Abstract: Exceptional clinical responses produced by the first chimeric antigen receptor T [CAR‐T] cell therapies, and their entry into commercial markets prompted a logarithmic increase in the number of next generation CAR‐T clinical trials. As a result, there is a growing interest in understanding the analytical approaches utilized for reliable monitoring of these “living” drugs, and the challenges encountered during their clinical development. Multiparametric flow cytometry (MFC) assays have played a crucial role in understanding the phenotype and function of first approved CAR‐T therapies. Herein, three main areas for monitoring CAR‐T therapies in clinical trials are discussed: (1) analytical considerations critical for development of MFC assays for the reliable enumeration of CAR‐T levels, (2) operational challenges associated with clinical trial sampling and transportation, and (3) differential cellular kinetics observed by MFC and qPCR analyses and their relationship with efficacy (measurable residual disease levels). Initial experiences described here may enable design of fit‐for‐purpose tools and help to more rapidly advance the development of next generation CAR‐T therapies.

Abstract: The BOLERO-2 phase III trial compared the combination of everolimus and exemestane to placebo and exemestane in 724 postmenopausal women with hormone receptor-positive, HER2-negative advanced breast cancer. Results showed significant improvement in progression-free survival, response rate, and clinical benefit rate. Although significant benefit has been observed in all prospectively defined subgroups, variations were seen among patients. As the first step to delineate the molecular determinants of sensitivity to everolimus and interactions between estrogen receptor and mTOR pathways, we used next-generation sequencing technology to comprehensively assess the genetic alterations in archival tumor specimens. Formalin-fixed, paraffin-embedded archival tumor samples were obtained from 496 patients. DNA was extracted from 348 samples of sufficient quantity and 230 samples (representing approximately 1/3 of all patients) qualified for the analysis. No indication of sampling bias was observed when the data were assessed and compared against several covariates as well as to the full trial data. The coding regions of 182 cancer-related genes were analyzed using an Illumina HiSeq 2000. Mutations and copy number variations were evaluated in 230 samples for which DNA extraction, library construction, and hybrid capture were successful and sequencing depth was sufficient (250-1500×). Alterations predicted to be germline events were discarded. One hundred seventy-three genes were found to be altered in at least one tumor sample. The number of alterations per sample varied from 1 to 24 (average 9 ± 4). Sequence variations (N = 1565) consisted primarily of point mutations (85%), followed by deletions (9%) and insertions (6%). 225 of 230 (98%) patients had at least two sequence variations. The most frequently mutated genes included PIK3CA (49%), TP53 (24%), and ARID1A (16%). ESR1 and IGF1R mutations were detected in 9% and 4% of the cohort, respectively. PIK3CA and AKT1 (6%) mutations were found to be mutually exclusive. Copy-number alterations comprised of 524 amplifications and 27 bi-allelic deletions. The most frequently amplified genes included CCND1 (32%) and FGFR1 (18%). Specific rearrangements assayed in 14 genes occurred infrequently (9% of the samples). These results demonstrated the feasibility of performing large-scale next-generation sequencing in a global phase III clinical trial. High alteration frequencies were observed in specific genes, resulting in activated PI3K, FGFR1, and estrogen receptor pathways, corroborating the rationale of concomitant targeting of the mTOR and estrogen receptor pathways. The results generate testable hypotheses for the development of combinations of new targeted therapies in this patient population. Citation Format: Jose Baselga, Martine Piccart, Hope Rugo, David Chen, Howard A. Burris, Mario Campone, Shinzaburo Noguchi, Alejandra Perez, Inas Deleu, Mikhail Shtivelband, Louise Provencher, Adnan Derti, Alan Huang, Rob McDonald, Creton Kalfoglou, Douglas Robinson, Tetiana Taran, Tarek Sahmoud, David Lebwohl, Gabriel N. Hortobagyi. Assessment of genetic alterations using next-generation sequencing in postmenopausal women with hormone receptor-positive, HER2-negative advanced breast cancer: results from the BOLERO-2 phase III trial. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4564. doi:10.1158/1538-7445.AM2013-4564

Abstract: 10619 Background: The efficacy and safety of everolimus, an oral mTOR inhibitor, was assessed in two randomized, double-blind, placebo-controlled, phase 3 trials: EXIST-1 (NCT00789828) and EXIST-2 (NCT00790400). EXIST-1 examined everolimus for the treatment of subependymal giant cell astrocytoma (SEGA) associated with TSC and EXIST-2 for the treatment of renal angiomyolipoma (AML) associated with either TSC or sporadic lymphangioleiomyomatosis. In each instance, everolimus was superior to placebo for the primary endpoints, SEGA and renal AML response rates. Inhibitors of mTOR have antiangiogenic effects on tumor growth in vitro and in vivo. Methods: Patients were randomized to receive everolimus (n=78) starting at 4.5 mg/m 2 /day (target trough, 5-15 ng/mL) or placebo (n=39) in EXIST-1 and 10 mg/day everolimus (n=79) or placebo (n=39) in EXIST-2. Plasma samples were taken at baseline and pre-dosing on day 1 of weeks 4, 12, 24, 36, and 48 of treatment. Angiogenic markers of interest were vascular endothelial growth factor (VEGF)-A and -D, placental growth factor (PlGF), soluble VEGF receptor-1 (sVEGFR1), soluble VEGF receptor 2 (sVEGFR2), c-Kit, and collagen type IV. Results: Compared with placebo, a sustained ~30% and ~60% increase in VEGF-A was observed in the everolimus arm of EXIST-1 and EXIST-2, respectively. A concomitant decrease in collagen type IV (~25% EXIST-1; ~45% EXIST-2) and sVEGFR2 (~25% both trials) was also observed in the everolimus arm. A sustained decrease (~60%) in VEGF-D was observed in the everolimus arm of EXIST-2, but not EXIST-1. In both studies, no change was observed in PlGF, sVEGFR1, or c-Kit plasma concentrations in the everolimus arm or any biomarkers evaluated in the placebo arm. Baseline sVEGFR2 and VEGF-D were ~40% and ~4-fold higher, respectively, while VEGF-A was ~50% lower in EXIST-2 compared with EXIST-1. A similar baseline plasma concentration for the other biomarkers was noted in both studies. Conclusions: Patients presenting with SEGA or renal AML associated with TSC had a reduction in plasma concentrations of sVEGFR2, collagen type IV, and VEGF-D (AML only) and an increase in VEGF-A. Everolimus may have antiangiogenic properties in TSC patients.

Abstract: The antiviral activities of intracellularly expressed antisense RNAs complementary to the human immunodeficiency virus type 1 (HIV-1) pol , vif , and env genes and the 3′ long terminal repeat (LTR) sequence were evaluated in this comparative study. Retroviral vectors expressing the antisense RNAs as part of the Moloney murine leukemia virus LTR promoter-directed retroviral transcript were constructed. The CD4 + T-cell line CEM-SS was transduced with retroviral constructs, and Northern blot analyses showed high steady-state antisense RNA expression levels. The most efficient inhibition of HIV-1 replication was observed with the env antisense RNA, followed by the pol complementary sequence, leading to 2- to 3-log 10 reductions in p24 antigen production even at high inoculation doses (4 × 10 4 50% tissue culture infective doses) of the HIV-1 strain HXB3. The strong antiviral effect correlated with a reduction of HIV-1 steady-state RNA levels, and with intracellular Tat protein production, suggesting that antisense transcripts act at an early step of HIV-1 replication. A lower steady-state antisense RNA level was detected in transduced primary CD4 + lymphocytes than in CEM-SS cells. Nevertheless, replication of the HIV-1 JR-CSF isolate was reduced with both the pol and env antisense RNA. Intracellularly expressed antisense sequences demonstrated more pronounced antiviral efficacy than the trans -dominant RevM10 protein, making these antisense RNAs a promising gene therapy strategy for HIV-1.

Abstract: Introduction Detection of minimal residual disease (MRD) is an important predictor of patient outcome following treatment of B-cell acute lymphoblastic leukemia (B-ALL). We assessed concordance between two MRD assays, with different assay sensitivities, to determine which MRD detection method could support early relapse detection. Immunoglobulin next generation sequencing (Ig NGS) and flow cytometry (FC) were tested in samples from two clinical trials ELIANA (NCT02435849) and ENSIGN (NCT02228096) for pediatric relapsed and refractory B-ALL patients treated with tisagenlecleucel. We also assessed whether using blood with Ig NGS would be comparable to BM testing with FC. Finally we analyzed whether clonal evolution, as detected by Ig NGS, occurred during of the course of therapy for both CD19+ and CD19- relapse patients. Methods In this analysis, bone marrow and peripheral blood specimens at screening (pre-tisagenlecleucel infusion), post-infusion and relapse were tested. Ig NGS was performed in 300 samples from 88 patients. 237 samples from 83 patients also had FC MRD results available. MRD was measured on fresh blood and bone marrow using a 3-tube FC assay (CD10, CD19, CD13, CD20, CD22, CD33, CD34, CD38, CD45, CD58, CD123). The FC MRD assay has a lower limit of sensitivity of 0.01% of white blood cells. Ig NGS detection of MRD was performed using the Adaptive Biotechnology's NGS MRD assay. MRD quantitative values, along with the qualitative MRD calls at each assay sensitivity level (10-4, 10-5 and 10-6) were reported. At baseline, 85 out of 88 samples had informative clones. Results and Conclusions To examine the comparability of flow cytometry and Ig NGS methods in assessing MRD, baseline and post-treatment samples were tested. Baseline samples, which had a high disease burden, showed 100% MRD concordance between both assays. However, post-treatment, where the leukemic burden was dramatically reduced, Ig NGS detected a greater number of MRD positive samples compared to FC, at each sensitivity level tested (10-4, 10-5 and 10-6). At the highest sensitivity level of 10-6, Ig NGS was able to detect 18% more MRD positive post-treatment samples. Importantly, Ig NGS was able to detect MRD positivity 1-4 months ahead of clinical relapse in a small number of relapsed patients, whether relapse was CD19+ or CD19-. This may provide an important window of opportunity for pre-emptive treatment while a patients' tumor burden is still low. In B-ALL, it has previously been described that MRD levels can be one to three logs lower in blood compared to bone marrow (VanDongen JJ et al. Blood 2015). Our results support these findings whereby MRD burden in bone marrow was higher than in blood using both FC and Ig NGS. We next set out to determine if the increased sensitivity afforded by the Ig NGS assay could provide a level of MRD detection in the blood comparable to FC in the bone marrow. In patients with matching data available, Ig NGS was able to detect more MRD positive blood samples than FC MRD positive bone marrow samples. This suggests that monitoring of MRD using Ig NGS in the blood holds the potential to be used as a surrogate for FC MRD in bone marrow. The relationship between MRD and prognosis was examined. Patients who were MRD negative by both Ig NGS and FC at the end of first month post-infusion had better progression-free survival and overall survival compared to those with detectable MRD. Tumor clonality will be further analyzed to understand sub-clone composition at baseline and clonal evolution following tisagenlecleucel treatment. Taken together, these results highlight the importance of using a highly sensitive assay, such as Ig NGS, when monitoring for MRD. MRD detection by Ig NGS holds the potential to identify early response/relapse in patients, which could provide a window of opportunity for additional intervention before morphological relapse. Ongoing studies with larger patient groups will provide further information on the applicability of Ig NGS MRD detection and its association with long-term outcome in tisagenlecleucel-treated pediatric r/r B-ALL patients. Disclosures Pulsipher: Novartis: Consultancy, Honoraria, Speakers Bureau; CSL Behring: Consultancy; Amgen: Honoraria; Adaptive Biotech: Consultancy, Research Funding. Han:Novartis Pharmaceuticals Corporation: Employment, Equity Ownership. Quigley:Novartis Pharmaceuticals Corporation: Employment. Kari:Adaptimmune LLC: Other: previous employment within 2 years; Novartis Pharmaceuticals Corporation: Employment. Rives:Shire: Consultancy, Other: Symposia, advisory boards ; Amgen: Consultancy, Other: advisory board ; Novartis Pharmaceuticals Corporation: Consultancy, Other: Symposia, advisory boards ; Jazz Pharma: Consultancy, Other: Symposia, advisory boards . Laetsch:Bayer: Consultancy; Eli Lilly: Consultancy; Pfizer: Equity Ownership; Novartis Pharmaceuticals Corporation: Consultancy; Loxo Oncology: Consultancy. Myers:Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Research Funding, Speakers Bureau. Qayed:Novartis: Consultancy. Stefanski:Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Speakers Bureau. Baruchel:Shire: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; Servier: Consultancy; Roche: Consultancy; Jazz Pharmaceuticals: Consultancy, Honoraria, Other: Travel, accommodations or expenses; Celgene: Consultancy. Bader:Cellgene: Consultancy; Riemser: Research Funding; Medac: Patents & Royalties, Research Funding; Neovii: Research Funding; Novartis: Consultancy, Speakers Bureau. Yi:Novartis Pharmaceuticals Corporation: Employment. Kalfoglou:Novartis Pharmaceuticals Corporation: Employment. Robins:Adaptive Biotechnologies: Consultancy, Employment, Equity Ownership, Patents & Royalties. Yusko:Adaptive Biotechnologies: Employment, Equity Ownership. Görgün:Novartis Pharmaceuticals Corporation: Employment. Bleickardt:Novartis Pharmaceuticals Corporation: Employment. Wong:Novartis Pharmaceuticals Corporation: Employment, Equity Ownership. Grupp:Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy; Adaptimmune: Consultancy; University of Pennsylvania: Patents & Royalties.

Abstract: The potential of hematopoietic stem cells (HSCs) from human immunodeficiency virus type-1 (HIV-1)–infected individuals, eg, self-renewal and multilineage differentiative capacity, might be perturbed due to the underlying disease. In this study, we assessed the HSC activity in the CD34+Thy-1+ cell population of peripheral blood stem cells (PBSCs) of three asymptomatic HIV-1–infected individuals after granulocyte colony-stimulating factor (G-CSF; 10 μg/kg/d) mobilization. On day 4 of G-CSF treatment, 0.8% to 1% of the total blood mononuclear cells were CD34+. Leukapheresis followed by a two-step cell isolation process yielded a CD34+Thy-1+ cell population of high purity (76% to 92% CD34+Thy-1+ cells). This cell population showed no evidence of HIV-1–containing cells based on a semiquantitative HIV-1 DNA polymerase chain reaction. Furthermore, the purified cells showed normal hematopoietic potential in in vitro clonogenic assays. Successful gene transfer into committed progenitor cells (colony-forming units-cells) and more primitive stem/progenitor cells (long-term culture colony-forming cells) could be shown after amphotropic retroviral transduction. These data provide evidence that the CD34+Thy-1+ stem cell compartment can be mobilized and enriched in early stage HIV-1–infected patients. Furthermore, successful transduction of this cell population as a prerequisite for stem cell-based clinical gene therapy protocols was demonstrated.