ALBERT

Description: Adults and children with high-risk CRLF2-rearranged acute lymphoblastic leukemia (ALL) respond poorly to current cytotoxic chemotherapy and suffer unacceptably high rates of relapse, supporting the need to use alternative therapies. CRLF2 encodes the thymic stromal lymphopoietin (TSLP) receptor, which activates cell signaling in normal lymphocytes on binding its ligand, TSLP. We hypothesized that aberrant cell signaling occurs in CRLF2-rearranged ALL and can be targeted by signal transduction inhibitors of this pathway. In a large number of primary CRLF2-rearranged ALL samples, we observed increased basal levels of pJAK2, pSTAT5, and pS6. We thus characterized the biochemical sequelae of CRLF2 and JAK alterations in CRLF2-rearranged ALL primary patient samples via analysis of TSLP-mediated signal transduction. TSLP stimulation of these leukemias further induced robust JAK/STAT and PI3K/mTOR pathway signaling. JAK inhibition abrogated phosphorylation of JAK/STAT and, surprisingly, of PI3K/mTOR pathway members, suggesting an interconnection between these signaling networks and providing a rationale for testing JAK inhibitors in clinical trials. The PI3K/mTOR pathway inhibitors rapamycin, PI103, and PP242 also inhibited activated signal transduction and translational machinery proteins of the PI3K/mTOR pathway, suggesting that signal transduction inhibitors targeting this pathway also may have therapeutic relevance for patients with CRLF2-rearranged ALL and merit further preclinical testing.

Description: Abstract 2529 Genomic alterations involving the CRLF2 gene lead to over-expression of intact CRLF2 and have significant prognostic value in pediatric BCP-ALL. Not only do patients with these lesions have inferior outcomes, they also have a very high frequency of JAK1 and JAK2 mutations and may be candidates for targeted therapies. The two major CRLF2 lesions include cryptic translocations that produce IgH@-CRLF2 and interstitial deletions of the pseudoautosomal region of X/Y causing P2RY8-CRLF2 fusion. Both lesions can be detected by fluorescence in situ hybridization (FISH), and genomic PCR or RT-PCR can identify P2RY8-CRLF2. To develop rapid and inexpensive assays for detection/screening of these events, we developed a flow cytometry based method to measure CRLF2 expression and compared this assay to quantitative RT-PCR (qPCR) measurement of CRLF2 expression by evaluating their performance in an unselected cohort of 279 newly diagnosed pediatric BCP-ALL patients consecutively enrolled on the COG AALL03B1 biology/classification study between 10/30/09-5/1/10. Flow cytometry was performed first in real time on diagnostic specimens shipped to a central COG reference laboratory and then residual diagnostic material was shipped to a separate laboratory for RNA isolation and qPCR analysis. Of the 279 cases analyzed by flow, 257 (92%) yielded sufficient RNA quality and quantity for qPCR analysis. In our previous studies with qPCR and CRLF2 it was shown that CRLF2 lesions occurred only among those cases with the highest expression (ΔCt 〈 8). In order to assure that we identified all cases with CRLF2 lesions, we performed FISH and P2RY8-CRLF2 PCR on all cases with qPCR expression ΔCt 〈 10 (n = 109) and an additional 14 cases with a flow blast/lymph CRLF2 mean fluorescence intensity (MFI) ratio 〉1.15. Of these 123 cases, 11 were determined by FISH to have the IGH@-CRLF2 translocation and 15 were shown to have P2RY8-CRLF2 fusions by PCR. Figure 1 shows the locations of these genomic lesion-positive cases among the qPCR (panel A) and flow cytometry (panel B) CRLF2 expression data. The overall frequency of CRLF2 lesions among these patients is 10.1% (assuming all lesions were identified among the highest expressing cases) and, surprisingly, the frequencies of IgH@-CRLF2 and P2RY8-CRLF2 were very similar (4.3% and 5.8%, respectively). With both methods, the 11 IgH@-CRLF2 cases were found to be the highest expressing (among the top 12 cases by qPCR and 16 cases by flow). Receiver operating curve analysis of each method identified cutoffs with excellent performance: qPCR cutoff CRLF2 ΔCt = 5.47 with 96.9% specificity and 88.5% sensitivity; flow cutoff MFI CRLF2 ratio of 2.04 with 95.9% specificity and 92.3% sensitivity. The broader dynamic range of the qPCR assay may be necessary for the identification of poor risk cases with high CRLF2 expression that lack genomic lesions, however both methods are rapid, highly effective and very comparable for finding ALL cases that harbor CRLF2 genomic lesions, and suitable for incorporation in large scale clinical trials. Figure 1. qPCR and Flow Cytometry Results for CRLF2 Expression. Panel A (qPCR ΔCt values) and Panel B (log2 blast/lymphocyte ratios) plot the expression values for the 123 patients with the highest CRLF2 expression. Panel B plots the log2 blast/lymphocyte ratio for CRLF2 expression determined by flow cytometry. Small dots show the expression for each patient while the large diamonds highlight cases proven to have CRLF2 lesions either by FISH (IGH@-CRLF2) or PCR (P2RY8-CRLF2). Each unit of expression represents a two-fold difference in intensity. Figure 1. qPCR and Flow Cytometry Results for CRLF2 Expression. Panel A (qPCR ΔCt values) and Panel B (log2 blast/lymphocyte ratios) plot the expression values for the 123 patients with the highest CRLF2 expression. Panel B plots the log2 blast/lymphocyte ratio for CRLF2 expression determined by flow cytometry. Small dots show the expression for each patient while the large diamonds highlight cases proven to have CRLF2 lesions either by FISH (IGH@-CRLF2) or PCR (P2RY8-CRLF2). Each unit of expression represents a two-fold difference in intensity. Disclosures: No relevant conflicts of interest to declare.

Description: In a Plenary Paper, Young et al describe impressive favorable outcomes of emicizumab prophylaxis in children with hemophilia A and factor VIII inhibitors, reporting a 99% reduction in annualized bleeding, with 77% of patients having no treated bleeding events.

Description: Abstract 410 Collaborative genomic profiling efforts though the National Cancer Institute's TARGET Initiative and the Children's Oncology Group have identified CRLF2 and JAK mutations in a subset of children with high-risk acute lymphoblastic leukemia (ALL), but few biochemical studies have assessed the functional sequelae of these genetic alterations. CRLF2 encodes the thymic stromal lymphopoietin (TSLP) receptor chain, which heterodimerizes with the IL-7 receptor alpha chain (IL-7Rα). Children with high CRLF2-expressing ALL detected by gene expression profiling have high rates of minimal residual disease at end-induction (Day 29), and approximately 70% of these patients ultimately relapse (Harvey et al., Blood 2010). We hypothesize that characterization of aberrant signaling networks in these leukemias will facilitate identification of potential targets for small molecule inhibitor therapies. Using phosphoflow cytometry, we analyzed the phosphorylation status of key signaling molecules after stimulation with TSLP, IL-7, or pervanadate (an irreversible proximal membrane phosphatase inhibitor used as a positive control) in 2 human ALL cell lines with CRLF2 and JAK2 mutations and in 43 fresh or cryopreserved diagnostic primary patient samples, 27 of which overexpressed CRLF2 through P2RY8-CRLF2 fusion or CRLF2-IgH translocation and 16 of which did not have CRLF2 or JAK mutations (controls). Cells were rested in serum-free media for 60 minutes at 37°C, then stimulated with TSLP, IL-7, or pervanadate for 30 minutes to induce signaling. Cells were also exposed to the JAK inhibitor XL019 (Exelixis) for 60 minutes and/or subsequently stimulated with the aforementioned cytokines or pervanadate to determine the effects of JAK inhibition on signaling. Cells were then processed for phosphoflow cytometry according to our previously published methodologies (Kotecha et al., Cancer Cell 2008). High CRLF2-expressing leukemias (n = 27) with or without concomitant JAK mutations demonstrated strong surface staining of the TSLP receptor, as well as CD10, CD19, and CD127 (IL-7Rα). In vitro stimulation of leukemic blasts with TSLP elicited phosphorylation of STAT5 and S6, but not ERK 1/2, in leukemias with JAK and/or CRLF2 alterations. Control leukemias without CRLF2 and JAK mutations (n=16) did not stain for the TSLP receptor, and TSLP stimulation did not elicit phosphosignaling through the JAK/STAT, PI3K, or MAPK pathways. STAT5 and S6 phosphorylation in the high CRLF2-expressing leukemias was further abrogated by in vitro JAK inhibition with XL019. Surprisingly, despite flow cytometric staining for CD127, stimulation with IL-7 did not elicit phosphosignaling through these epitopes in high CRLF2-expressing or control leukemic blasts, although it did predictably phosphorylate STAT5 in control T and non-blast B cells contained within the primary patient leukemia samples. These results suggest that the JAK/STAT and PI3K pathways, but not the MAPK pathway, are involved in TSLP receptor signaling in high CRLF2-expressing ALL +/− JAK mutations and may represent druggable targets. Phosphoflow cytometry is an efficient method of interrogating intracellular signaling at a single-cell level in primary human samples and, furthermore, can be used for rapid identification of patients at time of leukemia diagnosis with high CRLF2-expressing ALL who exhibit the TSLP phosphosignature. Therapy for this subset of high-risk patients might be modified to include a targeted therapeutic (such as a JAK inhibitor) to improve initial treatment responses and, ultimately, to enhance long-term survival. To this end, we have developed a Children's Oncology Group Phase I clinical trial of JAK inhibition for patients with relapsed or refractory leukemias (including those with CRLF2 and JAK mutations) and will validate the use of phosphoflow cytometry and other biologic assays to assess in vivo target inhibition during therapy. We ultimately envision incorporation of JAK inhibitor therapy into a systemic chemotherapy backbone for patients with high CRLF2-expressing ALL. Disclosures: No relevant conflicts of interest to declare.

Description: Sarit Assouline and Won Seog Kim contributed equally. Introduction: Follicular lymphoma (FL) is considered an indolent yet incurable disease characterized by recurrent relapses: disease-free intervals shorten, and refractoriness increases with each relapse. Patients (pts) with FL who have received at least two prior systemic therapies typically have a poor prognosis. This is particularly true for those who have progression of disease within 24 months of front-line treatment (POD24), or are refractory to multiple agent classes; such patients are left with limited treatment options. Mosunetuzumab is a full-length, fully humanized immunoglobulin G1 CD20/CD3 bispecific antibody that redirects T cells to engage and eliminate malignant B cells. GO29781 (NCT02500407) is an ongoing open-label, multicenter, Phase I/Ib, dose-escalation and expansion study evaluating the safety, efficacy and pharmacokinetics of mosunetuzumab in pts with relapsed/refractory (R/R) B-cell lymphoma. Here, we present updated clinical data from pts with R/R FL treated with mosunetuzumab after at least two prior systemic therapies. Methods: Data are presented from Group B, in which pts received intravenous mosunetuzumab monotherapy as step-up doses in Cycle 1 on Days 1 and 8 and the target dose administered on Day 15. Mosunetuzumab was given on Day 1 of each subsequent 21-day cycle for 8 cycles in pts with a complete response (CR), and up to 17 cycles in those with a partial response (PR) or stable disease (SD). Results: As of January 21, 2020, 62 pts with FL (with at least two prior systemic therapies), received mosunetuzumab at dose levels between 0.4/1.0/2.8mg and 1/2/13.5mg (Cycle 1 Day 1/8/15 dose levels). The median age was 59 (range 27-85) years, and median number of prior therapies was 3 (range 2-11). Thirty-three pts (53%) were refractory to both a prior anti-CD20 antibody and an alkylating agent (double refractory), 30 (48%) had POD24, and four (6%) had received prior chimeric antigen receptor T-cell (CAR-T) therapy. The overall response rate (ORR) was 68% (42/62), with 31 pts (50%) achieving CR (Figure). Consistent CR rates were observed in high-risk pt populations, including those with double refractory disease (18/33 [55%]), POD24 (16/30 [53%]), PI3Ki refractory (7/9 [78%]), and those who received prior CAR-T therapy (2/4 [50%]). With a median time on study of 14.4 months, 26 pts (62% of all responders; including 74% of pts who achieved CR) remained in remission at the data cut-off. The median duration of response (DOR) was 20.4 months (95% CI: 11.7 months, upper limit not reached) for all 42 responders. The median PFS was 11.8 months (95% CI: 7.3-21.9 months). Adverse events (AEs) were reported in 60 pts (97%); serious adverse events (SAE) were reported in 22 pts (35%). The most frequently reported (〉10% of pts) grade (Gr) 3 or higher AEs included hypophosphatemia (23%; transient and clinically asymptomatic) and neutropenia (21%; with a low rate of febrile neutropenia [2%]). Overall, 14 pts (23%) experienced CRS; in four pts, CRS was classified as a SAE. CRS events were reversible, mostly of Gr 1 or 2 (Gr 1, n=11; Gr 2, n=2; Gr 3, n=1; Lee, et al. Blood 2014), and predominantly occurred during Cycle 1. No patient required tocilizumab, intensive care unit admission or use of vasopressors for CRS management. Neurologic AEs (NAEs; defined by any AEs reported as Preferred Terms in SOC Nervous System Disorders and SOC Psychiatric Disorders) were observed in 28 pts (45%); all were Gr 1 (n=18) or 2 (n=10). The most commonly reported NAEs were headache (24%), insomnia (15%) and dizziness (11%). No Gr ≥3 NAEs or serious NAEs were reported. Conclusions: Fixed-duration mosunetuzumab monotherapy results in high response rates and durable disease control with a tolerable safety profile in heavily pretreated patients with FL, including known high-risk subgroups. Updated pharmacodynamics and biomarker data will be presented at the meeting. Disclosures Assouline: BeiGene: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Takeda: Research Funding; Pfizer: Consultancy, Honoraria; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria, Speakers Bureau. Kim:Mundipharma: Research Funding; Donga: Research Funding; Kyowa Kirn: Research Funding; Celltrion: Research Funding; JJ: Research Funding; Pfizer: Research Funding; F. Hoffmann-La Roche: Research Funding. Sehn:Genentech, Inc.: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; Seattle Genetics: Consultancy, Honoraria; Teva: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Servier: Consultancy, Honoraria; Chugai: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Apobiologix: Consultancy, Honoraria; Acerta: Consultancy, Honoraria; Merck: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Kite: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Lundbeck: Consultancy, Honoraria; TG therapeutics: Consultancy, Honoraria; Verastem Oncology: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; MorphoSys: Consultancy, Honoraria. Schuster:AlloGene, AstraZeneca, BeiGene, Genentech, Inc./ F. Hoffmann-La Roche, Juno/Celgene, Loxo Oncology, Nordic Nanovector, Novartis, Tessa Therapeutics: Consultancy, Honoraria; Novartis, Genentech, Inc./ F. Hoffmann-La Roche: Research Funding. Cheah:Celgene, F. Hoffmann-La Roche, Abbvie, MSD: Research Funding; Celgene, F. Hoffmann-La Roche, MSD, Janssen, Gilead, Ascentage Pharma, Acerta, Loxo Oncology, TG therapeutics: Honoraria. Nastoupil:Karus Therapeutics: Research Funding; Gilead/KITE: Honoraria; TG Therapeutics: Honoraria, Research Funding; Merck: Research Funding; Novartis: Honoraria, Research Funding; Gamida Cell: Honoraria; Bayer: Honoraria; Pfizer: Honoraria, Research Funding; LAM Therapeutics: Research Funding; Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Genentech, Inc.: Honoraria, Research Funding. Shadman:Fred Hutchinson / University of Washington: Current Employment; Abbvie, Genentech, Inc., AstraZeneca, Sound Biologics , Pharmacyclics, Verastem, ADC therapeutics, Beigene, Cellectar, BMS, Mophosys and Atara Biotherapeutics: Consultancy; Mustang Bio, Celgene, Pharmacyclics, Gilead, Genentech, Inc., Abbvie, TG therapeutics, Beigene, AstraZeneca, Sunesis: Research Funding. Yoon:Amgen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Kyowahako Kirin: Research Funding; Janssen: Consultancy; F. Hoffmann-La Roche: Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland., Research Funding; YuhanPharma: Research Funding. Matasar:Daiichi Sankyo: Consultancy; IGM Biosciences: Research Funding; Immunovaccine Technologies: Honoraria, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; Teva: Consultancy; Rocket Medical: Consultancy, Research Funding; Seattle Genetics: Consultancy, Honoraria, Research Funding; GlaxoSmithKline: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; Juno Therapeutics: Consultancy; Takeda: Consultancy, Honoraria; Genentech, Inc.: Consultancy, Honoraria, Research Funding; Merck: Consultancy; Bayer: Consultancy, Honoraria, Research Funding. Diefenbach:Trillium: Research Funding; Millenium/Takeda: Research Funding; Merck: Consultancy, Research Funding; MEI: Research Funding; LAM Therapeutics: Research Funding; Incyte: Research Funding; Genentech, Inc.: Consultancy, Research Funding; Denovo: Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Seattle Genetics: Consultancy, Research Funding. Gregory:Janssen: Consultancy; F. Hoffmann-La Roche, Genentech, Inc., MSD, AbbVie, BeiGene, AstraZeneca, Celgene, BMS: Research Funding; F. Hoffmann-La Roche, Novartis, AbbVie: Speakers Bureau; F. Hoffmann-La Roche, Novartis, Sandoz, Gilead, AbbVie, MSD: Honoraria; F. Hoffmann-La Roche, Novartis, Sandoz, Gilead: Membership on an entity's Board of Directors or advisory committees. Bartlett:BMS/Celgene: Research Funding; Merck: Research Funding; Kite, a Gilead Company: Research Funding; Immune Design: Research Funding; Janssen: Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium: Research Funding; Pharmacyclics: Research Funding; Seattle Genetics: Consultancy, Research Funding; Affimed Therapeutics: Research Funding; Autolus: Research Funding; Acerta: Consultancy; Roche/Genentech: Consultancy, Research Funding; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; BTG: Consultancy; ADC Therapeutics: Consultancy; Forty Seven: Research Funding. Wei:Genentech, Inc.: Current Employment; F. Hoffmann-La Roche: Current equity holder in publicly-traded company. Doral:Genentech, Inc.: Current Employment; F. Hoffmann-La Roche: Current equity holder in publicly-traded company. Yin:Genentech, Inc.: Current Employment, Current equity holder in publicly-traded company. Negricea:F. Hoffmann-La Roche: Current Employment. Li:F. Hoffmann-La Roche: Current Employment, Current equity holder in publicly-traded company; Genentech, Inc.: Current Employment. Penuel:Genentech, Inc./ F. Hoffmann-La Roche: Current Employment; F. Hoffmann-La Roche: Current equity holder in publicly-traded company. Huang:F. Hoffmann-La Roche: Current Employment. Budde:AstraZeneca: Speakers Bureau; Merck, Amgen, AstraZeneca, Mustang Therapeutics: Research Funding; F. Hoffmann-La Roche, Kite Pharma: Consultancy. OffLabel Disclosure: Mosunetuzumab (RG7828) is a full-length, fully humanized immunoglobulin G1 (IgG1) bispecific antibody targeting both CD3 (on the surface of T cells) and CD20 (on the surface of B cells). Mosunetuzumab redirects T cells to engage and eliminate malignant B cells. Mosunetuzumab is an investigational agent.