ALBERT

Description: We used next generation sequencing (NGS) of the immunoglobulin genes to evaluate minimal residual disease (MRD) in 153 specimens from 32 patients with newly diagnosed adult B cell ALL enrolled in the phase II SWOG S0333 multi-center study. We used the clonoSEQTM assay developed by Adaptive Biotechnologies that detects 1 leukemic cell in a background of 1 million nucleated cells and focuses in the B cell receptor (Ig). Initially, a set of pre-study specimens was sequenced in order to identify the precise sequence of the VDJ or DJ fragments. Clones representing more than 5% of the total repertoire of IgH molecules profiled were considered potentially leukemic. The follow-up specimen IgH repertoire sequences were compared to the diagnostic clonal ones and the leukemic marker sequence(s) previously identified were searched for explicitly. At least one Ig clonotype was detected in 29/32 (91%) cases analyzed. The 3 remaining cases were reviewed, and in 2 cases the specimens available for NGS had been reported as having no blasts by morphology. The leukemic clonal sequence was a complete VDJ rearrangement in 17/32 patients (53%), an incomplete DJ rearrangement in 8/32 patients (25%), and in 3/32 cases both VDJ and DJ rearrangements coexisted. One patient had a kappa light chain rearrangement. 17/32 (53%) cases contained more than one IgH rearrangement at diagnosis (median=2, range: 1 - 4). One of our patients is a potential case of therapy driven clonal selection. He presented at diagnosis with two related clones, one representing 75% of VDJ sequences and the second one 18%. At relapse, the second clone was responsible for most of the VDJ sequences (95%). The NGS results were compared to the MRD results detected by multiparameter flow cytometry (MFC) in 66 specimens analyzed by both methods. The concordance between the methods in the qualitative determination of the presence or absence of leukemia was 82% (54/66). In 12 specimens (18%) MRD was detected by sequencing but not by MFC. One specimen had MRD detected at very low levels by MFC and was negative by NGS. Our study includes 54 paired bone marrow (BM) and peripheral blood (PB) specimens. The median values of leukemia detected by NGS were 6-fold higher in BM than PB (range: 0.38 - 821-fold). Twenty-five pairs show no detectable MRD in either specimen. MRD was still detectable in 20 of the remaining 29 PB cases (for one of the pairs the BM specimen was negative). In 6/9 (67%) pairs of samples with disease detectable in BM but not in PB by NGS, no MRD was detected by MFC in the BM specimen. Lastly, outcome analysis was conducted in 21/32 patients with specimen available for MRD studies at the time of registration to second induction. Patients without MRD by NGS had a 5-year relapse free survival (RFS) of approximately 80%, while patients with MRD positive by both NGS and flow have the poorest outcome (p = 0.003) (see Figure). Patients with MRD detectable only by NGS have and intermediate RFS (p = 0.078, and p = 0.04 when compared to patients with MRD negative by both techniques, and patients with leukemia detected both by NGS and flow respectively). These results suggest that MRD detection by immunoglobulin gene sequencing is a very sensitive technique, and may identify patients with an excellent survival. Moreover, the increased sensitivity of the method may allow peripheral blood testing to supplement routine marrow sampling for MRD determination. Figure 1 Figure 1. Disclosures Williamson: Adaptive Biotechnologies: Employment, Equity Ownership. Kirsch:Adaptive Biotechnologies: Employment, Equity Ownership. Robins:Adaptive Biotechnologies: Consultancy, Equity Ownership.

Description: Abstract 2191 Background: Treatment protocols for newly diagnosed AML typically use age (often 60 years) alone to restrict eligibility to either younger or older patients. Implied in this practice is the assumption that age is the principal predictor of therapeutic failure in AML due to either early treatment-related mortality (TRM) or resistance to therapy in patients who do not incur TRM. Yet, clinical observation and previous studies indicate that other prognostic factors modulate the effect of age on TRM and resistance, suggesting that age as sole or primary criterion for treatment allocation may be suboptimal. Methods: To test this hypothesis in newly-diagnosed non-APL AML, we quantified the relative effects of age and other covariates using 1,127 patients (median age: 57 years) treated on Southwest Oncology Group (SWOG) trials from 1986–2009 and 1,604 patients (median age: 61 years) treated on various protocols at M.D. Anderson Cancer Center (MDA) from 2000–2008. We calculated weekly hazard rates (the probability of death in week × given that the patient was alive at the beginning of the week) for both cohorts overall and in various age subgroups. We used the area under the receiver operator characteristic curve (AUC) to quantify the effects of covariates for prediction of TRM and resistance (no TRM but patient does not enter CR or relapses within 1 year of CR), where an AUC of 1 indicates that a covariate is perfect at prediction while an AUC of 0.5 indicates no prediction (i.e. it is no better than flipping a coin). Results: Despite the use of different treatment protocols, survival in the SWOG and MDA cohorts was virtually superimposable. In both cohorts, the maximum weekly hazard occurred at weeks 3 and 4 from start of treatment, after which it decreased. The maximum hazard was relatively independent of age and remained between weeks 3 and 5 in patients age 70 years, respectively. The existence of such a discrete cut-point suggested that patients who die early are qualitatively distinct and prompted us to examine the relative effect of age and other covariates in patients who (a) died within the first 30 days of treatment (our empirically-based definition of TRM, 9% of MDA and 12 % of SWOG patients, respectively) and (b) survived at least 30 days but did not enter complete remission or relapsed within 1 year (“resistant”, 43% of MDA and 59% of SWOG patients, respectively). A model including age alone to predict early mortality had an AUC of 0.67, while a model including performance status (PS) alone had an AUC of 0.72. By comparison, a more refined model hat included PS, age, platelet count, cytogenetics, secondary AML, albumin, white blood cell count, peripheral blood blast count, and LDH yielded an AUC of 0.86. Elimination of age resulted in a model with an AUC that was only minimally lower (0.85). Prediction of resistance was more difficult with a model including age, secondary AML, cytogenetics, peripheral blood blasts, race, hemoglobin, and marrow neutrophils giving an AUC of only 0.70. Elimination of age had little effect (AUC 0.67) while age alone gave an AUC of 0.64. Conclusion: Age alone appears inadequate in predicting resistance and particularly TRM. The use of models based on several covariates improves predictive ability, but the ability to predict resistance is still limited, suggesting the value of randomized trials to assess treatment designed to reduce resistance. The observation that elimination of age has little effect on the predictive ability of such models, suggests that age is primarily a surrogate for other covariates. Disclosures: No relevant conflicts of interest to declare.

Description: Background Adults with newly diagnosed or relapsed acute myeloid leukemia (AML) and high-risk myelodysplastic syndromes (MDS) typically receive intensive chemotherapy to achieve disease remission. Generally, these patients remain hospitalized until blood count recovery due to the risk for infections and bleeding during pancytopenia. However, a recent small pilot study at our institution suggested that early discharge (ED) following induction/salvage therapy for MDS/AML may be safe and may reduce cost. We therefore conducted a phase 2 study to test this care strategy in a larger cohort of patients (NCT01235572). Methods Patients aged 18-75 years with high-risk MDS or AML (other than acute promyelocytic leukemia) were enrolled before or during induction or salvage chemotherapy and provided with outpatient care teaching. Patients were considered eligible for ED if they fulfilled the following medical and logistic criteria: ECOG performance status of 0-1, adequate organ function; no active bleeding; agreeable to close outpatient follow-up; reliable caregiver; and residency within 60 minutes of the outpatient clinic. Patients who met medical but not logistic criteria served as inpatient controls. If readmitted, ED was again possible if all medical/logistic criteria were met. Patients remained on protocol until blood count recovery, additional chemotherapy was administered, or a maximum of 45 days. Our goal was to compare the number of early deaths, healthcare costs and resource utilization among ED patients versus inpatient controls. Safety was monitored with early stopping if the early death rate was 〉7% in the ED group, with a predefined interim analysis after 30 patients. All data are provided as median (range). Results One hundred and seven eligible patients were enrolled over a 2-year period. Two patients died during chemotherapy. Twenty-seven patients failed to meet medical ED criteria after completion of chemotherapy and were taken off study, mostly due to poor performance status. Eighteen patients, age 51.4 (22-70) years, met medical but not logistic ED criteria and served as controls; they were followed for 14 (9-41) days, with 7 patients taken off study at the time of hospital discharge before blood count recovery. Sixty patients, age 51.6 (22-71) years, met all ED criteria and were discharged upon completion of induction (n=19) or salvage (n=41) chemotherapy for AML (n=50) or MDS (n=10). A median number of 1 (1-3) readmission occurred in 53 of these patients, primarily for neutropenic fever; 8 patients were readmitted twice and 3 patients were readmitted 3 times prior to coming off study. Overall, ED patients spent 12.8 (0-38) and 7.5 (0-33) days as out- and inpatients, respectively, for a total of 62.7% (0-100%) of the study time spent as outpatients. ED patients required 0.41 (0-1.9) clinic visits and 0.13 (0-0.66) physician visits per outpatient day. Duration of IV antibiotics was similar in ED and control patients (10 [0-40] vs 12 [0-40] days; p=0.38) as was number of red blood cell transfusions (0.27 [0.0-0.94] vs 0.29 [0.08-0.548] units/study day; p=0.21). In contrast, ED patients required fewer platelet transfusions (0.21 [0.09-1.25] vs 0.33 [0.21-0.80] units/ study day; p=0.02). Six patients in the ED group required between 1-6 days of intensive care unit (ICU) care (p= 0.17 for the difference in ICU days between discharges and controls). Three deaths occurred in the ED group during the study period: two of sepsis (2 and 7 days after readmission), and one of fungal sinusitis (11 days after readmission). The median daily total professional and facility charges, dated from the day of re-evaluation until removal from protocol, were significantly lower for patients discharged early compared to inpatient controls: $3,871 [$360.86-$13,361] vs $6,283 [$4,868-$11,898], p

Description: Monosomal karyotype (MK), defined as 2 or more monosomies, or a single monosomy in the presence of structural abnormalities, has recently been reported as identifying a distinct subset of acute myeloid leukemia (AML) patients with an extremely poor prognosis. In an effort to confirm this observation, we analyzed the prognostic impact of MK in 1344 AML patients between the ages of 16 and 88 years treated on Southwest Oncology Group protocols. MK was found in 176 (13%) patients. The proportion of patients with MK increased with age, being present in 4% of patients age 30 or younger, but in 20% of those over age 60. Ninety-eight percent of MK cases were within the unfavorable cytogenetic risk category and comprised 40% of this group. The complete remission rate in patients with unfavorable cytogenetics without MK was 34% versus 18% with MK (P 〈 .01). The 4-year overall survival of patients with unfavorable cytogenetics but without MK was 13% in contrast to a 4-year survival of only 3% with MK (P 〈 .01). Thus, MK defines a sizeable subset of patients with unfavorable cytogenetics who have a particularly poor prognosis.

Description: Key Points Coexpression of NUP98/NSD1 and FLT3/ITD in AML is associated with very low complete remission rates and poor survival. It is the interaction between NUP98/NSD1 and FLT3/ITD that determines poor outcome in NUP98/NSD1-associated AML.

Description: Background The cellular origin of human acute myeloid leukemia (AML) remains controversial. Limited G6PD-based X chromosome inactivation (XCI) studies suggested heterogeneity of leukemic stem cells (LSCs), with evidence of some AMLs resulting from multipotent CD34+/CD33- stem cells and others emerging from, or predominantly involving, lineage-committed CD34+/CD33+ myeloid precursors. So far, the difficulty of growing stem/progenitor cells from primary AML specimens has provided a major challenge for precise study of human LSCs and clinical implications of their heterogeneity. To overcome this, we developed a novel co-culture method to support long-term in vitro growth of primary AML specimens together with a highly sensitive XCI assay for isolated immature progenitor cells. Methods Diagnostic bone marrow specimens from 50 adult females with newly diagnosed AML were obtained from the SWOG Sample Repository. All patients received standard induction therapies without the CD33-targeting immunoconjugate, gemtuzumab ozogamicin. Human umbilical vein endothelial cells, transduced with a lentiviral construct encoding the open reading frame 1 of the early region 4 of adenovirus to provide growth factor/serum-independence, were co-cultured with aliquots of flow cytometrically-isolated CD34+/CD33- and CD34+/CD33+ cells in medium containing the aryl hydrocarbon receptor antagonist, StemReginin-1. Cultures were replenished with fresh medium weekly and kept in hypoxia for up to 8 weeks, and defined fractions plated after 4, 6, and 8 weeks for colony-forming cell assays. Resulting colony forming units-granulocyte and/or macrophage (CFU-GMs) were harvested and individually assessed for XCI via methylation-sensitive PCR-based analysis of a polymorphic short tandem repeat (STR) in the X-linked human androgen-receptor gene (HUMARA). Specimen growth was considered if it yielded ≥5 CFU-GMs after ≥4 weeks of co-culture. Specimens with 〉75% disease-specific CFU-GMs were considered clonal. Results Among the 50 specimens, 45 (90%) were informative with regard to HUMARA STR, allowing clonal assessment of isolated cell progeny. Sixteen (35.6%) yielded CFU-GMs derived from CD34+/CD33- cells after long-term (≥4 weeks) co-culture sufficient for clonal analyses, whereas the remaining samples yielded either no growth (n=22) or insufficient CFU-GM growth (n=8) from this starting cell population. Five of the 16 specimens with sufficient CFU-GM growth derived from CD34+/CD33- cells (31.3%) yielded colonies consistent with a polyclonal growth pattern, and 3 samples without sufficient CFU-GM growth derived from CD34+/CD33- cells yielded colonies from unsorted cells that were consistent with a polyclonal growth pattern; these 8 leukemias were cytogenetically classified as favorable (n=3) or intermediate-risk (n=5, including 2 with normal karyotype and NPM1 but not FLT3/ITD mutation). In contrast, 11 specimens yielded CFU-GMs growth derived from CD34+/CD33- with a pattern consistent with clonal growth. The complete remission rate was slightly but statistically non-significantly higher among patients whose specimens yielded polyclonal CFU-GMs derived from CD34+/CD33- cells relative to those with clonal progenitor growth (87.5% vs. 63.6%, P=0.34). More importantly, however, the overall and relapse-free survival of patients whose specimens yielded polyclonal CFU-GMs derived from CD34+/CD33- or unsorted cells was significantly longer than that for patients whose specimens yielded a clonal progenitor growth pattern after long-term culture of CD34+/CD33- cells (P=0.0006 and P=0.0054, respectively; Figure 1); restriction of this analysis to the 16 specimens with sufficient CFU-GM growth from CD34+/CD33- cells did not change this outcome difference (e.g. hazard ratio for overall survival for restricted dataset: 0.13 [95% confidence interval: 0.06-0.72] vs 0.08 [0.04-0.44]). Conclusion Our novel co-culture method allows for the study of progenitor/stem cell involvement in a significant portion of human AMLs. Our data suggest that a subset of adult AMLs emerges from, or predominantly involves, only lineage-committed CD34+/CD33+ myeloid precursors but not the less mature CD34+/CD33- precursors. This subset, which is comprised of AMLs with diverse cytogenetic/molecular abnormalities, is characterized by an excellent outcome with intensive chemotherapy. Disclosures: No relevant conflicts of interest to declare.

Description: Background Intensive chemotherapy for adults with newly diagnosed acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS) is associated with a considerable risk of morbidity and mortality, commonly due to infections and bleeding. Little information is available on pre-treatment risk factors that predispose to these complications. Here, we investigated the relationship between baseline peripheral blood counts and (a) subsequent adverse events and (b) duration of neutropenia following administration of induction chemotherapy at our institution over the last decade for newly diagnosed AML/MDS undergoing curative-intent induction chemotherapy. Patients and Methods We retrospectively analyzed 205 consecutive adults receiving 7 + 3-like treatment regimens. Daily blood counts were recorded from initiation of chemotherapy until the day of neutrophil recovery (defined as an absolute neutrophil count [ANC] greater than or equal to 500 cells/µL). Adverse events (fever, infection, bacteremia, transfer to the intensive care unit, and death) were recorded until day 35 or administration of additional chemotherapy, whichever came first. Cytogenetic risk was classified based on revised MRC/NCRI criteria. Associations between baseline patient characteristics and adverse events were assessed using Kaplan-Meier survival curves with a log-rank test (for two groups) or log-rank test for trend (for three or more groups); patients requiring salvage therapy for persistent disease were censored on the day such therapy was initiated. Cox proportional hazards models were used to estimate the hazard ratio (HR) for the associations between age, gender, disease type, cytogenetic risk and adverse outcomes in univariate and multivariate analyses. Results The median age of our study cohort was 53 (range: 18 to 81) years. Sixty-six percent of the patients had de novo AML. Cytogenetic risk was favorable in 14%, intermediate in 60%, and adverse in 24%. Among all patients, the complete remission (CR) rate was 75%, while 2% died before response could be assessed (range: day 13 to 28) and 23% were refractory. When analyzed as a continuous variable, the baseline neutrophil count was statistically significantly associated with fever (HR=0.97 [95% confidence interval: 0.95-0.999] for each increase of 1000 cells/µL, P=0.04) and infection (HR=0.92 [0.87-0.98], P=0.01), but not bacteremia (HR=0.95 [0.89-1.01], P=0.10) or requirement for intensive care unit (ICU) care (HR=1.00 [0.93-1.07], P=0.99). For subsequent analyses, we dichotomized the study cohort based on pre-treatment ANC and compared patients with baseline ANC

Description: Abstract 2590 Background FLT3-internal tandem duplication (ITD) is found in about 30% of patients with acute myeloid leukemia (AML) at diagnosis and confers a high risk of relapse. Thus allogeneic hematopoietic transplant (HCT) is recommended for these patients in first complete remission (CR) and after HCT they become candidates for trials of FLT3-ITD inhibitors (such as quizartinib) to prevent relapse. However at referral to tertiary centers after reaching CR, FLT3-ITD status at diagnosis is often unknown, complicating decisions about HCT. FLT3-ITDs are known to be associated with a normal karyotype (NK), translocation 6;9 and a high white blood cell (WBC) count, and we hypothesized that assessment of likely FLT3-ITD status at diagnosis in patients presenting in CR not tested at diagnosis would be improved by examining these covariates simultaneously. Methods Our initial analysis included 434 adult patients with newly diagnosed AML (excluding APL) treated on three SWOG trials (S9031, S9333, and S0106) in whom FLT3-ITD status (positive/negative) was established at diagnosis. Univariate and then multivariate analyses were used to identify covariates independently associated with FLT3-ITD positivity. The relative abilities of these to predict FLT3-ITD positivity were quantified using the area under the receiver operator characteristic curve (AUC); an AUC of 1.0 denotes perfect prediction, whereas an AUC of 0.5 is analogous to a coin flip. The log odds ratios (ORs) from the multivariate models were used to assign a score to each covariate and scores were summed; such that the higher the score, the greater is the likelihood of the FLT3-ITD positivity at diagnosis. We tested the performance of the scoring system in 2 newly-diagnosed populations that had not contributed to the system's development and in whom FLT3-ITD status at diagnosis was known: (a) 210 patients treated at FHCRC (Fred Hutchinson Cancer Research Center) and (b) 1,401 patients treated at MDACC (M.D. Anderson Cancer Center). Covariates examined were: age, sex, performance status (PS), WBC count, platelet count, bone marrow blast percentage, secondary AML, and cytogenetic risk (using SWOG/Eastern Cooperative Oncology Group criteria). Results FLT3- ITD was present in 101 of the 434 SWOG patients (23%) in the scoring system development population. The log OR were rounded to the nearest half point to create the scoring system. Only WBC 〉 20,000 (reference, WBC 〈 20,000) and cytogenetics (reference, normal) had non-zero scores, which are summarized below: Scores less than −0.5 were called low, ≥−0.5 and

Description: Background Fluorescence in situ hybridization (FISH) is an invaluable tool for complementing conventional karyotype analysis in the diagnosis of leukemia. FISH panels are often requested by clinicians, along with karyotype analysis, for molecular determination of clinically actionable abnormalities. For most cooperative group clinical trials, unlike karyotype analysis, FISH has not been required to determine eligibility or risk stratification. Yet no previous studies have shown how often FISH results might alter the cytogenetic risk category determined by karyotype analysis. Furthermore, FISH panels which may include 5∼10 probe sets are costly while karyotype analysis for new diagnosis of myeloid leukemia is routine and highly informative. Therefore we aim to determine the frequency of cytogenetic risk alteration by FISH testing to serve as an evidence base for future study design and best clinical practice. Patients and Methods Our study set includes all leukemia patients enrolled in SWOG leukemia studies from 2002 to 2012 with both cytogenetic and FISH results available at the time of initial diagnosis (n=248), representing 12% of total patients enrolled in these studies. There are 169 patients with acute myeloid leukemia (AML), 22 of whom had acute promyelocytic leukemia (APL); 45 with chronic myeloid leukemia (CML-CP); and 34 with acute lymphocytic leukemia (ALL). The age range was 19-87 (median 51) years. Patients received various therapies based on the study protocols. All cytogenetic and FISH results were submitted from SWOG-approved laboratories and were reviewed by the SWOG Cytogenetics Committee. Risk categorization was based on combined SWOG and MRC criteria. Results There was great variation in the number and the type of FISH probes used for the workup of new diagnosis of leukemia (range 1-12 per patient). The most frequent FISH findings with added value were cryptic translocations, including amplification and/or rearrangement of MYC (mean overall survival 8.2 months without remission) and rearrangement of MLL. In the submitted studies, there were no misses for subtle chromosome abnormalities by karyotype analysis, including the inv(16), although FISH clarified deletion 16q instead of inv(16) occasionally. FISH and karyotype analyses were consistent for large chromosome abnormalities such as deletions of 5q and 7q, t(8;21), and trisomy 8. Of all 248 patients, 47 (19.0%) had favorable cytogenetics, 133 (53.6%) intermediate, 64 (25.8%) unfavorable, and 4 (1.6%) unknown. Of the 147 non-APL AML patients, 23 (15.6%) had favorable cytogenetics, 79 (53.7%) intermediate [72 (49.0%) after FISH], and 64 (43.5%) unfavorable [71 (48.3%) after FISH]. Overall, eight patients (3.2%) demonstrated alterations in risk categorization after FISH analysis: 7 from intermediate to unfavorable among AML patients, and 1 from favorable to intermediate among ALL patients. Of the 45 CML patients, karyotyping adequately identified all t(9;22), and 3 patients demonstrated additional secondary abnormalities. FISH identified 5 patients with submicroscopic deletions on the derivative 9 (which was once considered a higher-risk feature but appears to have been overcome by therapy with tyrosine kinase inhibitors). Of the 34 ALL patients, 16 (47.1%) had unfavorable cytogenetics; 12 (35.3%) intermediate; and the remaining 2 showed favorable hyperdiploidy, one of which was reclassified as intermediate due to detection of structural rearrangements by FISH. Conclusion The frequency of cytogenetic risk alteration by FISH after karyotyping analysis for new diagnosis of leukemia was low, supporting current clinical trial designs that do not require extensive FISH panel testing for protocol eligibility. However, FISH clearly adds value in detecting cases with cryptic rearrangements, which can contribute to altered risk stratification. It may be helpful to ascertain subtle rearrangements by FISH, such as inv(3), inv(16), t(6;9), MLL (11q23) rearrangement, and MYC (8q24) amplification and rearrangement. This work was supported by PHS Cooperative Agreement grants awarded by the National Cancer Institute, CA32102 and CA38926. Disclosures: No relevant conflicts of interest to declare.

Description: Background: Because of the concern for therapeutic resistance and excessive toxicity or even treatment-related mortality (TRM), many medically unfit patients do not receive AML-directed therapy, although evidence suggests that outcomes are improved if essentially all of these patients are offered some form of chemotherapy rather than given supportive care only. Here, we evaluated the potential value of attenuated doses of CPX-351, a liposomal formulation of daunorubicin and cytarabine, in medically unfit patients with newly diagnosed AML or high-risk MDS (≥10% blasts). In earlier trials, CPX-351 appeared to afford superior outcomes in high-risk AML patients with a wide therapeutic window suggesting that reduced dose treatment may be helpful in this population. Methods: Patients aged ≥18 years with untreated AML or high-risk MDS were eligible if they had a TRM score of ≥13.1, corresponding to an expected TRM (i.e. death within 28 days of treatment initiation) of 31% with standard induction chemotherapy. Bilirubin was to be