ALBERT

Description: Background:Leukemia cutis (LC) occurs in 10-30% of AML cases and may be a marker of poor prognosis. However, outside of monocytic AML (FAB M4/M5), no clinical or genetic predictors of LC are known. Recently, a number of somatic molecular mutations have been described in AML. Using amplicon-based next-generation sequencing (NGS) of a panel of recurrent, hematologic malignancy-associated mutations, we sought to determine potential molecular markers associated with the development of LC. Methods: A cohort of non-M3 AML patients treated at the University of Pennsylvania was identified in which NGS had been performed on either leukemic blasts obtained during clinical care or from the institutional tissue bank.Average read depth for 33 hematologic malignancy-associated genes was approximately 3000X, minimal depth was 250x, and reporting frequency cutoff for variants was 5%. Mutations were reported as pathogenic or variants of uncertain significance (VUS, further sub-classified internally as likely disease associated, VUS, or likely benign) based on the University’s Center for Personalized Diagnostics (CPD) review of publically available data; only pathogenic or likely disease-associated mutations were included in this analysis. A database maintained by dermatopathology was reviewed to identify cases of leukemia cutis at any time during the disease course. Independent dermatopathology review was obtained for indeterminate cases. Association between presence of each of the 3 most common molecular mutations (FLT3-ITD, DNMT3A, and NPM1) and development of LC was assessed by logistic regression, with adjustment for FAB M4/M5, as appropriate. The association between presence of a molecular mutation in different functional classes (tumor suppressors, activated signaling, chromatin modifiers, transcription factors, splicing machinery) and the development of LC was also assessed. Results:279 adult patients with AML with known molecular genotype were identified. Molecular profile was determined from AML diagnosis in (243, 88%) with the remainder undergoing assessment after prior therapy (relapsed or refractory). 56% were male with median age of 60 years (range 18-87) and median WBC count at diagnosis of 22 K/uL (range 0.4 -388 K/uL; 17% ≥100K/uL). The majority of patients had intermediate cytogenetic risk (12% favorable, 59% intermediate, 23% unfavorable, 6% unknown) and 41% of patients had FAB M4/M5 AML (9% unknown). The three most common mutations were NPM1 (29%), DNMT3A (25%), and FLT3-ITD (23%). NPM1mutations were enriched in patients with FAB M4/M5 AML (41% vs 23%, p=0.003). Leukemia cutis was present in 26 (9%) of patients. NPM1 mutant status was present in 14 of 26 cases of leukemia cutis (OR 3.17, 95% CI 1.40-7.20, p=0.006). No association was detected for LC and the presence of mutant FLT3-ITD (OR 1.27, p=0.613), mutant DNMT3A (OR 1.7, p=0.224), or a mutation in any functional class of AML mutations (all p-values NS). The impact of NPM1 mutant status remained significant after adjustment for association with M4/M5 AML (OR 3.91, p=0.005). As the histologic subtype of AML might modify the association between NPM1 mutations and leukemia cutis, we next examined the impact of NPM1 mutant status on patients with FAB M4/M5 AML and non-M4/M5 AML. Among patients with M4/M5 AML, 10/12 (80%) patients with LC were NPM1 mutant compared to 32/91 (35%) without LC suggesting that the presence of mutated NPM1 was significantly associated with the development of LC (OR 9.22, p=0.006). Among patients with non-M4/M5 AML, 3/9 (33%) of patients with leukemia cutis were NPM1 mutant compared to 32/142 (22.5%) without LC indicating no association in the non-M4/M5 subgroup (OR 1.72, p=0.461). Interestingly, M4/M5 AML was not associated with LC in the NPM1 WT cohort (OR 0.65, p=0.6). Conclusion: Using NGS, we identify a novel association between NPM1 mutation status and the presence of leukemia cutis, particularly within monocytic AML. Confirmation of these observations in a larger dataset is planned. Our data suggest potential cellular effects of NPM1 mutation affecting homing of leukemic blasts to skin and support the World Health Organization’s provisional classification of NPM1-mutated AML as a distinct biologic entity. Disclosures No relevant conflicts of interest to declare.

Description: Key Points Serum 2HG analysis by LC-MS can accurately identify patients with AML with and without IDH mutations. Oncometabolite testing of serum 2HG is indicated as a diagnostic, prognostic, and therapeutic monitoring tool in AML.

Description: Allogeneic blood or bone marrow transplant (BMT) can be a curative treatment for many children and adolescents with acute leukemia. With advances in unrelated donor transplant, others and we have shown that unrelated donor BMT can have similar survival to matched sibling BMT. There are several reports describing outcomes after matched related donor transplantation among various ethnic groups. Thus far, there are no published studies comparing outcomes among ethnic groups after alternative donor transplantation. Anecdotally, however, there have been concerns regarding outcomes among racial and ethnic groups, especially African-Americans. In order to address this question, we utilized our institutional database to analyze survival among children and adolescents receiving an alternative donor BMT at Children’s Hospital of Wisconsin from 1988-present. We compared survival in Caucasians and African-Americans undergoing unrelated donor and mismatched related donor transplantation (including haploidentical donors). One hundred and twenty four Caucasians underwent matched and mismatched unrelated donor transplantation compared to 11 African Americans. The 2-year probabilities of overall survival were significantly better for Caucasians at 53% (95% CI 44–62) than for African Americans, 18% (95% CI 2–45), p=0.01. Fifty-four Caucasians and 9 African Americans received mismatched family donor transplantation. Corresponding probabilities of overall 2-year survival were 38% (95% CI 25–51) and 30% (95% CI 5–64), respectively. Interestingly, our data show no statistically significant difference in survival after mismatched related donor transplantation between the Caucasian and African-American cohorts. Our data should be interpreted cautiously as the number of African Americans transplanted at our institution is few. Additionally, our analysis is limited by our inability to adjust for disease status at transplantation, HLA disparity and other known risk factors that may impact survival. Nevertheless these observations from a single institution cannot be ignored and warrant further analysis in a larger cohort such that outcomes after transplantation may be adjusted appropriately for relevant risk factors. We believe that a national database/registry study will have the numbers necessary to answer the questions that need to be asked regarding outcomes with alternative donor transplantation in the African-American population. We also believe that as cell processing and supportive care technologies improve mismatched family member transplantation outcomes, these advances could have a significant impact in improving leukemia-free survival for African-American children and adolescents.

Description: Abstract 2248 HPC, Apheresis products collected for autologous use must be cryopreserved prior to hematopoietic progenitor cell transplantation. Improved recovery of cryopreserved cord blood cells was demonstrated by Rubinstein et al. (PNAS:92, 10119, 1995) when the thawed cells were diluted with an equal volume of a solution containing 2.5% (wt/vol) of human serum albumin (HSA) and 5% (wt/vol) Dextran 40, the cells centrifuged, the supernatant removed, and the cells restored to their original volume with the same Dextran/Albumin solution as compared to thawing the product without dilution or washing. This method has the additional advantage of allowing cells to be thawed under controlled conditions within the laboratory and greatly reduces reactions to DMSO. Several variations in this procedure have been published including one employed at our center that has been used to thaw all cryopreserved products since 1996 in which 5% HSA and 10% Dextran 40 was added sequentially to the thawed cells each at 1/2 the frozen volume. The cells and solution were mixed and after 10 minutes of incubation the bag was filled to capacity (300 mL total) with Dextran 40 prior to centrifugation. Reconstitution to the original volume for infusion was also by sequential addition of HSA and Dextran 40 (Method 1). A variation of this method is to use a premixed solution of 2.5% HSA and 5% Dextran 40 both before and after centrifugation (Method 2). A third method, which is currently recommended by the Blood and Marrow Transplantation Clinical Trials Network (BMT-CTN) for thawing HPC, Cord Blood is similar to method 2 but uses a higher concentration of HSA resulting in 4.2% HSA in the premixed Dextran/Albumin solution (Method 3). We directly compared the three methods using autologous products (n=3) that were no longer needed for infusion and that were each frozen in multiple bags to assess viability (7-AAD method), along with recovery of viable total nucleated cells (TNC), CD3+ T-cells, and CD34+ cells. No significant differences were seen for any of the outcomes between method 2 and method 3. However, both method 2 and method 3 resulted in better overall viability, viable TNC recovery and viable recovery of CD34+ cells and CD3+ T-cells than method 1. Starting in January 2010 we modified our standard thawing procedure to method 3 (to conform to CTN requirements) and have compared the results of 96 bags thawed using this method with 173 bags thawed using method 1. Thaw viability (Trypan blue method) using method 1 was 72.7%±11.8% compared to 77.5%±9.1% for method 3, p=0.0005 and viable cell recovery was 62.1%±12.6% versus 68.5%±10.2%, p=

Description: Allogeneic hematopoietic progenitor cell transplantation (HPCT) is the optimal treatment for several hematological malignancies, aplasia or other disorders of the hematopoietic or immune system. Post HPCT, patients undergo a prolonged period of immune incompetence during which they are highly susceptible to viral and fungal infections. Epstein Barr Virus (EBV), Adenovirus (Ad) and Aspergillus fumigates are among the most common and lethal infections during this period. We have described the use of overlapping pools of pentadecapeptides pulsed onto monocyte-derived dendritic cells (DC) to generate cytolytic T-cell (CTL) lines specific for an A. fumigatus-derived antigen, Asp f16 (Ramadan et. al., 2005, J Clin Immunol139:257; Ramadan et. al., 200 J Clin Immunol 140:81). We have further demonstrated in 5 donors, the generation of CTL lines recognizing peptides contained within the conserved region of the Adenovirus hexon protein using a similar approach (Keever-Taylor, et. al, Cytotherapy 8, Suppl 1, 122). To reduce the need for large numbers of DC to prime and expand the CTL cultures, we modified our procedures to use peptide pool (pp)-pulsed EBV-transformed B lymphoblastoid cell lines (BLCL) as antigen presenting cells (APC) following 2–3 rounds of initial priming with pp-DC. This approach results in lines that are predominately reactive to Asp f16 or Ad hexon, and which are moderately reactive to EBV. We expanded our studies to determine the feasibility of generating CTL lines that recognize all three infectious agents. Peripheral blood lymphocytes (PBL) from donor RD0309 were primed weekly ×3 with autologous DC (10:1 PBL to DC ratio) pulsed with a pool of both Asp f16 (104 individual peptides) and Ad hexon (105 individual peptides) pentadecapeptides, then switched to Asp/Ad-pulsed BLCL as APC. The cultures were screened one week following restimulation for lysis of non-pulsed (np-BLCL) and Asp f16-pp-pulsed (Asp-BLCL) and Ad hex-pp-pulsed (Ad-BLCL) BLCL targets. Weak reactivity to Ad-BLCL was detected after 3 primings with DC alone that increased following one round of Asp/Ad-BLCL priming. Asp f16-specific CTL activity was not clearly detected until one week after 3DC plus 3BLCL primings, at which time there was also clear reactivity to np-BLCL targets (26.4% np-BLCL, 37% Asp-BLCL, and 53.9% Ad-BLCL at 50:1), indicating CTL activity to all three infectious agents. With an additional priming, Aspergillus specific reactivity exceeded that towards Adenovirus and EBV-specific reactivity declined. These data show that a pool of overlapping peptides from two different antigens presented on DC followed by BLCL as APC can result in multi-specific cell lines that can be potentially used in adoptive immunotherapy protocols for the treatment or prevention of common opportunistic infections following HPCT.

Description: We demonstrated that partially T-cell depleted unrelated donor HSCT for Severe Aplastic Anemia (SAA) is a reasonable treatment option for children and young adults who fail immune suppression therapy. (Margolis et al., 1996, Br J Haematol.)We now report, long term follow up data for 40 patients transplanted for SAA between the years 1986 to 2002. The patient group consisted of 22 males and 18 females, ranging in age from 0.5–24.3 (median 8.5) yrs. Retrospective molecular HLA typing shows that donors were matched for nine patients, and mismatched for 31. Patients were conditioned with cytosine arabinoside, cyclophosphamide, and total body irradiation, as previously described. Some patients additionally received ATG to promote engraftment. The marrow product underwent partial T-cell depletion using an antibody and complement process as described. in the original report. GVHD prevention was with cyclosporine. Three patients did not engraft. All three with non-engraftment died within 60 days of BMT from infectious and hemorrhagic complications. Since employing ATG as part of the conditioning regimen, all patients have engrafted. Of the 37 patients who engrafted the median time to an ANC〉500 was 16 (range 8–25) days. Eight patients developed Grade II AGvHD, 1 grade III and 2 grade IV. Of 29 evaluable patients, 12 developed limited chronic GVHD, and 3 developed extensive CGvHD. Twenty-one patients are currently surviving with a follow-up of 4 to 19 yrs. (median 12.7 yrs.). Overall survival is 52% at 12 yrs. Of the 19 patients that died, causes of death included infection n=7, (PCP n=1, CMV n=2, Aspergillus n=1, Adenovirus n=2, PTLD n=1); GVHD n=2; Graft failure n=3; Multiorgan system failure n=2; ARDS n=1; Hemorrhage n=2; VOD n=1; Secondary malignancy n=1 (Hodgkin’s disease n=1). Of the 21 surviving patients, all patients have a Karnofsky score ≥ 90%. The late effects in our survivors include two secondary malignancies (osteosarcoma and vaginal carcinoma in situ); cataracts n=11; growth retardation n=11; gonadal dysfunction n=6; hypothyroidism n=5; cognitive problems n=4; musculoskeletal problems (AVN, osteoporosis) n=7; hyperlipidemia n=3; and renal disease n=2. One patient had a subsequent pregnancy that resulted in a preterm delivery at 26 weeks and a neonatal death. Our experience, now with long follow-up, shows that an intensive conditioning regimen to prevent graft rejection, coupled with partial T-cell depletion of an unrelated donor bone marrow graft to decrease the risk of GVHD, provides for durable survival with an acceptable incidence of acute and chronic GVHD. The relatively low incidence of GVHD is notable in view of the number of patients with donors who had identified HLA disparity. However, there are long-term risks associated with this regimen including secondary malignancies, delayed growth and development, metabolic problems, and musculoskeletal problems. We are encouraged by the recently reported short-term results using regimens for this disease which avoid or limit the use of TBI. Recognizing that many patients have donors with HLA disparity, which increases the risks of graft rejection and GVHD, we believe that combining partial T cell depletion with advanced immunomagnetic methods of graft manipulation and a fludarabine based regimen may allow us to balance the risks of graft rejection, GVHD, and late-effects that are unique to patients with SAA.

Description: Introduction: Salvage immunochemotherapy (IC) followed by high-dose chemotherapy with autologous stem cell transplantation (autoSCT) is standard-of-care second-line therapy (2L) for patients with relapsed or refractory (R/R) diffuse large B cell lymphoma (DLBCL) deemed fit for autoSCT as per the CORAL study (J Clin Oncol. 2010 Sep 20;28(27):4184-90). Optimal therapeutic management of patients with R/R DLBCL who are autoSCT-ineligible is unknown. Here we describe the real-world outcomes of patients with R/R DLBCL who receive palliative intent 2L therapy in community and academic settings and do not receive autoSCT. Methods: This analysis includes de-identified patients from the nationwide Flatiron Health electronic health record-derived database with a histologic diagnosis of DLBCL and R/R disease after frontline IC who do not undergo autoSCT and receive treatment with either bendamustine-based therapy, gemcitabine-based therapy, lenalidomide, or ibrutinib. Patients receiving rituximab/ifosfamide/carboplatin/etoposide (R-ICE) and high-dose cytarabine-containing second-line therapies were excluded. Event free survival (EFS) was defined as the interval between the start of current therapy and start of subsequent therapy if needed, last follow-up on current therapy, or death on therapy. Overall survival (OS) was defined as the time between start of current therapy and death or last follow-up while alive. Results: A total of 250 patients were eligible for inclusion in 2L. Eight patients received autoSCT after gemcitabine therapy and were excluded from this analysis. Clinicopathologic characteristics at time of diagnosis include 56% male, 87% age 〉60, 55% ECOG performance status 〉1, 87% stage III-IV disease, 78% IPI 〉2, 56% germinal center (GCB) of those with cell of origin testing performed, 9% cMYC rearrangement positive when tested, and 29% transformed from indolent disease. A total of 106, 78, 36, and 22 patients received bendamustine, gemcitabine, lenalidomide, and ibrutinib, respectively. For all patients, median EFS was 5.1 months and median OS was 14.3 months in 2L. Median EFS was 7.6, 2.4, 9.1, and 4.2 months, and median OS was 16.0, 9.4, 16.3, and 11 months for bendamustine, gemcitabine, lenalidomide, and ibrutinib in 2L, respectively. Patients receiving bendamustine and lenalidomide demonstrated significantly improved EFS compared to those receiving gemcitabine (p=0.001 and 0.01, respectively), see Figure 1. We observed no difference in EFS (p=0.40) or OS (p=0.89) between lenalidomide and bendamustine in 2L. Univariate analysis demonstrated receipt of gemcitabine, ECOG PS〉1, and IPI 〉2 to have statistically significant increased hazard for treatment failure and ECOG PS〉1 to have an increased hazard for death in 2L relative to the reference group. Multivariate analysis demonstrated receipt of gemcitabine (HR 1.57, p=0.03 95% CI: 1.04 - 2.37) and ECOG PS〉1 (HR 1.61, p=0.02 95% CI: 1.09-2.38) were associated with an increased hazard for treatment failure in 2L. Median EFS for patients on lenalidomide was 6.7 and 8 months (p=0.26), and median OS was 13.9 and 12.2 months (p =0.48) for patients with nonGCB and GCB cell of origin, respectively. Conclusions: For patients with R/R DLBCL treated with palliative therapy in the 2L, bendamustine- and lenalidomide-based therapies resulted in significantly longer EFS compared to gemcitabine therapy. Although we cannot exclude the possibility that some patients received gemcitabine in 2L with the original intent to proceed with autoSCT, this does not contest our results as this therapy remains inferior to bendamustine and lenalidomide even if given to a potentially more fit patient population. Analysis shows no difference in outcomes by cell of origin if receiving lenalidomide in 2L. These findings may serve as benchmarks for outcomes following receipt of these therapies in the non-investigational setting and suggest both bendamustine and lenalidomide may be considered reasonable standard-of-care therapies for patients unfit for autoSCT in the 2L setting. Figure 1 Disclosures Landsburg: Celgene: Membership on an entity's Board of Directors or advisory committees; Triphase: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Curis, INC: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Curis, INC: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Seattle Genetics: Speakers Bureau; Seattle Genetics: Speakers Bureau; Triphase: Research Funding; Takeda: Research Funding; Takeda: Research Funding. OffLabel Disclosure: Outcomes with lenalidomide and ibrutinib in patients with relapsed/refractory DLBCL will be discussed.

Description: Abstract 4306 VOD is a serious and potentially life-threatening complication of HPCT as a result of liver injury from the effect of chemotherapy and/or radiation. The reported incidence rate in pediatric HPCT patients varies widely from 5% to 40%. Previous studies have shown the beneficial effects of post-transplant pharmacological therapies such as ursodeoxyholic acid (ursodiol), heparin, and defibrotide at preventing VOD. However, the combined effect of heparin and ursodiol prophylaxis in preventing VOD in pediatric patients has yet to be determined. This study evaluated retrospectively whether there was a benefit of such combined therapy in pediatric HPCT patients. Our center adopted as standard practice for all HPCT patients the initiation of low dose heparin at 4 units/kg/hour with the commencement of conditioning for HPCT until day +28 post transplant. In 2003, we combined ursodiol 10 mg/kg TID to start with HPCT conditioning and to continue until day + 100 post transplant with low dose heparin through day + 28 for all pediatric HPCT patients. We performed a retrospective chart review and compared the characteristics and the incidence of VOD in patients who underwent transplantation from 1996-2002 and received heparin alone compared to 2003-2008 when the patients received the combination of heparin and ursodiol prophylaxis. Patients were identified through medical records with the ICD diagnosis of VOD. The medical records were reviewed and those patients who did not meet the Baltimore criteria for the diagnosis of VOD were excluded. Only patients who developed VOD with their first transplants were included. Group I = Heparin (216) Group II = Heparin + Ursodiol (220) Allogeneic 187 (86.5%) 160 (72.7%) Autologous 29 (13.5%) 60 (27.3%) Median Age 9 yrs 8 yrs Male 123 (57%) 135 (62%) Female 93 (43%) 85 (38%) Non-malignant 34 (15.7%) 50 (22.8%) Hematologic malignancy 143 (66.2%) 109 (49.5%) Non-hematologic malignancy 39 (18.1%) 61 (27.7%) # VOD 13 5 The 100 day incidence of VOD was 0.0605 (SE 0.01618) in group 1 and 0.0227 (SE 0.01002) in group 2. The difference is 0.0377 (SE 0.0190) and based on a standard normal distribution with a p = 0.0473. The estimated risk of VOD for patients receiving Heparin + Ursodiol is 0.94 (risk or hazard ratio) that of the risk with Heparin alone, with a 95% confidence interval of (0.918, 0.960). This represents about a 6% reduction in risk for those receiving Heparin + Ursodiol. The day 100 survival in the VOD patients was 6 out of 13 in group 1 and 3 out of 5 in group 2. In conclusion, low dose heparin and ursodiol prophylaxis appears to be an effective strategy in VOD prevention in pediatric patients. The combination appeared to be more effective than heparin alone. However, this study is limited in that it is retrospective in nature. Disclosures: Off Label Use: heparin and ursodiol as VOD prophylaxis.

Description: Key Points Alternative donor HCT can be performed in patients with FA without using radiation. All 26 patients younger than 10 years of age undergoing HCT for marrow failure using lower-dose busulfan-containing regimen survived.