ALBERT

Description: Abstract 2596 Background: Patients with acute myeloid leukemia (AML) receiving induction or salvage chemotherapy often undergo bone marrow evaluation around day 14 to determine early treatment response and guide decision-making regarding need for immediate re-induction therapy. However, the parameters that define whether or not to initiate such therapy are seemingly not always objective. Thus we sought to define what factors influence physicians' decisions to pursue a second course of induction chemotherapy in AML. Methods: We retrospectively reviewed 211 patients with newly diagnosed, relapsed, or refractory AML who received induction chemotherapy at the University of Washington /Fred Hutchinson Cancer Research Center from January 2008 to May 2012. 178 had a marrow aspirate and/or biopsy between days 13 to 17 after start of chemotherapy and were included in our “day 14” analysis. Bone marrows were assessed both morphologically and by multi-parameter flow cytometry (MFC). We used the Wilcoxon signed rank test and Fisher exact test to compare patient age, pre-treatment cytogenetics (SWOG criteria), newly diagnosed vs. relapsed or refractory AML, day 14 marrow cellularity, day 14 blast count by morphology or MFC, and induction regimen given on the first course (ara-C-containing vs. not) among patients who did and did not receive a second course of induction chemotherapy within 1 week of the “day 14” marrow. The second course could either be the same or different than the first course. Results: 81% of the 178 patients had fewer than 10% blasts by morphology and MFC on day 14. None of these patients received course 2 within 1 week. 34 patients (19%) had greater than 10% blasts on the day 14 marrow by morphology or flow cytometry, of whom 18 (53%; 95% confidence interval 36–70%) received course 2 prior to day 21 and 16 (47%; 95% CI 30–64%) patients did not. As the decision of whether or not to begin course 2 within 1 week varied once blasts counts were 〉10%, we analyzed the 34 patients with 〉10% blasts between day 13–17 to assess what factors influenced the decision to begin a second course of treatment. The median age in those who received and did not receive course 2 within 1 week was 60 and 50.5, respectively (p=0.269). The fraction of newly diagnosed AML (as opposed to relapsed or refractory disease) in the two groups was 83% and 69%, respectively (p=0.429). The blast count by morphology was 69.5% in those who did vs. 24.4% in those who did not receive a second course within 1 week of the day 14 marrow (p=0.001) and by flow cytometry were 63% and 10%, respectively (p10% blasts in a day 14 marrow and 56% who received a higher intensity regimen on course 1 did not begin a second course during the next week despite National Comprehensive Cancer Network (NCCN) guidelines that they do. We are studying whether this reflects objective patient and marrow characteristics, such as change in blast count from pre-treatment to day 14 marrow, or other more subjective factors in clinical practice. Disclosures: Becker: Sanofi: Research Funding.

Description: Lithium (Li) is a GSK3 inhibitor that induces differentiation and anti-proliferation of AML cells in vitro. Pre-clinical studies demonstrated GSK3 inhibition leads to increased expression of the receptor of tretinoin, the retinoic acid receptor. We conducted a phase I trial of Li in combination with tretinoin for non-promyelocytic AML based on pre-clinical studies demonstrating synergistic AML cell differentiation using the two-drug combination. We report laboratory correlative data demonstrating AML cell differentiation using the combination of Li and tretinoin. Methods. 12 relapsed or refractory AML patients were enrolled. Li was administered at 300mg three times daily (twice daily in patients age ≥60) continuously and adjusted to meet the target Li plasma concentration of 0.6-1mEq/L; tretinoin doses were 22.5 mg/m2/day (dose level 1) and 45 mg/m2/day (dose level 2) administered on days 1-7 and 15-21 of a 28-day cycle. Li was given for a 3-day lead-in period prior to cycle 1. 7 patients were evaluable for laboratory correlative studies. Blood and/or bone marrow samples were collected at baseline, day 1 and day 8 of cycles 1 and 2 and at the end of the study period. Specimens were processed only if ≥5% leukemic cells were available for analysis. Immunophenotyping was performed by multi-parameter flow cytometry using fluorochrome-labeled antibodies to CD11b, CD14 and CD15 as markers for monocytic and/or granulocytic differentiation. Cells were concurrently stained for CD34, CD117 and CD38 for identification of the leukemic blast and stem cell population (CD34+CD38-). After fixation and permeabilization using a formaldehyde-based solution, intracellular staining was performed for phosphorylated Serine-9 GSK3β (pGSK3β) as a marker for GSK3 inhibition. Li directly binds GSK3 and causes reversible enzyme inhibition by Ser9 phosphorylation. Results. The target serum concentration of Li was achieved in all patients and found to inhibit GSK3 in leukemic cells. pGSK3β measured by mean fluorescence intensity (MFI) increased by a median of 1067 (168-1738) by day 30 in 4 patients with samples available for analysis; the earliest increase in pGSK3β MFI occurred at day 1 after the Li lead-in period. Six subjects (86%) demonstrated differentiation as defined by ≥10% increase in a cell population expressing at least one of the surface markers associated with monocytic or granulocytic differentiation, with median increase 27.4% (range 10.0-59.2%). Maximum increase occurred at median day 30 (range 8-60) of the study period. Three patients (50%) exhibited differentiation in both the CD34+ or CD117+ blast population and in non-blast cells. Two subjects (33%) had increased differentiation in the non-blast population only, and 1 (17%) had increased differentiation in the blast population only. Three patients had clinically stable disease (defined as no increase in blasts for ≥4 weeks from study start) and showed a median increase in differentiated cells of 27.4% (17.2-37.6%) compared to a 14.8% (10.0-19.6%) increase in the 3 patients with progressive disease (p=0.44). Four patients had day 60 bone marrow available for analysis; in 3 patients, there was a median of 29.3% (range 12-32%) increase in cells expressing at least one differentiation surface marker in the CD34+ or CD117+ blast population. In 6 subjects with evaluable sample, a median relative reduction of 67% (37-98%) of the AML stem cell population (CD34+ CD38-) was seen in the blood at day 8 to 60. Conclusions. The combination of lithium and tretinoin induces differentiation in leukemic cells. Unlike most cytotoxic AML therapies which target rapidly dividing cells, this strategy may be effective in targeting the AML stem cell population. Response to differentiation therapy was variable, suggestive of the heterogeneity of the disease. Li induced a modest effect on GSK3 inhibition at clinically achievable plasma concentrations. Though this study demonstrated the ability to inhibit GSK3 in leukemic blasts and induce differentiation, higher levels of GSK3 inhibition are likely necessary for greater clinical efficacy. As Li concentrations cannot be increased significantly due to drug toxicity, future studies investigating the use of more potent GSK3 inhibitors in AML are needed to further investigate this strategy. Disclosures Wald: Invenio Therapeutics: Equity Ownership.

Description: Background: Patients with acute myeloid leukemia (AML) admitted to the ICU have high in-hospital mortality, ranging from 40-70% (Halpern A, JAMA Oncology, 2017, 3; 374 and Thakkar SG, Cancer, 2008, 112; 2233). A model that considers AML-specific as well as ICU-specific variables could be of great value to identify critically ill patients likely to survive beyond hospital discharge and would help providers frame goals of care discussions. The AML Composite Model (AML-CM) was recently developed to predict early as well as late mortality after diagnosis of AML (Sorror ML, JAMA Oncology, 2017, 3:1675). The AML-CM incorporates AML cytogenetic risk, age, and comorbidity burden. It is unknown whether the AML-CM scores calculated at diagnosis of AML could add prognostic value to variables collected at the time of admission to ICU. To this end, we investigated the predictive value of AML-CM scores at diagnosis in addition to other risk factors including traditional ICU markers of illness severity collected from the time of ICU admission for prediction of 90-day mortality in patients with AML. Methods: This is a retrospective study of 218 patients with AML admitted to an ICU at one of two affiliated referral hospitals at any time during or after the initiation of chemotherapy between 2008 and 2017. We used factors from three time points: 1) initial diagnosis: sex, race, and AML-CM; 2) time of ICU admission: age, presence of relapsed or refractory disease, history of hematopoietic stem cell transplant (HCT), presence of neutropenia with ANC

Description: After allogeneic hematopoietic cell transplantation (HCT), a relatively small number of donor hematopoietic cells must reconstitute the entire recipient hematopoietic system, while the donor is left with a near normal pool of hematopoietic cells. We hypothesized that the increased replicative demand on donor cells in the recipient after allogeneic HCT will accelerate telomere shortening and magnify the genetic alterations that are associated with normal aging, including clonal hematopoiesis. We aimed to compare mutation frequency in genes associated with clonal hematopoiesis of indeterminant potential (CHIP) and myeloid diseases between donors and recipients, with a focus on transplant pairs with older donors. We obtained contemporary blood samples from 10 related donor-recipient pairs surviving a median of 36.6 years (range 6.6-45.7 years) after HCT. Information on donor-recipient pairs is summarized in Table 1. Variant libraries were prepared from bulk peripheral blood mononuclear cells (PBMCs) using Archer Dx VariantPlex Myeloid panel of 75 myeloid disease-associated genes (ArcherDx, Boulder, CO). Sequencing was completed on the Illumina HiSeq system. Variants with allelic frequency (AF) ≥2% were detected in donors (median number of variants 50.5, range 35-107) and recipients (median number of variants 50, range 32-109). In all pairs, there was significant overlap in the variants detected, although some were unique to donors or recipients (Figure 1). Two of the 3 donor-recipient pairs with 〉25 years difference in donor age (84 and 71 years) and recipient age (47 and 42 years) showed an increase in the shared variant AF in the recipient in DNMT3A (nonsense and frameshift mutations) of 5 to 18% and 5 to 16%, respectively. All other shared variants in CHIP-associated genes (DNMT3A, ASXL1, TET2) detected in 6 pairs did not show significant difference in AF (Table 2). Among other shared variants of non-CHIP genes, 7 pairs showed mutations with ≥5% difference in AF, but the difference was small (mean difference 5.3%, range 4.5-7.4%) (Table 3). In conclusion, our results suggest that even decades after transplantation and high replicative demand, most donor variants, at the level of detection of this assay, do not preferentially expand in the recipient. Donor-recipient pairs with older donors and ~30-year difference in age with the recipient showed a modest expansion in DNMT3A clones. Future studies will compare contemporary samples to historical samples from the time of transplant. Disclosures Radich: Novartis: Other: RNA Sequencing; TwinStrand Biosciences: Research Funding.

Description: Hematopoietic cell transplantation (HCT) is used in the treatment of hematologic malignancies (HM). Clonal hematopoiesis of indeterminate potential (CHIP) is the age-associated process whereby healthy individuals accumulate low-frequency clones driven by mutations in genes recurrently mutated in myeloid cancers; CHIP is associated with an increased risk of HM and cardiovascular disease. It has been hypothesized that CHIP clones present in an HCT donor might disproportionately proliferate in the recipient due to a relative growth advantage during immune reconstitution. Here we use Duplex Sequencing (DS) to investigate, years after HCT, whether the clonal makeup of CHIP in prior donors differs from that of the donor-derived hematopoietic system long after allogeneic transfer to a recipient. DS is an ultra-sensitive NGS error-correction technology that relies on sequencing of the individual paired strands of original source DNA molecules to dramatically reduce technical background compared to other NGS methodologies. Here we performed DS on 10 pairs of donor/recipient peripheral blood DNA samples collected 7-46 years post-transplant, as well as DNA from a healthy young control. We used a sequencing panel comprising 29 genes recurrently mutated in acute myeloid leukemia (AML) and generated mean DS molecular depth of 27,580x per sample, with maximum DS depth reaching 45,402x. We detected an average of 356 and 283 total variants per donor and recipient, respectively, and 462 total variants in the control. These included 291 single-count exonic variants (SEV) in the control and an average of 215 SEVs in donors (Fig. A). To enrich for CHIP (clonal) variants, we filtered out single-count (non-clonal) events, as well as intronic (non-splice site) variants and germline SNPs. Whereas the young control had only 5 clonal exonic variants (CEV), meaning nearly all of the variants in the control were SEVs, SNPs or intronic (Fig. A,B), HCT donors and recipients respectively averaged 44 and 38 CEVs. 91% of CEVs were non-synonymous (Fig. B), which is higher than expected by chance. The average age of the donors was 36 years old at the time of HCT (range 12-84), and the oldest donor (M1) had the most CEVs. 24/29 genes in the panel exhibited CEVs in the control or HCT donors (Fig. C). DNMT3A, TET2, BRINP3 (FAM5C) and ASXL1 had the greatest number of CEVs and this imbalance was not explained purely by gene size. The young control sample had an overall somatic mutation frequency (MF) of 4.7x10-7. HCT donors and recipients had average MFs of 1.0x10-6 and 1.7x10-6, respectively. 154 CEVs were shared in at least 1 donor-recipient pair, with the majority present at variant allele frequencies (VAFs) 10-fold, all in DNMT3A, TET2 and RAD21. Only 1 CEV decreased 〉10-fold: TP53 R273H (4.9x10-4 to 4.2x10-5). Analysis of the same 10 donor-recipient pairs using a commercial sequencing service found that, with a VAF cutoff of 2% and filtering for SNPs, only 7 shared CEVs could be detected that increased or decreased in any recipient. Our ultra-high accuracy DS data indicates that the structure of CHIP is more polyclonal than has been previously reported. In a handful of older healthy donors we detected dozens of concurrent CHIP mutations, with a relative gene distribution nearly identical to that previously reported in studies on thousands of patients using less sensitive method. We observed a similar age-dependent effect. Furthermore, our findings indicate that the clonal dynamics of CHIP clones between HCT donors and recipients are substantially more complex than previously known. Nearly all somatic variants shared between HCT donors and recipients were observed at