ALBERT

Description: Despite the well-established success of ABL1 tyrosine kinase inhibitors (TKIs) in the treatment of patients with chronic myeloid leukemia (CML), approximately 20% of patients treated with frontline imatinib develop resistance by 5 years on therapy. The majority (~60%) of such resistant cases are explained by acquired mutations within the BCR-ABL1 kinase domain that compromise inhibitor binding, and nearly all of these mutations are effectively targeted by one or more of the 2nd and 3rd generation ABL1 kinase inhibitors. In contrast, the remaining ~40% of imatinib-resistant cases harbor no explanatory BCR-ABL1 kinase domain mutation, presumably attributable to BCR-ABL1 kinase-independent mechanisms. We hypothesized that resistance in these patients results from acquired auxiliary molecular aberrations which persistently activate signaling pathways downstream despite inhibition of BCR-ABL1 kinase activity. To identify such mechanisms, we performed whole exome sequencing and RNA sequencing on a cohort of 135 CML patients comprising the following subgroups: newly diagnosed/TKI naïve (n=28), BCR-ABL1 kinase-dependent resistance (n=31), and BCR-ABL1 kinase-independent resistance (n=65), and TKI-induced remission (n=7). Resistant patients were required to have demonstrated clinical resistance to one or more ABL1 kinase inhibitors in the form of suboptimal response or loss of cytogenetic response; the subtype of resistance was defined based on the presence or not of an explanatory BCR-ABL1 kinase domain mutation at the time of resistance. The majority of samples collected were from patients with chronic phase CML (n=97), although smaller cohorts of accelerated phase CML, blast crisis CML, and Ph+ ALL were also profiled (n=20, 19, and 9, respectively). Among the 44,413 protein-altering and 902 splice site variants detected across the 120 WES samples, there were on average 908 missense, 146 truncation and 69 splice variants per sample. Genes with truncation and missense variants were compared between BCR-ABL1 kinase-independent and -dependent resistant chronic phase samples. A total of 44 genes were seen with a frequency difference of at least 10%, including PLEKHG5 and NKD2 (30% and 28% difference, respectively), which are involved in regulation of NF-kB and Wnt signaling. Consistent with previous reports, we also detected EZH2 and TET2 as exclusively mutated in the BCR-ABL1 kinase-independent resistance patients (6% and 3%, respectively). Further analyses stratifying variants among resistant patients according to specific ABL1 kinase inhibitor therapy failed and comparing, where available, serial samples from pre- and post-treatment for clonal expansion are underway. Additionally, sufficient material was available to perform ex vivo small-molecule inhibitor screening for 48 patient specimens, the resultant data of which was used to generate putative effective drug target profiles and integrated with exome sequencing variants to prioritize variants of functional relevance (HitWalker; Bottomly et al., Bioinformatics 2013). Among 23 patient samples exhibiting BCR-ABL1 kinase-independent resistance, the mutated genes most frequently ranked in the top 10 functional-prioritized variants were: ABL1 (which included non-kinase domain variants; 34.7%), MAP3K1, MUC4, FGF20 (each 17.4%), ARHGEF15, MEF2A, EPHA8, TYRO3, BMP2K, and IRS1 (each 13.0%). Notably, the top six candidates are members of the neutrophin (ABL1, MAP3K1, and IRS1), EPHA forward (EPHA8, ARHGEF15), and p38 MAPK signaling pathways (MAP3K1 and MEF2A). Taken together, these findings suggest that several of the same pathogenic molecular abnormalities seen in other myeloid malignancies are also present in CML patients with BCR-ABL1 kinase-independent resistance, including a subset which align to persistent re-activation of signaling pathways involved in CML disease pathogenesis and progression. As such, genetic and/or functional profiling of these patients in the clinic may translate to actionable candidates for combination therapy to maximize disease control and improve patient outcomes. Disclosures Agarwal: CTI BioPharma Corp: Research Funding. Radich:Novartis: Consultancy, Research Funding; BMS: Consultancy; Ariad: Consultancy. Deininger:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Research Funding; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; CTI BioPharma Corp.: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees. Druker:Pfizer: Patents & Royalties; Dana-Farber Cancer Institute: Patents & Royalties: Millipore royalties via Dana-Farber Cancer Institute; Curis: Patents & Royalties; Array: Patents & Royalties; CTI: Consultancy, Equity Ownership; Pfizer: Patents & Royalties; Curis: Patents & Royalties; Array: Patents & Royalties; Dana-Farber Cancer Institute: Patents & Royalties: Millipore royalties via Dana-Farber Cancer Institute; Oncotide Pharmaceuticals: Research Funding; Novartis: Research Funding; BMS: Research Funding; ARIAD: Patents & Royalties: inventor royalties paid by Oregon Health & Science University for licenses, Research Funding; Roche: Consultancy; Gilead Sciences: Consultancy, Other: travel, accommodations, expenses; D3 Oncology Solutions: Consultancy; AstraZeneca: Consultancy; Ambit BioSciences: Consultancy; Agios: Honoraria; MolecularMD: Consultancy, Equity Ownership, Patents & Royalties; Lorus: Consultancy, Equity Ownership; Cylene: Consultancy, Equity Ownership.

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 286 It is well established that deregulation of the PI3K signaling pathway plays an important role in the etiology of human malignancies including those of hematologic origin. In 30–50% of solid tumors, oncogenic activation of the PI3K pathway is the result of gain-of-function mutations in the PI3K p110α isoform or due to the loss-of-function of the PTEN phosphatase that is responsible for PI3K downregulation. In B cell malignancies these mutations are rarely observed in spite of the fact that PI3K pathway activation is commonly observed and often essential for tumor cell growth and survival. In this case, PI3K pathway activation has been shown to result from constitutive B cell receptor (BCR) activation and/or from exposure to survival factors present in the microenvironment. The activation of the PI3K pathway by cell surface receptors is directly mediated by the Class I isoforms (α, β, δ, and γ), however, their relative contribution in B cell tumors is unknown. Interestingly, genetic and pharmacological approaches that specifically inactivate the p110δ isoform have demonstrated its important role in normal B cell signaling in response to multiple factors including antigen, CD40L, BAFF, SDF-1 and CXCR13 making it an attractive target for therapeutic intervention in B cell malignancies. CAL-101 is an oral p110δ specific inhibitor which is currently being evaluated in a phase I clinical trial for the treatment of patients with select hematologic malignancies. This compound is a novel potent p110δ inhibitor with an IC50 of 2.5 nM against purified p110δ and EC50 of 65 nM in p110δ-mediated basophil activation in whole blood. CAL-101 demonstrates 300-, 200-, and 40-fold selectivity over the other class I family members (α, β, and, γ respectively) and no activity against class II and III PI3K family members or other PI3K-related proteins including mTOR and DNA-PK. Furthermore, a kinome-wide screen failed to detect activity against any additional kinases. To investigate the potential role of p110δ in B cell hematologic tumors we screened a wide range of leukemia and lymphoma cell lines for constitutive PI3K pathway activation. The expression of p110δ was observed in 〉90% of these cell lines and in many cases was accompanied by constitutive Akt phosphorylation. In this context, CAL-101 was able to reduce p-Akt levels and block additional downstream effectors such as p-S6, and GSK-3β in cells that represent a range of tumor types including follicular lymphoma, acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma, and mantle cell lymphoma (MCL). Furthermore, treatment with CAL-101 resulted in growth suppression and induction of apoptosis which was accompanied by PARP and caspase-3 cleavage. Growing evidence suggests that signals from the microenviroment are essential for the expansion, homing, and survival of malignant B cells, in addition to promoting resistance to conventional drug therapy. To investigate the potential role p110δ plays during B cell signaling via interactions with the microenvironment, we examined invoked stimulation of leukemia and lymphoma cell lines with CXCL12, CXCL13, BAFF, or BCR crosslinking in the presence or absence of CAL-101. Stimulation with either chemokines or growth factors resulted in the phosphorylation of Akt which was inhibited by CAL-101 in a dose dependent manner. Moreover, p110δ inhibition with CAL-101 inhibits the chemotaxis of ALL and MCL cell lines to CXCL12. These studies have now been extended to the analysis of primary patient B-ALL and MCL cells to further establish preclinical proof of concept for therapeutic application of CAL-101. In summary, CAL-101 is a potent and selective p110δ kinase inhibitor with broad anti-tumor activity against cancer cells of hematologic origin. Our results demonstrate that selective inhibition of p110δ with CAL-101 inhibits malignant B cell growth, survival, and migration. Furthermore, p110δ inhibition may enhance the effect of cytotoxic drugs or overcome cell mediated drug resistance by inhibiting the protective signals of the microenviroment, providing a rational for combination therapy. These data suggest that p110δ may play an important role in regulating signals between malignant B cells and their microenvironment thereby providing the preclinical rationale for clinical evaluation of CAL-101 alone or in combination with cytotoxics or biologics in patients with hematologic malignancies. Disclosures: Lannutti: Calistoga Pharmaceuticals: Employment. Meadows:Calistoga Pharmaceuticals: Employment. Kashishian:Calistoga Pharmaceuticals: Employment. Steiner:Calistoga Pharmaceuticals: Employment. Johnson:Calistoga Pharmaceuticals: Research Funding. Giese:Calistoga Pharmaceuticals: Employment.

Description: Abstract 2743 CD52 is a cell surface glycoprotein of unknown function that is expressed in B and T lymphocytes, macrophages, and monocytes, but is not expressed in normal hematopoietic stem/progenitor cells. CD52 is also expressed in chronic lymphocytic leukemia (CLL), B-cell acute lymphoblastic leukemia (ALL), and some cases of T-ALL. Alemtuzumab, a recombinant humanized monoclonal antibody, targets CD52 and is used to treat CLL. In contrast to normal hematopoietic stem/progenitor cells, CD52 expression has been described in acute myeloid leukemia (AML) and in blast crisis (BC) chronic myeloid leukemia (CML). Based on these observations we were curious whether CD52 expression distinguished normal from malignant or more mature from immature stem/progenitors cells, and whether these cells were sensitive to alemtuzumab. CD52 expression was examined in three blast cell populations (CD34+/CD38-, CD34+/CD38+, and CD34-) in patients with myeloid (44) and lymphoid (18) neoplasms, and normal patients (6). In normal hematopoietic cells, stems cells are enriched in the first population; more mature cells are characterized by increasing CD38 expression and loss of CD34 expression. In AML and CML leukemia stem cells may arise within either CD34+ population and possibly in the CD34- population. Relative to normal lymphocytes average CD52 expression could be characterized as low to moderate. Using an expression cutoff of 〉 20%, in contrast to normal patients, CD52 was detected in at least one of three blast populations in almost all patients. Using a more stringent cutoff of 〉 50%, CD52 was expressed in CD34+/CD38- cells in 7/11 B-ALL and 6/7 T-ALL cases and was concordantly expressed in the other two populations. Using the same criteria in myeloid malignancies (Table 1), expression occurred more frequently in AML, AML arising from myelodysplastic syndrome (MDS), and BC CML. In AML and AML arising from MDS, CD52 was expressed in the 34+/38- population in 7/15 cases (47%) and 4/7 cases (57%), respectively; it was expressed in both BC CML patients. In AML and BC CML patients, CD52 was expressed at similar levels in the CD34+/CD38+ fraction. No clear association between CD52 expression and cytogenetic abnormalities was found. We then examined whether CD52 expression differentiated normal from malignant blasts (CD34+/CD38- and CD34+/CD38+) in two CML myeloid BC patients. FISH and quantitative PCR demonstrated that BCR-ABL was expressed in all 4 populations, which were also morphologically distinct. Colony forming unit (CFU) assays demonstrated a significantly decreased ability to form CFU (on average 5–20 fold decrease) in CD52+/CD34+/CD38- CML cells suggesting CD52 cells may be more mature. Lastly and not previously described, we found that several BC CML cell lines express CD52, and complement-mediated cell cytotoxicity was similar in the highest expressing cell lines to that seen in EHEB (B-CLL) cells known to be targeted by alemtuzumab. Thus, alemtuzumab may have clinical efficacy in BC CML. In conclusion, CD52 is expressed on blast populations enriched for leukemic stem cells. Whether the absence or presence of CD52 more precisely segregates a leukemia stem cell containing population currently remains unknown and requires functional testing in a murine model. Our preliminary experiments in CML suggest CD52 may not differentiate between normal and malignant stem/progenitor cells. However, CD52 expression may distinguish normal and malignant stem cell populations in cases where CD52 and CD38 are more highly expressed. The observation that CD52 expression is increased in acute vs. chronic leukemias raises the intriguing possibility that CD52, if not directly involved, may be a marker for genes or pathways contributing to the block in differentiation seen with progression to acute leukemia. Furthermore, given that CD52 expression is heterogeneous in chronic disorders, it is possible that CD52 expression within these populations may correlate with poor prognosis or impending leukemic conversion. Table 1. The proportion of patients (44) expressing CD52 at levels 〉 50% in 3 blast populations. Three populations were present in most, but not all patients. Gray shading indicates chronic myeloid diseases. MPN is myeloproliferative neoplasm; NOS is not otherwise specified; ET is essential thrombocythemia; CMML is chronic myelomonocytic leukemia; and an arrow represents progressed to. Disclosure: Oehler: Pfizer: Research Funding. Radich:Novartis: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria.

Description: Non-myeloablative conditioning regimens and the progress made in conventional transplantation have broadened the age restrictions for recipients of hematopoietic stem cell transplantation (HCT). The increasing age of the patient has naturally increased the age of the donor population. As aging is a risk factor for malignancies, and there is a variable period of time in which malignancies are asymptomatic, the odds of transferring malignant or pre-malignant clones in the process of HCT might increase. We uncovered 8 cases of clonal abnormalities with immunophenotypic or molecular aberrations, characteristic of specific hematologic malignancies found in HCT recipients arising from donor hematologic cells over a decade (1990–2001). The median age for the donors in our series is 44.5 years (range: 36–70 years). In 5 out of the 8 cases, the clone with malignant characteristics was detected both in donor and recipient, and in 3 cases only in the recipient (in 2 cases the donor was not tested). In 3 of the 8 HCT recipients, leukemia of donor origin prompted therapeutic intervention (a second transplant in two cases), while the others have remained under observation, as the aberrant cell clones of donor origin have not resulted in overt hematologic malignancies. In 4 cases, the abnormal clones were B cell in lineage, and have not yet evolved into disease, neither in the patients nor in 2 of the 4 donors studied. In case number 4 the donor developed CLL two years after the HCT, and the aberrant clone was detected with IgH VDJ specific PCR from an aliquot of the donor marrow preserved from the marrow harvest. In cases 5, 6, and 7, the time from the transplant to the detection of the clonal abnormality spanned more than one year. The donor of case 5 was tested and did not present the abnormality detected in his recipient (no information was obtained from donors in cases 6 and 7). In cases 4 and 8, the second malignancies were first detected in the donor. While the recipient in our fourth case has not developed any disease signs or symptoms, the recipient in our eighth case did develop clinical disease shortly after the transplant. The selection bias in discovering these cases makes it difficult to estimate a true prevalence of donor-derived malignancies in our transplant population. However, we speculate that the broadening of age limits for transplantation, with the associated increase of the donor age, has set the stage for the increased appearance of age-related donor malignancies. Increased sensitivity techniques to detect aberrant populations, such as flow cytometry, should perhaps be considered for routine pre-transplant screening of older donors. Donor Tumor Cases Pt. Patient Disease Lab Abnormality / Days post-transplant Clinical disease? / Last follow up Donor age Detected in donor? /Disease in donor? Donor last follow up ND=not done, NFU=no follow up 1 MF MCL/55d No/3.5 m 56 Yes/No 2m 2 MDS CLL/28d No/24 m 70 Yes/No NFU 3 CLL MZL/294d No/55 m 57 Yes/Yes 55m 4 AML CLL/10y No/120 m 45 Yes/Yes 120m 5 Renall Cell Carcinoma AML/15m Yes/21 m 42 No/No 21m 6 CML MDS/4y No/96 m 44 ND/NFU NFU 7 CML MDS/4y Yes/114m 44 ND/NFU NFU 8 ALL ALL/130d Yes/6m 36 Yes/Yes 65m

Description: Abstract 3094 Poster Board III-31 Clofarabine (Clo) has demonstrated single agent activity in acute lymphoblastic leukemia (ALL) and acute myeloid leukemia. Therefore, efforts have been undertaken to combine Clo with other antileukemic agents. Clo and cytarabine (Cy) target two different sites in DNA, have synergistic activity in vivo, and have non-overlapping toxicities, making this combination potentially promising for the treatment of relapsed/ refractory ALL. The maximum tolerated dose of this combination has been previously determined. The goals of this study were to: (1) evaluate the complete remission rate with and without complete count recovery (CR and CRi) of patients with relapsed/ refractory ALL treated with Clo/Cy; (2) to assess expression of connective tissue growth factor (CTGF) and nucleoside transporters in patients. Methods Patients were treated at SWOG institutions from February 2007 through July 2008. Clo was supplied by Genzyme. This protocol was reviewed and approved through each institution's review board. Eligibility criteria included: age ≥ 16 years, relapsed or refractory ALL (excluding Burkitts or mixed lineage leukemia), Zubrod performance status 0-2. CTGF expression was analyzed by triplicate RT-PCR on pre-treatment samples. Expression of the nucleoside transporters (hENT1, hCNT3, dCK) was analyzed by immunohistochemistry. All patients received treatment with Clo 40 mg/m2/d and Cy 1 g/m2/d on Days 1-5. Response was assessed between Days 28-35. Patients with a partial remission (a 50% decrease in marrow blast percentage) could receive re-induction therapy. CR was defined as 〈 5% marrow blasts, neutrophils 〉 1,000/μL, platelets ≥ 100,000/μL, and no evidence of extramedullary disease. CRi was defined the same as CR but the platelet count could be 〈 100,000/μL. Patients with a CR could receive one cycle of consolidation therapy. Patients were to be accrued in 2 stages. In the first stage, 20 patients were to be registered. If at least 2 CR/ CRis were observed, an additional 15 patients were to be accrued (87% power to conclude that an agent with a response rate of 30% warrants further study). Results The study met criteria to proceed to the second stage. Thirty-seven patients were enrolled. One patient is excluded from the analysis because treatment was not started due to high liver function tests after registration. Of the 36 evaluable patients, the median age was 41 years (range: 20-68), median WBC 5200/μL (range 900-93,700), and 23 patients (64%) were male. The median time from initial diagnosis to registration was 14 months (range 1-52 months). Nineteen patients (53%) were in first relapse, 7 (19%) in subsequent relapse, 1 (3%) with an unknown number of relapses, and 9 (25%) refractory. Two patients had received prior allogeneic BMT. Thirty-two patients have died, with 10 deaths occurring during protocol treatment and 7 of these deaths attributable to treatment [infection (3), pleural effusion (1) DIC (1), hypotension/ renal failure/ cardiac arrhythmia (1), multiorgan failure (1)]. Three patients received re-induction, and 1 patient received consolidation therapy. The CR/CRi rate was 17% (95% CI 6-33%), and median overall survival 3 months. At least 8 patients proceeded to BMT after completing protocol therapy. The number of patients accrued to this study was relatively small and the CR rate was low. Therefore, it is unlikely that any statistical differences in CR rate would be found, with respect to expression of various biologic correlates. However, there was a trend for patients with higher expression of CTGF having an inferior OS (p=0.13). In addition, although 70% of patients had high levels of hENT1 expression, only 33% and 44% of patients respectively had high levels of hCNT3 and dCK, which could serve as a mechanism of resistance to nucleoside analog therapy. Conclusion The CR/CRi rate in this study was modest. This regimen does not exhibit sufficient activity to warrant further testing unless it was significantly altered. However, this was a heavily pre-treated, poor risk population of patients. Of interest, low expression of nucleoside transporters and high expression of CTGF may predict response and OS. Larger prospective studies will be needed to confirm this. Therapies directly targeting these latter pathways or working independently of these pathways may help improve the prognosis of patients if these results are confirmed. Disclosures Advani: Genzyme: Honoraria, Research Funding. Off Label Use: The combination of clofarabine/ cytarabine is investigational. Radich:Novartis: Research Funding. Lancet:Genzyme: Consultancy. Stuart:Genzyme: Research Funding.

Description: In many parts of the world, diagnosis and monitoring of CML patients is limited by the availability and cost of molecular testing. In countries without molecular diagnostic capabilities, blood samples can be shipped to central labs, but this is both hampered by sample degradation, and the high costs of shipping. This study explores the method of directly spotting peripheral blood onto a paper template (dried blood spots), with subsequent shipping, RNA extraction, and BCR-ABL testing. Methods: Blood Spots and Shipment. We received dried blood spots from Australia and African countries by mail or courier, and blood from CML patients from our institution were also used for these experiments. 200μL of blood (PB) was pipetted onto Whatman 503 Protein Saver Cards (PSC; Sigma-Aldrich), where each card contains four 50μL spots. Cards were allowed to dry for at least 24 hours at room temperature. For mailing, PSCs were sealed into glassine envelopes with a packet of desiccant, and then placed inside a mailing envelope following DOT and IATA regulation for shipping non-regulated, exempt human specimens. RNA Extraction from Cards and %BCR-ABL determination. Blood spots were incubated with proteinase K followed by RNA isolation using RNeasy Mini Kits (Qiagen). Extracted RNA was quantified using a NanoDrop spectrometer (Thermo Scientific). %BCR-ABL was determined using the automated Cepheid GeneXpert platform or manual two-step quantitative RT-PCR on the 7900HT Fast Real-Time PCR System (Applied Biosystems). Results: Bench top time course: To test for effects of long transit times on RNA quality, we performed a time course study of cards at room temperature (RT) with 5 samples. For each sample, multiple cards were spotted with PB. The cards were then allowed to sit at RT for predetermined amounts of time, up to 42 days, before extracting RNA. We measured RNA integrity for one of the specimens (CML # 5) and found rapid degradation with the RIN number going from 8.7 for the fresh blood to 2.8 after 28 days on the card. However the amplification for both BCR-ABL and ABL differed less than one cycle between the fresh blood and the last time point by manual qRT-PCR (BCR-ABL Ct = 23.63 for fresh blood and 24.06 for day 28 PSC; ABL Ct = 26.69 for fresh blood and 27.64 for day 28 PSC). Figure 1 shows the results of the time course experiment for the 5 samples as a plot of ΔCt versus time in days. BCR-ABL qRT-PCR concordance studies: We compared the %BCR-ABL results obtained in fresh specimen at the institution sending the sample with the %BCR-ABL results we obtained from RNA extracted from PSC using the Cepheid GeneXpert. Paired evaluable results were available for 9 samples with a median WBC = 9.8 x 109/L (range: 3.37x109/L – 85.5x109/L). Samples were 8 to 49 days old at the time of extraction. The amount of RNA input into the GeneXpert reaction ranged from 38.75ng to 1μg. The %BCR-ABL detected ranged from 0.37% to 27% (see Table). The mean absolute difference between fresh blood and PSC BCR-ABL% is 2%; the relative mean percent change for BCR-ABL, using fresh blood as the reference is 13.1% (S.D., 31.2), P = 0.24. Conclusions and future directions: Dried blood spots are relatively inexpensive method to transport blood that preserves enough RNA stability to allow highly accurate BCR-ABL detection, when compared to results performed on an identical platform using fresh peripheral blood samples. Further studies are undergoing to accurately determine the sensitivity of this method and the feasibility of using regular mail for inexpensive transport of specimens. Table 1IDWBC (1000/μL)Sample Age at Spotting (Days)Sample Age at RNA extraction (Days)RNA ng/μlVolume GeneXPert (μL)Paper %BCR-ABL (IS)GeneXpertFresh Blood % BCR-ABL (IS) GeneXpertI1na010426349naI224.101311092745I38009181544naI47.4285102.4*3.1I55.50495241.92I63.61307.4225912I785.5130102102439I812.212912.415128.8I9na1281.5250.37*0.71I103.370273257.85.7I1115.912731102325I126.612714.415na2.3 *%BCR-ABL was manually calculated due to late ABL Cts because of low starting material. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Description: BACKGROUND: HyperCVAD is used commonly in adult ALL but can be difficult for older or infirm patients (pts) to tolerate (JCO 2000, p. 547; Cancer 2008, p. 2097). At our center, only 50% who receive it reach a minimal residual disease negative (MRD-) state within 90 days (Am J Hematol 2018, p. 546). Lower-intensity options with ABL tyrosine kinase inhibitors (TKIs) yield high response rates in Philadelphia chromosome (Ph)+ disease, but long-term efficacy may be limited (Blood 2016, p. 774; ASH 2017, #99). DA-EPOCH is effective and relatively well-tolerated in high-grade lymphomas (NEJM 2013, p. 1915; Blood 2014, p. 2354). This prompted us to study DA-EPOCH as initial treatment of adult ALL. METHODS: Pts ≥ 18 years old with newly-diagnosed ALL were eligible if they were not a candidate for pediatric-inspired therapy (e.g., Ph+, age ≥ 40) and had adequate liver and kidney function. All pts signed IRB-approved consent forms. This single-center trial is registered on clinicaltrials.gov (NCT03023046) and supported by NCI 5P30 CA015704-41. DA-EPOCH was given as initially described (Blood 2002, p. 2685). Doses of myelosuppressive agents were adjusted based on the hematologic nadir after the previous cycle, but pts could start the next cycle before day 21 once ANC ≥ 1K/μL and platelets (plt) ≥ 50K/μL, and (after 1st 5 pts treated) dose-reduction was deferred if cytopenias reflected ALL. If Ph+, imatinib (IM) 600 mg or dasatinib (DAS) 100 mg daily on Days 1-14 of each cycle was added. If CD20+, rituximab (R) 375 mg/m2 was given once per cycle. All pts received G-CSF and intrathecal prophylaxis. Response was determined by morphology (morph), multiparameter flow cytometry (MFC) and (for Ph+) BCR-ABL RT-PCR on bone marrow aspirate (BMA). When MRD- by MFC (MFC-), high-throughput sequencing (HTS)-based MRD testing was performed as described previously (Clin Cancer Res 2014, p. 4540). BMA was performed after cycle 1, then repeated as needed to confirm MFC- or persistence. Up to 8 cycles could be given followed by maintenance (POMP ± TKI) or hematopoietic cell transplant (HCT) at physician's discretion, though morph CR after ≤ 2 cycles was needed to continue. The primary endpoint was rate of MFC- after ≤ 4 cycles (i.e., 〈 90 days), defined by EuroFlow criteria (Leukemia 2010, p. 521). Using a historical rate with hyperCVAD of 50% (cited above) and defining success if observed rate is 70%, a Simon 2-stage design with α = 0.09 and 80% power led to the following: total sample size of 28 pts, stop enrollment if ≤ 7 of 15 were MFC- after ≤ 4 cycles, and judge the regimen of interest if ≥ 18 of 28 were MFC- after ≤ 4 cycles. Follow-up provided to 7/10/18. RESULTS: 18 pts have received ≥ 1 cycle (Table). 15 (83%) achieved morph CR after ≤ 2 cycles. 8 of 14 (57%; 95% CI 0.29-0.82) evaluable for the primary endpoint were MFC- after ≤ 4 cycles; 3 pts remain too early to evaluate; 1 pt was removed after 2 cycles to continue DA-EPOCH closer to home. 2 Ph+ pts received IM, and 10 received DAS. 7 of 9 (78%) evaluable Ph+ pts were MFC- after ≤ 4 cycles, 3 after 1 cycle. 6 Ph+ pts (5 p190 and 1 p210; 67%) were MRD- by PCR after ≤ 4 cycles. 1 of 5 evaluable Ph- pts (20%) reached MFC- after 1 cycle. MRD by HTS was sought in 5 MFC- patients: clonal sequence was identifiable pre-treatment in 4, and 3 (75%) achieved MRD- by HTS, including 2 after only 1 cycle (both Ph+). 16 (89%) pts developed Grade (Gr) 3+ non-hematologic toxicity at some point throughout treatment, with febrile neutropenia (7 Gr 3), hypofibrinogenemia (4 Gr 3), and GI bleed (2 Gr 3, 1 Gr 5) the most common. 1 pt (6%) died in morph CR after Cycle 3; no other pts stopped due to toxicity. Median cycle length was 21 days (range: 16-27). Once beyond Cycle 2, only 7 of 28 total cycles (25%) yielded ANC 〈 500/μL for ≥ 1 week or any plt 〈 25K/μL (i.e., dose-reduction per the DA-EPOCH paradigm). 5 pts (28%) have undergone HCT, 3 of whom were in CR1 after DA-EPOCH (all Ph+). 5 in CR after DA-EPOCH have relapsed (33%), 1 of whom was post-HCT. No deaths from ALL have occurred. Median and 6-mo EFS are 8 mo and 61% (respectively). Median OS is not reached, and 6-mo OS is 94% (Figure). CONCLUSIONS: DA-EPOCH yields deep remissions (including by HTS) in newly-diagnosed ALL, particularly with TKI for Ph+ disease. Toxicity is manageable and rarely leads to discontinuation or delay. Because of the relatively high rate of early MFC-, the interim stopping rule for efficacy was not met, and enrollment continues. Updated results (including genomic profiling by RNAseq) will be presented. Disclosures Cassaday: Kite Pharma: Research Funding; Pfizer: Consultancy, Research Funding; Merck: Research Funding; Amgen: Consultancy, Research Funding; Incyte: Research Funding; Jazz Pharmaceuticals: Consultancy; Adaptive Biotechnologies: Consultancy; Seattle Genetics: Other: Spouse Employment, Research Funding. Shustov:Seattle Genetics: Research Funding. Becker:GlycoMimetics: Research Funding. Radich:TwinStrand Biosciences: Research Funding.

Description: The Y chromosome encodes male-specific minor histocompatibility (H-Y) antigens that stimulate T- and B-lymphocyte responses after sex-mismatched allogeneic hematopoietic cell transplantation (HCT). A CD8+ cytotoxic T lymphocyte (CTL) clone that recognizes a novel HLA-B*2705–restricted H-Y antigen encoded by the DDX3Y gene was isolated from a male who had received a hematopoietic cell graft from his human leukocyte antigen (HLA)–identical sister. The antigenic peptide is a decamer that differs from the homologous DDX3X-encoded peptide at 4 positions. Expression of DDX3Y and of the H-Y epitope that it encodes was examined by quantitative polymerase chain reaction (PCR) and by CTL recognition assays. Expression of DDX3Y is detected in all myeloid and lymphoid leukemic cells that carry an intact Y chromosome. Moreover, the DDX3Y-encoded H-Y epitope is presented on the surface of both myeloid and lymphoid leukemic cells from male HLA-B*2705+ patients. DDX3Y-specific CTLs prevent engraftment of human acute leukemia in nonobese diabetic/severe combined immune deficient mice, demonstrating that the DDX3Y-encoded H-Y antigen is also expressed in leukemic stem cells. These results demonstrate that CD8+ T-cell responses against DDX3Y have the potential to contribute to graft-versus-leukemia (GVL) activity after female into male allogeneic HCT. This study is registered at http://clinicaltrials.gov as NCT00107354.