shorter time from last chemotherapy to collection (CTC) has been found to negatively ALC at pre-pheresis and leads to poor outcome in relapsed diffuse large B cell lymphoma. We hypothesized that in relapsed/refractory classical Hodgkin lymphoma, CTC affects the ALC on the date of collection. Retrospective review of all adult patients who underwent autologous HCT (2007-2017) for histologically confirmed classical Hodgkin lymphoma at the Markey Cancer Center- University of Kentucky was done. Exclusion criteria included lack of salvage chemotherapy data, and those undergoing allogeneic HCT. Descriptive statistics were done for baseline characteristics. Median ALC was compared using Mann-Whitney U test. Kaplan-Meier curve with log rank testing was used to compare progression free survival (PFS) as well as overall survival (OS). Statistical analysis was done on SPSS 24. A total of 44 patients met the inclusion criteria. The median age was 34 (range 18-63) yrs. Male to female ratio was 2:1. The most common histology was nodular sclerosis (60%). The first line chemotherapy was ABVD in 83%. The most commonly used salvage regimens were Ifosfamide, Gemcitabine, and Vinorelbine (IGEV) in (55%) and Ifosfamide, Carboplatin and Etoposide (ICE) in (30%). Stem cell harvest was done from peripheral blood only using Cytoxan+GCSF (45%) and GCSF (18%). GCF+Plerixafor was used in 36% of the patients. Most of the patients (93%) collected a median of (5.5 × 106 CD 34 cells/kg) in 3 days. The Median CTC was 43 (range 26-91) days. Patients were then divided into two cohorts, Short CTC (6 weeks or less) and long CTC (more than 6 weeks). Baseline characteristics were balanced between the two groups (see table 1). There was no statistical difference in the median ALC (2.35 vs 1.47, p=0.226) (figure a), or CD34 cell count (6.06 vs 4.5, p=0.184) (figure b). At a median follow up time of 17 months (range 0-116 months), the progression free survival between the two groups were comparable (46 mo vs not reached, p=0.423) (figure c). Overall survival did not differ significantly (not reached for both groups, p=0.894) (figure d). In our single institutional experience, time to collection of peripheral stem cell harvest did not affect ALC or outcome following salvage chemotherapy followed by autologous HCT in classical Hodgkin lymphoma. Other prognostic markers need exploration to improve outcome of relapse/refractory classical Hodgkin lymphoma.

Abstract Introduction: Low socioeconomic status (SES) has been shown to shorten survival in classical Hodgkin lymphoma (cHL). We sought to determine if differences in outcome of cHL varied according to SES and geographic setting in a population-based analysis. Methods: All adult patients diagnosed in 2000-2014 with cHL were collected through Kentucky Cancer Registry (KCR). Baseline SES and clinical variables were collected. Pearson Chi-square, log-rank, and cox regression tests were utilized. To minimize selection bias, cases with less than 6 months survival after cancer diagnosis were excluded. Results: A total of 1075 cHL patients were included, of which 605 (56.3%) were early stage vs. 405 (37.7%) were advanced stage. Most patients were under age 50 (63.2%) with a male predominance (55.4%). Nodular sclerosis was the most common histology (57.6%). Therapy consisted of chemotherapy alone in 729 (67.8%) patients, while 222 (20.7%) had received both chemotherapy and radiation. 633 (58.8%) lived in metro areas. Metro-area residents were more likely to come from non-Appalachian counties (93.7%), have more advanced education (76.8%) and have private insurance (60.1%) than non-metro-area residents (41.2%) (p<0.0001). There were no differences in age, gender, stage and histology at presentation according to geographic setting. Patients living in metro-area had a better OS by bivariate analysis (p=0.01) (Figure 1). A multivariate analysis identified age <50, early stage, privately insured, non-lymphocyte-depleted histology, and treatment with combined chemo-radiation as independent factors favoring an improved OS. Conclusion: Our study shows that despite the absence of differences in presenting clinical variables based on SES or geographic setting, significant discrepancies in outcome are observed. This disparity in outcomes may have been affected by differences in access to or quality of care delivery and may widen with the introduction of more costly novel interventions. Disclosures No relevant conflicts of interest to declare.

OBJECTIVES:Allogeneic hematopoietic stem cell transplant (HCT) continues to evolve with the treatment in higher risk patient population. This practice mandates stringent update and validation of risk stratification prior to undergoing such a complex and potentially fatal procedure. We examined the adoption of the new comorbidity index (HCT-CI/Age) proposed by the Seattle group after the addition of age variable and compared it to the pre-transplant assessment of mortality (PAM) that already incorporates age as part of its evaluation criteria. METHODS:A retrospective analysis of adult patients who underwent HCT at our institution from January 2010 through August 2014 was performed. Kaplan-Meier's curve, log-rank tests, Cox model and Pearson correlation was used in the analysis. RESULTS:Of the 114 patients that underwent allogeneic transplant in our institution, 75.4% were ≥40 years old. More than 58% had a DLCO ≤80%. Although scores were positively correlated (correlation coefficient 0.43, p < 0.001), HCT-CI/Age more accurately predicted 2-year overall survival (OS) and non-relapse mortality (NRM) in patients with lower (0-4) and higher (5-7) scores (52% and 36% versus 24% and 76%, p = 0.004, 0.003 respectively). PAM score did not reach statistical significance for difference in OS nor NRM between the low (<24) and high-risk (≥24) groups (p = 0.19 for both). CONCLUSIONS:Despite our small sample population, HCT-CI/Age was more discriminative to identify patients with poor outcome that might benefit from intensified management strategies or other therapeutic approaches rather than allogeneic HCT.

Introduction: Early stage classical Hodgkin lymphoma (cHL) carries good prognosis. Patients with B symptoms have been found to have unfavorable risk. Due to overall good outcome, studies are focusing on minimizing toxicity by omission of consolidative radiation. The aim of our study is to review outcome of this cohort in a population based analysis. Methods: All early stage (stage I and II) cHL adult patients diagnosed 2005-2014 were collected through Kentucky Cancer Registry (KCR). Patients reported to have had B symptoms (Unexplained fevers >38°C; drenching night sweats; or weight loss >10% of body weight within 6 months prior to diagnosis) were included in the current study. Baseline characteristics as well as survival outcome were compared between chemotherapy alone and combined chemo-radiation treatment. Pearson Chi-square, log-rank, and cox regression tests were used in the analysis. To minimize selection bias, events during the first 6 months of therapy were censored. 10-year survival data were then compared to Surveillance, Epidemiology and End Results (SEER) registry. Results: A total of 130 adult patients were included in the study; 76 patients got chemotherapy alone and 54 patients got chemo-radiation therapy. Median age was 35 (ranged 18-88). Most patients were younger than 50 year-old (76.9%). Most patients had nodular sclerosis (N=91) while 29 patients had unknown histology. There was no statistical difference in pretreatment features between the group receiving chemotherapy and those who received chemo-radiation (See table). There was no difference in 10-years overall survival between the chemotherapy group (73%) and chemo-radiation (80%) (p=0.830) (See figure). When adjusting for multivariate analysis, age younger than 50 was the only statistically significant variable affecting survival with a HR 0.17 (95% CI: 0.058-0.505). Only 4 (5.3%) patients developed second primary cancer in chemotherapy alone versus 1 (1.9%) in chemo-radiation therapy (p =0.4). Compared to overall survival in SEER database, younger patients ( Conclusion: Our study shows lack of benefit of the combined approach for the management of unfavorable prognosis classical Hodgkin lymphoma in a population based analysis. Younger patients had worse outcome when compared to SEER registry while older age group had worse prognosis with the available therapies and might benefit from alternative interventions. Download : Download high-res image (95KB) Download : Download full-size image Disclosures No relevant conflicts of interest to declare.

Introduction: Lenalidomide, bortezomib, and dexamethasone is a standard of care in the treatment of fit multiple myeloma patients due to high efficacy, with ORR exceeding 90% in the first-line setting (Richardson et al. 2010). The initial RVD regimen utilized a 21-day cycle with bortezomib administered IV 1.3 mg/m2 days 1, 4, 8, and 11; lenalidomide 25 mg administered days 1-14; and dexamethasone administered 160 to 320 mg per cycle. However, toxicity may be treatment-limiting, with bortezomib-induced neuropathy affecting up to 80% of patients. Modifications to RVD to optimize tolerance include reducing the bortezomib dose to a weekly schedule (1.6 mg/m2 IV or 1.3 mg/m2SC) and extending the cycle to 28 or 35 days (Broijl et al. 2016; O'Donnell et al. 2014). Notably, those regimens were studied in transplant-ineligible or relapsed/refractory patients. Based on regimens like CyBorD (which utilize weekly SC administration of bortezomib, are well-tolerated and are efficacious), we modified the standard 21-day RVD regimen to include 3 doses of weekly bortezomib in an attempt to preserve efficacy while minimizing toxicity. We present a retrospective analysis of both fit and transplant-ineligible patients treated at our institution using “Louisville RVD.”

Hematopoietic stem cells (HSC) have extensively been studied in mice to show their ability to self-renew and differentiate. By proving full blood regeneration after lethally-irradiated mice were injected with human HSC, the premise of stem cell transplant in humans started. This therapy opened the door to treatments ranging from marrow failures, sickle cells disease, and leukemias. To this day, it is unclear if a single hematopoietic stem cell is responsible for the entire marrow recover or if HSC have a finite lifespan. In the following case report, we will demonstrate the ability of HCS to engraft and restore marrow function in a human to the point of being able to serve as a donor for a subsequent successful donor for someone else. Case-Report: A 49 year-old man with AML achieved a CR with HD Ara-C induction in 2011. He refused consolidation chemotherapy at the time, but agreed to dose-intensive therapy with high-dose Ara-C followed by autologous hematopoietic cell infusion performed Jun 2012. He relapsed in Dec 2012 and was reinduced with HD Ara-C/idarubicin, achieving a second CR. Allogeneic hematopoietic cell transplant was proposed. Of note, the patient had been the HLA matched donor for his brother 29 years prior. After no suitable donors were found, his brother who had received his stem cells years back, was considered. Genetic markers that distinguished the patient and his brother were identified by testing buccal mucosa cells and proved the brother was still 100% donor (current patient). His brother agreed to be the donor after confirming he remained in remission. The patient received conditioning treatment followed by hematopoietic cells from his brother in Jul 2013. He engrafted with no GvHD. Bone marrow at 1 year post-transplant showed continued CR, 100% donor. This patient with an allogeneic-autologous hematopoietic cell transplant remains in remission 3 years out. Discussion: This unique scenario demonstrates how a recipient of a transplant was able to serve as a donor almost 30 years later. Not only did the second recipient have full engraftment from multipotent stem cells, but he has remained in a complete remission for the past 3 years. The capacity of long term hematopoietic reconstitution (LT-HSC) is indirectly demonstrated in this case. In addition, a case like this poses the question of whether transplant recipients who are in remission and currently not considered eligible donors, would be a good option in extreme circumstances. Another question that arises from this unique case and would merit further investigation is the length of remission achieved. Why was the first transplant (autologous) short lived yet the second transplant (using own stem cells harbored in his brother) achieve such a durable remission. Where these stem cells affected by the donor's bone marrow microenvironment while being in his brother?

Abstract Introduction We conducted a study to see the activity of hypomethylating agent (HMA) in relapsed refractory lymphoma. In parallel, we studied KIR expression and promoter methylation before and after treatment with HMA. Allele-specific stochastic KIR expression is maintained in mature natural killer (NK) cells by a combination of DNA methylation and histone modification, with DNA methylation being dominant. The aim of this part of the study is to understand how KIR gene expression changes in response to epigenetic therapy, so that we then can manipulate NK cells to optimize the effect of chemo-immunotherapy of neoplastic diseases. Methods Blood samples were collected from patients enrolled in an open-labeled phase I trial of the combination of azacitidine with cyclophosphamide, vincristine and rituximab in relapsed/refractory lymphoma. Regimen: Azacitidine on days one through five (starting at 25mg/m2), followed by oral cyclophosphamide at 300 mg/m2 on days six through nine, vincristine 1.4 mg/m2 day eight, and rituximab 375 mg/m2 day eight. Each cycle was to be repeated every 21 days, up to eight cycles if participants continued to benefit from therapy. Sampling: Between 10-20uL whole blood was collected from eligible patients before commencement of therapy and on Cycle 1 Day 5, Cycle 2 Day 1 and Cycle 3 Day 1. Peripheral blood mononuclear cells (PBMC) were isolated immediately ex vivo using Lymphocyte Separation Media (Lonza), and were divided into two aliquots: one for flowcytometry and the other for pyrosequencing (Epigendx, Hopkinton, MA). Samples from each time point were stained with fluorescently-labelled antibodies to CD3, CD16, CD56, CD14, CD20 and four different KIRs. For calculating the number of KIRs per NK cell, we used a gating strategy in which CD56dim NK cells are identified, and then the number of KIR expression is counted on those by sequential gating. The percentages of monocytes and lymphocytes among the PBMC population were also determined using CD14 and CD20 antibodies. Monocytes were further characterized according to their CD16 expression into Classical (CD14highCD16negative), Intermediate (CD14highCD16low), and Non-classical (CD14lowCD16high). Results PBMC from 10 patients were available for flow cytometric analysis and pyrosequencing. There were statistically significant differences (p=0.0089) in percent of DNA methylation determined by PBMC pyrosequencing, with significant decrease from Cycle 1 Day 1 to Cycle 1 Day 5 and then an increase from Cycle 1 day 5 to Cycle 2 Day 1. The decline in the percent of DNA methylation in PBMC at day 5 of cycle 1 of the chemotherapy was also found to be statistically significant (Figure 1; p=0.0202). There was a large variation in the types and numbers of KIR molecules expressed at baseline (Table 1) on CD56dim NK cells among the patients in the cohort studied, with no significant differences between the various time points and response to treatment. Despite an apparent trend of increasing percentages of non-classical and intermediate monocytes (at the expense of classical monocytes), the data was not statistically significant (Figure 2; p=0.06). We also noticed a trend of decreasing percentage of monocytes and increasing percentage of lymphocytes in the PBMC population with increasing treatment cycles. However, this was not statistically significant either. Conclusions KIR expression may play role in HMA induced tumor cell killing. In our study, no significant changes were seen in the number of KIR receptors on NK cells post-treatment with hypomethylating agent despite observing a reduction in global DNA methylation in PBMC. This could be due to a number of reasons, including the small size of the study, the rapid turnover of NK cells, or that the reduced methylation was not sufficient to induce changes in KIR expression. The significance of the increase in the number of non-classical and intermediate monocytes following treatment with HMA is not well understood and needs further investigation. (The study was supported by Celgene) Disclosures No relevant conflicts of interest to declare.

Six patients received platelet concentrate transfusions from their HIA-identical siblings. Platelet concentrates were administered either fresh, or after being frozen in 10% dimethylsulfoxide, at a slow controlled rate (1#{176}C/mm) or rapidly (-. 8#{176}C/mm) in the vapor-phase of a liquid nitrogen refrigerator. The median freeze-thaw loss was 1 3.5%. The mean 1 -hr and 20-hr corrected increments in platelet count were calculated for fresh platelet concentrates transfused before and after transfusion with

Multiple grading systems have been proposed to prognostically stratify patients by their acute graft versus host disease (aGvHD) following allogeneic hematopoietic cell transplant (HCT). Most recently MacMillan et al suggested a refined score, using the CIBMTR and Minnesota systems as framework. To our knowledge, this novel aGvHD risk-scoring system has not been verified in the context of T cell depletion as aGvHD mitigating strategy. Wet examined this in our patient population.