Disease progression in patients with the restrictive and mixed phenotype of Chronic Lung Allograft dysfunction—A retrospective analysis in five European centers to assess the feasibility of a therapeutic trial

Background Chronic Lung Allograft Dysfunction (CLAD) is a major obstacle for long term survival after lung transplantation (LTx). Besides Bronchiolitis Obliterans Syndrome, two other phenotypes of CLAD, restrictive allograft syndrome (RAS) and mixed phenotype, have been described. Trials to test in these conditions are desperately needed and analyzing natural outcome to plan such trials is essential. Methods We performed a retrospective analysis of functional outcome in bilateral LTx recipients with RAS and mixed phenotype, transplanted between 2009 and 2018 in five large European centers with follow- up spirometry up to 12 months after diagnosis. Based on these data, sample size and power calculations for randomized therapeutic trial was estimated using two imputation methods for missing values. Results Seventy patients were included (39 RAS and 31 mixed phenotype), median 3.1 years after LTx when CLAD was diagnosed. Eight, 13 and 25 patients died within 6, 9 and 12 months after diagnosis and a two patients underwent re-transplantation within 12 months leading to a graft survival of 89, 79 and 61% six, nine and 12 months after diagnosis, respectively. Observed FEV1 decline was 451 ml at 6 months and stabilized at 9 and 12 months, while FVC showed continuous decline. Using two methods of imputation, a progressive further decline after 6 months for FEV1 was noted. Conclusion The poor outcome of these two specific CLAD phenotypes suggests the urgent need for future therapeutic randomized trials. The number of missing values in a potential trial seems to be high and most frequently attributed to death. Survival may be used as an endpoint in clinical trials in these distinct phenotypes and imputation techniques are relevant if graft function is used as a surrogate of disease progression in future trials.

516 Prevalence of carpal tunnel syndrome in patients with transthyretin cardiac amyloidosis

Aims Carpal tunnel syndrome (CTS) represents an important red flag for transthyretin (ATTR) cardiac amyloidosis (CA). However, no large studies have investigated the prevalence of CTS in wild type ATTR (wtATTR) and hereditary ATTR (hATTR). To investigate the prevalence of CTS in patients with ATTR-CA, both wild type and hereditary, differentiating between monolateral and bilateral carpal tunnel syndrome. Methods and results 381 patients, 308 male and 73 female, with a definite diagnosis of ATTR CA have been evaluated. Among these, 230 patients with diagnosis of wild-type ATTR (wtATTR) and 151 patients with hereditary ATTR (hATTR) were identified. Patients with diagnosis of hATTR are sorted according to phenotype in cardiologic (43 patients) and mixed when both cardiologic and neurologic phenotype are observed (108 patients). Patients with neurological phenotype without CA were excluded. Overall, CTS is present in 57.6% of ATTR patients; A higher prevalence (P < 0.05) of CTS was observed in wtATTR (61.6%) respect to hATTR (51.7%). Monolateral isolated CTS is significantly frequent (P < 0.05) in patients with hATTR (35.1%) than in wtATTR (12.7%), on the contrary bilateral CTS is significantly more frequent (P < 0.05) in patients with wtATTR (48.5%) than in hATTR (16.6%). Among patients with hATTR, of the 43 patients with cardiologic phenotype, 18 patients (41.9%) have diagnosis of CTS, subdivided in 28% with monolateral CTS and 72% with bilateral CTS. Among hATTR patients with mixed phenotype, 55.6% have diagnosis of CTS, subdivided in 80% with monolateral isolated CTS and 20% with bilateral CTS. Among 151 patients with hATTR, monolateral isolated CTS is significantly more present in patients with mixed phenotype (80% vs. 27%, P < 0.001) while bilateral CTS is significantly more frequent in patients with cardiologic phenotype (72.2% vs. 20%, P < 0.001). Conclusions CTS particularly with bilateral involvement is a common finding in wtATTR patients than in hATTR patients. On the contrary, monolateral isolated CTS is significantly more frequent in patients with hATTR than in wtATTR. Among patients with hATTR, bilateral CTS is significantly more frequent in patients with cardiologic phenotype than mixed phenotype while monolateral isolated CTS is significantly more present in patients with mixed phenotype.

Standardization in the Diagnosis of Mixed Phenotype Acute Leukemia (MPAL): Semiquantitative, Universally Applicable Flow Cytometric Criteria for Immunophenotypic Lineage Assignment and Isolated MPO

Mixed phenotype acute leukemia (MPAL) is a rare group of acute leukemias defined by immunophenotypic expression of more than one hematopoietic cell lineage. The World Health Organization (WHO) diagnostic immunophenotypic criteria for MPAL rely on the intensity of lineage-defining antigen expression, predominantly a qualitative assessment, and are often difficult to apply to a phenotypically heterogeneous leukemia. Cases of MPAL defined by isolated myeloperoxidase (isoMPO) expression on otherwise typical acute lymphoblastic leukemia (ALL) are variably diagnosed as MPAL or ALL based on the incompletely defined criteria for assigning MPO expression. We hypothesized that quantitative criteria for antigen intensity could be developed and applied in a uniform manner across flow cytometry instruments, reagents, and analysis software to enable a consistent approach to diagnosing MPAL and better defining isoMPO. We previously reported on a multicenter cohort identified by respective institutions as MPAL with subsequent central review according to 2008 WHO criteria (Oberley et al 2020). Of these, 100 had suitable dot plots for reevaluation (89: B/Myeloid, 10: T/Myeloid, 1: B/T). Antigen expression was concurrently reviewed by two hematopathologists to reach consensus (BLW, AEK). The cohort was divided a priori into randomly selected training and testing cases (n=30/n=70). Classification criteria were generated in the training cohort for WHO lineage-defining antigen expression (myeloid: cMPO, CD64, CD14; B: CD19, T: cCD3) from 10 cases and then refined through review of an additional 20 cases. Positive antigen expression was classified as maximal intensity approaching that of the mature normal counterpart population (NCP) (cMPO: neutrophils; CD64, CD14 and CD11c: monocytes; CD19: B cells; cCD3: T cells) either by 1) range of expression recapitulating maturation of the NCP, irrespective of the percentage on the leukemic population (Figure 1A); or 2) uniform expression above background on a discrete (sub)population (Figure 1B). To account for technical variation within and among laboratories, maximal antigen intensity on the leukemic population was measured in 0.5 log increments and normalized to the maximal intensity of the NCP. An intensity of ≥50% of the NCP above background was defined as sufficient for MPAL lineage assignment and <50% consistent with isoMPO. This approach was then used to classify the remaining 70 cases. Using this approach, 41/98 (42%) cases previously classified as MPAL remained classified as MPAL: 31 as B/Myeloid (25 by maturational MPO expression, 6 by maturational CD64 and/or CD14 expression); 9 as T/Myeloid (8 by maturational MPO expression, 1 with maturational CD64, CD14 and CD11c expression); and 1 as B/T. No cases in the cohort showed uniform expression ≥50% of the NCP. The remaining 57 showed isolated low-level MPO expression (maximal intensity <50% of the NPC) on a uniform leukemic population or on a dichotomous subpopulation (isoMPO), 56 B/Myeloid and 1 T/Myeloid. Two cases of otherwise typical B-ALL without myeloid or monocytic antigen expression were reclassified as B-ALL, one of which showed low variable CD10 suggestive of DUX4-rearranged B-ALL. In comparison to our previously reported study of 2008 WHO-defined MPAL, 53/89 (60%) centrally-confirmed cases would be classified here as isoMPO. Conversely, five cases excluded under 2008 WHO central review would be reclassified as having sufficient antigen expression to qualify as MPAL (2 B/Myeloid, 3 T/Myeloid). Novel semiquantitative immunophenotypic criteria applied to a large cohort of acute leukemias originally diagnosed as MPAL was able to objectively identify a large subset as having dim, uniform MPO expression (isoMPO). Our approach emphasizes antigen expression recapitulating maturational expression similar to their non-leukemic cellular counterparts, normalizes absolute intensities to internal positive and negative control populations to minimize technical variability between observers and assays, and as per the 2017 WHO, does not require a specific percent threshold of positivity. While requiring validation, this is a critical first step toward establishing a reproducible delineation of these complex cases to practically implement the WHO classification to support treatment decisions and ongoing investigation into MPAL genomics and outcomes (available for this cohort by ASH). Figure 1 Figure 1. Disclosures Oberley: Caris LIfe Science: Current Employment. Orgel: Jazz Pharmaceuticals: Consultancy.

First-in-Human Clinical Study of a Novel CD7-Targeted Chimeric Antigen Receptor (CAR)-T Cell Therapy for Refractory/Relapsed Mixed Phenotype Acute Leukemia (MPAL)

Introduction Mixed phenotype acute leukemia (MPAL) is a rare subtype of acute leukemia with features of both acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). The blast cells of MPAL express multilineage immunophenotypic markers and may have a shared B/T/myeloid phenotype. CD7 is expressed by the leukemic blasts and malignant progenitor cells of approximately 30% of AML patients. MPAL is usually associated with a relatively poor prognosis which is even worse compared to either AML or ALL, and even among patients that undergo allogeneic hematopoietic stem cell transplantation (allo-HSCT), underscoring the need for new therapies for these patients. CAR-T cell therapy has shown to be efficacious with a tolerable safety profile in a subset of hematological malignancies. Here, we explored the safety and efficacy of CAR-T therapy in treating refractory/relapsed (r/r) MPAL patients with CD7-positive in a phase I dose-escalation clinical study (https://clinicaltrials.gov NCT04938115). Methods Peripheral blood (PB) mononuclear cells were obtained from patient themselves or patient transplant donors for those who relapsed post-transplant by leukapheresis. T-cells were purified using CD3+ magnetic beads or CD4+ and CD8+ magnetic beads. The second-generation CD7 CAR with a 4-1BB costimulatory domain was manufactured per the manufacturer's protocol. This phase I dose escalation study was initiated to explore the safety and efficacy of CD7 CAR-T in treating patients with MPAL. Prior to the CAR-T cells infusion, patients received systemic bridging chemotherapy due to rapid disease progression and then all patients received intravenous fludarabine (30mg/m 2/d) and cyclophosphamide (300mg/m 2/d) (FC) lymphodepleting chemotherapy for 3 consecutive days (Day -5 to Day -3). The median time from leukapheresis to CAR-T cell infusion was 14 days. Results From Nov. 2020 to June 2021, 5 adult acute leukemia patients with CD7-positive (4 MPAL, 1 FLT3-mutated AML) were enrolled and infused with CD7 CAR T-cells, one with a low-dose (1.5×10 5/kg), 2 with a medium dose (5.0×10 5/kg), and 3 with a high dose infusion (1.0×10 6/kg). The median transduction efficiency of products was 70.4% (range: 32.0%-96.9%). As of July 30, 2021 cut-off date, the median observation time was 37 days (range: 35-232 days). Characteristics of enrolled patients are shown in Table 1. The median age was 33 years (range: 21-37 years), and the median bone marrow (BM) blasts percentage by morphology was 28.0% (range: 2.5-51.5%) at enrollment. Four patients had prior allo-HSCT history including one who had extramedullary disease (EMD) who relapsed following a 2 nd transplant and had also received CD19 CAR-T cell therapy prior to enrollment in the current trial. The median interval period from prior transplant to CD7 CAR-T cell infusion was 16 months (range: 8-23 months). Following infusion, the median peak of circulating CD7 CAR-T cell was 1.17×10 5copies/μg (0.651~7.04×10 5copies/μg) genomic DNA which occurred around Day 21 (Day14 - Day21) and 59.2% (4.28%~74.58%) occurring on Day18 (Day11-Day21) by q-PCR and flow cytometry respectively (Fig.1). Four weeks post CD7 CAR-T cell infusion, 4/5 patients achieved complete remission (CR) or CR with incomplete blood recovery (CRi) in BM and all achieved minimal residual disease (MRD)-negative CR. One patient who relapsed post 2 nd transplant, also with EMD, achieved CR in BM, however, remission was not achieved in EMD through PET-CT evaluation on Day 35. One MRD-negative CR patient subsequently underwent consolidative allo-HSCT 120 days post CD7 CAR T-cell infusion and has remained in MRD-negative CR. Other 3 patients without consolidative allo-HSCT remained in MRD-negative CR till the last follow-up. All 5 patients developed cytokine release syndrome (CRS)-three of 5 patients had grade I CRS, one patient had grade II and one patient had grade III CRS. None of the patients had neurotoxicity. Among the 4 patients who had prior allo-HSCT, 1 developed grade I graft-versus-host disease post CAR-T therapy. Conclusion This study demonstrated that CD7-targeted CAR-T therapy offers an opportunity to achieve CR for CD7-positive MPAL patients even for those who relapsed post-transplant. Safety was manageable, however, more data on additional patients and longer observation times are needed to further evaluate the efficacy of CD7 CAR-T products. Figure 1 Figure 1. Disclosures Ba: SenlangBio: Current Employment.

A Comprehensive Cancer Center Experience with Hyper-CVAD Plus Ponatinib As Frontline Therapy for Adult Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia

Background Ponatinib, a third-generation BCR-ABL1 tyrosine kinase inhibitor (TKI), + hyper-CVAD showed remarkable activity against Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL), and may be superior to chemotherapy + earlier generation TKIs in terms of depth of remission, event-free survival (EFS), and overall survival (OS). However, this regimen's efficacy and tolerability have yet to be externally validated. Here, we summarize our real-world experience with ponatinib + hyper-CVAD for untreated Ph+ ALL and other Ph+ acute or blast phase leukemias. Methods We retrospectively analyzed all adults treated at the University of California, Davis (UCD) from March 2012 to May 2021 with ponatinib + hyper-CVAD upfront. The primary endpoints were 3-year OS and EFS. Secondary endpoints were complete molecular response (CMR), measurable residual disease (MRD) negativity by multiparameter flow cytometry (MFC), complete cytogenetic response (CCyR) rates, and adverse events (AEs). Time to event analyses were done via the Kaplan-Meier method. Patients alive were censored at their last follow-up date. Patients undergoing allogeneic hematopoietic cell transplant (HCT) after 6 months of achieving complete remission (CR) were censored at the time of HCT for the landmark analysis. Patients with missing data were excluded from the response analyses. Results We identified 13 Ph+ ALL patients who received ponatinib + hyper-CVAD for initial induction. The baseline characteristics for the Ph+ ALL patients are summarized in Table 1. The median follow-up was 16 months. The median number of hyper-CVAD cycles completed was 8 (range, 1-8) with ponatinib. Two patients proceeded to HCT in CR1, one at 3.5 months after starting induction, and due to difficulty controlling the patient's concurrent multiple myeloma prior to HCT and recovery from anti-neoplastic therapy, the second was delayed to 46 months after starting induction. The 3-year OS and EFS with ponatinib + hyper-CVAD were each 92% (95% confidence interval, 78.9-100) (Figure 1). Landmark analysis completed 6 months following CR showed a 3-year OS of 100% in patients treated with ponatinib + hyper-CVAD without HCT in first CR (CR1). The CMR, CCyR, and MRD-negativity by MFC rates with ponatinib were all 92.3% (12/13). The median time to CMR, CCyR, and MRD-negativity by MFC were 51 days, 22 days, and 53 days, respectively. Notable AEs with ponatinib include neutropenic fever (92%), bacterial infection (69%), transaminitis (38%), venous thromboembolism (31%), invasive fungal infection (15%), hemorrhage (15%), cerebrovascular accident (CVA) (15%), and tumor lysis syndrome (8%). One patient died during induction with ponatinib due to a bacterial infection. Two patients switched to a different TKI due to a CVA after 4 and 24 months. Only 2 patients did not complete 8 cycles of hyper-CVAD, due to death during induction (n=1) and proceeding to HCT after 3 cycles (n=1). As for similar Ph+ leukemias, 3 chronic myeloid leukemia with lymphoid blast crisis (CML-LBC), 1 CML with mixed phenotype blast crisis (CML-MPBC), and 1 with mixed phenotype acute leukemia (MPAL) were treated with ponatinib + hyper-CVAD. The MPAL patient achieved CMR within 55 days, while the CML-MPBC and 2 CML-LBC patients achieved CMR after HCT. The third CML-LBC patient is in CR with ongoing treatment. After median follow-up of 25 months, all 5 were alive, and only the MPAL patient relapsed 28 months after starting treatment and 1 year after HCT. Conclusion To our knowledge, this is the first report externally validating the efficacy and tolerability of ponatinib + hyper-CVAD for Ph+ ALL. We also show the feasibility of using this regimen in patients with Ph+ CML-LBC, CML-MPBC and MPAL. Despite the small sample size and retrospective nature, our study supports existing data demonstrating that this regimen challenges both the designation of Ph+ ALL as a high-risk disease and the trend to transplant in CR1. Our findings support that ponatinib + hyper-CVAD should be considered a standard of care for Ph+ ALL. Figure 1 Figure 1. Disclosures Kaesberg: Incyte: Speakers Bureau. Rosenberg: Takeda, Janssen: Speakers Bureau. Abedi: BMS/Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Speakers Bureau; Seattle Genetics: Speakers Bureau. Tuscano: Genentech, Pharamcyclics, Abbvie, BMS, Acrotech, Seattle Genetics, Takeda: Research Funding. Jonas: AbbVie, BMS, Genentech, GlycoMimetics, Jazz, Pfizer, Takeda, Treadwell: Consultancy; 47, AbbVie, Accelerated Medical Diagnostics, Amgen, AROG, Celgene, Daiichi Sankyo, F. Hoffmann-La Roche, Forma, Genentech/Roche, Gilead, GlycoMimetics, Hanmi, Immune-Onc, Incyte, Jazz, Loxo Oncology, Pfizer, Pharmacyclics, Sigma Tau, Treadwell: Research Funding; AbbVie: Other: Travel reimbursement. OffLabel Disclosure: Ponatinib is approved for Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ALL) that is resistant or intolerant to prior tyrosine kinase inhibitor therapy. In this study, we described outcomes with ponatinib in combination with hyperCVAD in the frontline setting, which is off-label.