Abstract: Hairy cell leukemia (HCL) is a rare B-cell malignancy, and there is a need for novel treatments for patients who do not benefit from purine analogs. Ibrutinib, an oral agent targeting Bruton tyrosine kinase in the B-cell receptor signaling pathway, is highly effective in several malignancies. Its activity in HCL was unknown, so we conducted a multisite phase 2 study of oral ibrutinib in patients with either relapsed classic or variant hairy cell leukemia. The primary outcome measure was the overall response rate (ORR) at 32 weeks, and we also assessed response at 48 weeks and best response during treatment. Key secondary objectives were characterization of toxicity and determination of progression-free survival (PFS) and overall survival (OS). Thirty-seven patients were enrolled at 2 different doses (24 at 420 mg, 13 at 840 mg). The median duration of follow-up was 3.5 years (range, 0-5.9 years). The ORR at 32 weeks was 24%, which increased to 36% at 48 weeks. The best ORR was 54%. The estimated 36-month PFS was 73% and OS was 85%. The most frequent adverse events were diarrhea (59%), fatigue (54%), myalgia (54%), and nausea (51%). Hematologic adverse events were common: anemia (43%), thrombocytopenia (41%), and neutropenia (35%). Ibrutinib can be safely administered to patients with HCL with objective responses and results in prolonged disease control. Although the initial primary outcome objective of the study was not met, the observation of objective responses in heavily pretreated patients coupled with a favorable PFS suggests that ibrutinib may be beneficial in these patients. This trial was registered at www.clinicaltrials.gov as #NCT01841723.

Abstract: The Bruton's Tyrosine Kinase (BTK) inhibitor ibrutinib is very effective in chronic lymphocytic leukemia (CLL). Resistance in CLL is at least in part mediated by acquired mutations in BTK or down-stream PLCG2. Here we describe the largest institutional cohort of CLL patients treated with ibrutinib, focusing on risk factors for relapse, prevalence of known resistance mutations, and development of a monitoring strategy to identify patients at risk for relapse. All 308 patients from The Ohio State University Comprehensive Cancer Center participating in 4 clinical trials of ibrutinib were included. Deep sequencing for BTK and PLCG2 was performed using Ion Torrent Personal Genome Machine and covered coding regions of both genes. Preclinical experiments used XLA cell lines (Coriell Institute) infected with lentiviral constructs containing empty vector, wild type BTK, or C481S BTK. With a median follow-up of 40.5 months (range 4-71 months), 136 patients remain on study, 14 have received transplant or therapy elsewhere, and 158 have discontinued. 83 patients experienced disease progression, classified as Richter's or PLL transformation (n=28) and progressive CLL (n=55). Using multivariable models, baseline risk factors for ibrutinib discontinuation due to transformation include complex karyotype (p 〈 0.01) and MYC abnormalities on FISH (p=0.051). Age 〈 65, del(17p) by FISH, and complex karyotype all associated with discontinuation due to CLL progression (p 〈 0.05). Relapse generally has a poor prognosis, with a median survival from ibrutinib discontinuation for patients with transformation and progressive CLL of 3.9 (95% CI 2.0 to 10.1) and 22.7 (95% CI 13.5 to not reached) months, respectively. 46 patients with progressive CLL had samples at relapse available for deep sequencing. Of these, 39 had mutations in BTK and/or PLCG2 (84.8%; 95% CI 71.1-93.7). Distribution of mutation included patients with BTK C481 mutation only (n=30), mutation in PLCG2 only (n=3) and both BTK/PLCG2 genes (n=6). Given the poor prognosis at relapse and presence of acquired mutations in the majority of progressive CLL patients, we were interested to evaluate whether the emergence of small clones of mutated cells could be used as a biomarker to predict relapse. For 15 patients with BTK or PLCG2 mutations, serial samples were available prior to relapse, and a clone of resistant cells could first be detected a median of 9.3 months prior to clinical relapse (95% CI: 7.6-11.7). Based on the finding that patients who relapsed on ibrutinib often had rapid progression and poor outcomes, we initiated a clinical-grade monitoring strategy in our institutional CLIA-certified molecular lab starting in November 2014. Mutational analysis of the entire coding regions of BTK and PLCG2 was performed on a cohort of 112 patients every 3 months. To date, 8 patients have clinically relapsed and all 8 had BTK C481S mutations with expansion of the clone prior to relapse. BTK C481S mutations of over 1% allelic frequency were detected in an additional 8 patients. All patients have had expansion of the resistant clones and all but one, who discontinued therapy without clinical relapse, have had increasing circulating CLL cells in the peripheral blood. Four of these 7 patients have also had increasing lymph node size on CT scan. In 11 patients who relapsed with BTK C481S, samples were available following discontinuation. In 5 patients treated subsequently with venetoclax, allelic fractions of C481S decreased dramatically or were eliminated, but in 6 patients treated with other agents, the mutant clone persisted. To investigate whether this was due to differences in biology between wild type and C481S BTK, we created XLA cell lines without BTK protein expression stably expressing BTK, or C481S BTK. C481S BTK showed enhanced BCR signaling as evidenced by pERK and cMYC expression, and enhanced migration compared to wild type BTK (p=0.04). This demonstrates for the first time that this acquired resistance mutation fundamentally alters the tumor cell biology. These data confirm our initial findings that relapse on ibrutinib is primarily mediated through mutations in BTK and PLCG2 and suggest that the presence of these mutations may have utility as an early biomarker to predict clinical relapse. Early identification of relapsing patients could improve outcomes by facilitating initiation of adjunct therapies such as venetoclax to eliminate the small resistant clone. Disclosures Woyach: Karyopharm: Research Funding; Morphosys: Research Funding; Acerta: Research Funding. Jones:Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics, LLC, an AbbVie Company: Membership on an entity's Board of Directors or advisory committees, Research Funding. Andritsos:Hairy Cell Leukemia Foundation: Research Funding. Awan:Innate Pharma: Research Funding; Pharmacyclics: Consultancy; Novartis Oncology: Consultancy. Lozanski:Beckman Coulter: Research Funding; Stemline Therapeutics Inc.: Research Funding; Boehringer Ingelheim: Research Funding; Genentech: Research Funding.

Abstract: Gene transfer for hemophilia A offers the potential for a one-time disease altering treatment, eliminating the risk of bleeds while freeing patients from the burden of lifelong chronic therapy. SPK-8011 consists of a bioengineered AAV capsid expressing B domain-deleted factor VIII (FVIII) under the control of a liver-specific promoter. In pre-clinical studies, we showed a dose-dependent increase in circulating FVIII levels in non-human primates infused with SPK-8011. We conducted a Phase I/II study of SPK-8011 in 12 men (ages 18-52 years) with severe (n=11) or moderately severe (n=1) hemophilia A. Prior to gene therapy, 8/12 subjects were on prophylaxis, and 4/12 received on-demand treatment. Subjects were enrolled in 1 of 3 dose cohorts, 5E11 vg/kg (n=2), 1E12(n=3), or 2E12(N=7). Safety analysis showed no inhibitor formation. A single serious adverse event (SAE) was reported, associated with an immune response to AAV capsid characterized by simultaneous decline in FVIII, transaminase elevation peaking at Grade 2, and development of positive IFN-g ELISPOTs to capsid was observed beginning at week 6.5 after vector infusion. The asymptomatic transaminase elevation did not respond promptly to initiation of oral steroids and the subject received two infusions of IV methylprednisolone in hospital, thereby fulfilling SAE criteria. The SAE has resolved. All vector doses led to expression of FVIII levels adequate to prevent bleeding and allow cessation of prophylaxis. Across the 12 subjects at 3 doses, there was a 97% reduction in annualized bleeding rate (ABR), and a 97% reduction in annualized infusion rate (AIR). In the 5E11 dose cohort, mean FVIII levels beginning 12 weeks post vector infusion are 13%, with no bleeding events, no elevated transaminase levels, no use of steroids, and stable FVIII expression out to 66 weeks (ongoing). In the 1E12 dose cohort, mean FVIII levels are 15% beginning at 12 weeks post-infusion and stable out to 46 weeks (ongoing). The first subject in the 1E12 dose infused a single dose of factor concentrate for a spontaneous joint bleed at day 159, and the second received multiple infusions for a traumatic bleed beginning at day 195. Declining FVIII levels triggered initiation of a course of tapering steroids in both subjects, at 12 and 7 weeks post vector infusion respectively, which led to stabilization of FVIII levels. The third subject has had no bleeding and did not receive factor infusions or steroids. In the 2E12 (highest) dose cohort, 5/7 subjects currently have FVIII levels 16-49%; their mean FVIII level beginning 12 weeks post-infusion is 30%. No bleeds have been reported among these subjects beginning 4 weeks post vector infusion. Additionally, 5/7 subjects in the 2E12 dose cohort received a course of steroids, initiated at 6-11 weeks post vector infusion, for one or more of the following: declining FVIII levels, rise in ALT above subject baseline, or elevated IFN-g ELISPOTs to AAV capsid. Steroid initiation normalized ALT levels and extinguished the ELISPOT signal in all cases; 2 subjects showed limited stabilization of FVIII levels, which fell to 〈 6% likely due to the immune response. For one of these, no bleeds have been reported through 12 weeks of follow up; the other has had 4 bleeds through 37 weeks of observation. Our data indicate that the kinetics of SPK-8011 expression are similar to those observed with investigational SPK-9001 for hemophilia B. All subjects demonstrated durable transgene expression for up to 66 weeks post vector administration (data cutoff 7/13/18). On cumulative follow up of 345 weeks, SPK-8011 demonstrated a favorable safety profile with no evidence of FVIII inhibitor formation, a single SAE, and 2/12 subjects who experienced ALT elevation above the upper limit of normal that resolved with steroid initiation. Data from the 5E11 (lowest) dose cohort are consistent with published natural history data indicating FVIII:C 12% is adequate to prevent spontaneous bleeding events. Given that 2 subjects in the 2E12 dose cohort lost some FVIII expression, which then stabilized on steroids, and 5/7 subjects in this cohort required steroids, prophylactic steroids may be warranted. We conclude that infusion of SPK-8011 in 12 subjects with severe or moderately severe hemophilia A resulted in safe, durable, dose-dependent FVIII expression resulting in an excellent preliminary efficacy profile with an overall 97% reduction in ABR and AIR. Disclosures High: Spark Therapeutics: Employment, Equity Ownership, Patents & Royalties. George:University of Pennsylvania: Equity Ownership; Pfizer: Consultancy. Ragni:CSL Behring: Research Funding; Alnylam: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sangamo: Research Funding; Shire: Research Funding; Biomarin: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novo Nordisk: Research Funding; Bioverativ: Consultancy, Research Funding; MOGAM: Membership on an entity's Board of Directors or advisory committees; SPARK: Consultancy, Research Funding. Croteau:Novo Nordisk: Consultancy; Octapharma: Consultancy, Honoraria, Research Funding; Pfizer: Research Funding; Spark Therapeutics: Research Funding; Tremeau Pharmaceuticals: Consultancy; Genetech: Consultancy, Research Funding; CSL-Behring: Consultancy; Catalyst Biosciences: Consultancy; Bioveritiv: Consultancy; Biomarin: Consultancy; Bayer: Consultancy; Baxalta/Shire: Consultancy, Research Funding. Joseney-Antoine:Spark Therapeutics: Employment. Macdougall:Spark Therapeutics: Employment. Tompkins:Spark Therapeutics: Employment. Hait:Spark Therapeutics: Employment. Couto:Spark Therapeutics: Employment. Bassiri:Spark Therapeutics: Employment. Valentino:Spark Therapeutics: Employment. Carr:Spark Therapeutics: Employment. Hui:Spark Therapeutics: Employment. Wachtel:Spark Therapeutics: Employment. Takefman:Spark Therapeutics: Employment. Mingozzi:Spark Therapeutics, Inc.: Employment. Anguela:Spark Therapeutics, Inc.: Employment. Reape:Spark Therapeutics: Employment.

Abstract: Autologous ex vivo hematopoietic stem cell gene therapy is particularly relevant in lysosomal storage diseases (LSD) as it offers the prospect of both a safe transplant, as observed in immune deficiency and hematologic illness, and an effective transplant, since it delivers greater enzyme levels to host tissues than is possible in allogeneic transplant. We report early data from such an approach in an allogeneic transplant refractory LSD, Mucopolysaccharidosis type IIIA (MPSIIIA, Sanfilippo syndrome). Background: MPSIIIA is a LSD caused by mutations in the SGSH gene leading to a deficiency of the enzyme N-sulfoglucosamine sulfohydrolase. As a result there is accumulation of heparan sulfate, with clinical manifestations of developmental delay, regression of previously acquired skills, hyperactivity, seizures and progressive cognitive decline leading to an early death at the end of the second decade of life. Unlike some other LSDs, MPSIIIA is unresponsive to allogeneic stem cell transplant (Hoogerbrugge et al., The Lancet 1995; Sivakumur & Wraith, Journal of inherited metabolic disease 1999), with the donor cells unable to deliver enough enzyme for clinically meaningful cross-correction. Significant pre-clinical work undertaken at the University of Manchester led to the design of the lentiviral vector containing the SGSH gene and a CD11b (myeloid) promoter. In murine studies MPSIIIA mice underwent stem cell mobilization and collection and stem cells were transduced with the lentiviral vector ex vivo. The mice received myeloablative busulfan before being infused with the autologous transduced stem cells. Enzyme expression in the brain was high with normalised heparan sulfate and improvement in the behavior of the mice (Sergijenko et al., Molecular Therapy 2013). Transduction and transplant of human CD34+ stem cells to humanized NSG mice demonstrated stable engraftment with no evidence of viral shedding or transformational potential (Ellison et al., Molecular Therapy-Methods & Clinical Development 2019), further adding to the safety profile and the translation of this work to the clinic. Study Design and Methods: This is a phase I/II safety and tolerability study. It is open-label and aims to recruit up to 5 patients. Patients enrolled into the trial are between the age of 3 and 24 months, have confirmed classical MPSIIIA (either by known genotypes, somatic features or family history) and have preserved neurocognitive function (DQ ≥80) before commencing the trial. Patients undergo stem cell mobilization and peripheral collection of CD34+ cells. The SGSH gene under the CD11b promoter is introduced by the lentiviral vector. Patients then receive myeloablative busulfan before infusion of the autologous transduced stem cells. Follow up takes place over 3 years. The primary end point is to assess the safety and tolerability of the transduced stem cell product. The primary efficacy endpoint is SGSH activity in leukocytes at 12 months. Several secondary and exploratory end points are also to be reported including neurocognitive outcomes. Current trial status: We report the preliminary data of the first treated patient recruited to the trial including the mobilization, transplant and sustained engraftment of gene-modified cells by vector copy number. Supra-physiological enzyme expression in multiple lineages - 150 fold increase above median in leukocytes and 200 fold increase above control in myeloid lineage - and substrate reduction in plasma, CSF and urine - reduced in urine to normal range by 3 months - has been observed. Disclosures Bigger: Orchard Therapeutics: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Research Funding. Thrasher:Orchard Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership; Rocket Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Generation bio: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership; 4Bio Capital: Consultancy, Membership on an entity's Board of Directors or advisory committees. Jones:Orchard Therapeutics: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees.

Abstract: True long-term nonprogressors (LTNPs)/elite controllers (ECs) maintain durable control over HIV replication without antiretroviral therapy. Herein we describe 4 unique persons who were distinct from conventional LTNPs/ECs in that they had extraordinarily low HIV burdens and comparatively weak immune responses. As a group, typical LTNPs/ECs have unequivocally reactive HIV-1 Western blots, viral loads below the lower threshold of clinical assays, low levels of persistent viral reservoirs, an over-representation of protective HLA alleles, and robust HIV-specific CD8+ T-cell responses. The 4 unique cases were distinguished from typical LTNPs/ECs based on weakly reactive Western blots, undetectable plasma viremia by a single copy assay, extremely low to undetectable HIV DNA levels, and difficult to isolate replication-competent virus. All 4 had at least one protective HLA allele and CD8+ T-cell responses that were disproportionately high for the low antigen levels but comparatively lower than those of typical LTNPs/ECs. These unique persons exhibit extraordinary suppression over HIV replication, therefore, higher-level control than has been demonstrated in previous studies of LTNPs/ECs. Additional insight into the full spectrum of immune-mediated suppression over HIV replication may enhance our understanding of the associated mechanisms, which should inform the design of efficacious HIV vaccines and immunotherapies.

Abstract: Heparin-induced thrombocytopenia (HIT) is a life-threatening, prothrombotic, antibody-mediated disorder. To maximize the likelihood of recovery, early and accurate diagnosis is critical. Widely available HIT assays, such as the platelet factor 4 (PF4) heparin enzyme-linked immunosorbent assay (ELISA) lack specificity, and the gold-standard carbon 14–labeled serotonin release assay (SRA) is of limited value for early patient management because it is available only through reference laboratories. Recent studies have demonstrated that pathogenic HIT antibodies selectively activate PF4-treated platelets and that a technically simpler assay, the PF4-dependent P-selectin expression assay (PEA), may provide an option for rapid and conclusive results. Based upon predefined criteria that combined 4Ts scores and HIT ELISA results, 409 consecutive adults suspected of having HIT were classified as disease positive, negative, or indeterminate. Patients deemed HIT indeterminate were considered disease negative in the primary analysis and disease positive in a sensitivity analysis. The ability of PEA and SRA to identify patients judged to have HIT was compared using receiver operating characteristic curve statistics. Using these predefined criteria, the diagnostic accuracy of PEA was high (area under the curve [AUC] , 0.94; 95% confidence interval [CI], 0.87-1.0) and similar to that of SRA (AUC, 0.91; 95% CI, 0.82-1.0). In sensitivity analysis, the AUCs of PEA and SRA were also similar at 0.88 (95% CI, 0.78-0.98) and 0.86 (95% CI, 0.77-0.96), respectively. The PEA, a technically simple nonradioactive assay that uses ∼20-fold fewer platelets compared with the SRA, had high accuracy for diagnosing HIT. Widespread use of the PEA may facilitate timely and more effective management of patients with suspected HIT.

Abstract: Introduction: Treatment for childhood ALL has evolved over the last five decades and transformed a once fatal disease to one with 5-year survival rates in excess of 90%. However, aging survivors of ALL are at risk for adverse health and social outcomes that significantly impact overall quality of life. Comprehensive clinical assessments of adults previously treated for childhood ALL are lacking, with most reports relying upon registry or self-reported data. To evaluate the impact of changes in therapy for ALL on the risk of late effects, we systematically screened and clinically assessed the largest cohort to date of adult survivors of childhood ALL treated over the last five decades. Our aim was to determine the occurrence of and risk factors for late chronic health conditions, neurocognitive deficits, and physical function limitations. Methods: History/physical examination, laboratory analysis, neurocognitive testing, and physical fitness were assessed among 934 (65.7% of eligible) survivors of pediatric ALL ≥ 18 years of age, treated at St. Jude Children’s Research Hospital ≥ 10 years ago, and participating in the SJLIFE cohort study. Chronic health conditions were graded per CTCAE criteria (v4.03). Neurocognitive function was measured in the domains of attention and executive function using standard clinical tests. Using national norms, age-adjusted z-scores were calculated. Mild impairment was defined as a z-score between -1 to -2, severe as ≤ -2. Aerobic function was measured with the 6-minute walk test [6MWT] (abnormal ≤ 490 meters) and mobility by the timed up and go test [TUG] (abnormal 〉 6 seconds to rise from a chair, walk 3 meters, return, and sit). Log-binomial linear models were used to evaluate relative risk (RR) and 95% confidence intervals (CI) for associations between treatment and outcome. Results: Survivors (50% female, 90% white) had a median age at diagnosis of 5.1 years (range 0.2-19.5), 31.2 years (18.4-59.7) at evaluation, and were 25.0 years (10.5-47.7) from diagnosis. Three hundred twenty-one (34.4%) received ≥ 24 Gy cranial radiation (CRT), 224 (24%) 〈 24 Gy, and 389 (41.6%) no CRT, of whom 373 were treated with high dose methotrexate (HDMTX). Nearly the entire cohort had at least one chronic condition (98%) and 59% a severe/life-threating condition (grades 3-4). The most common were obesity (70%), hypertension (70%), and peripheral sensory neuropathy (51.6%). Chronic conditions were more common in males (52% vs. 48%, p=0.003). By age 40, the cumulative prevalence of at least one chronic condition was 90% and 69% for a severe/life-threatening condition. After adjusting for age at diagnosis, age at evaluation, and gender, survivors exposed to CRT were more likely to have a severe/life-threatening condition (RR 1.3 95% CI 1.1-1.5) than those not exposed to CRT. One hundred sixty-eight survivors had 228 second neoplasms (121 malignant [79 non-melanoma skin cancers], 107 benign). Attention and executive function deficits (mild and severe) were identified across each category of CNS directed therapy (≥ 24 Gy CRT, 〈 24 Gy CRT, HDMTX) [table]. After adjustment for age at diagnosis and gender, those treated with CRT only were more likely to have mild (RR 1.4 95% CI 1.0-1.9) and severe (RR 2.2 95% CI 1.7-3.0) attention and executive function (RR 1.4 95% CI 1.1-1.8, RR 1.7 95% CI 1.4-2.2) deficits compared to those treated with HDMTX. Abnormal 6MWT was identified among 20.5% of the ALL survivors, 26.7% of those treated with CRT and 15.1% HDMTX. TUGS was abnormal in 35.5% of the cohort, 41.6% treated with CRT, 27.3% HDMTX. Adjusting for age at diagnosis, age at evaluation, gender, BMI, and educational attainment, CRT was significantly associated with impaired 6MWT (RR 1.8 95% CI 1.1-2.9) but not TUGS (RR 1.1 95% CI 0.9-1.4). Conclusions: Systematic evaluation identified a substantial number of medical conditions, deficits in attention and executive function, and functional impairments in adult survivors of childhood ALL at a young age. Removal of CRT has significantly reduced, but not eliminated, the occurrence of late effects. Maintaining health and quality of life in this population requires significant medical surveillance, counseling, and lifestyle modifications. Table CNS Directed Threapy Neurocognitive Deficit ≥ 24 Gy 〈 24 Gy HDMTX Attention Mild 19.3% 20.6% 17.1% Severe 31.6% 22.8% 14.1% Executive Function Mild 28.3% 28.2% 24.4% Severe 35.4% 24.1% 20.3% Disclosures No relevant conflicts of interest to declare.