Lancet Oncol Research Articles

Pulmonary hypertension secondary to valvular heart disease: a state-of-the-art review.

Pulmonary hypertension (PH) is a common disease affecting up to 1% of the population and at least 50% of patients diagnosed with heart failure (HF) (Hoeper et al. in Lancet Respir Med 4(4):306-322, 2016). It is estimated that PH is present in 15% to 60% of patients with valvular heart disease (VHD) which can result from an increase in pulmonary blood flow and subsequently in pulmonary venous congestion and pulmonary vascular resistance (PVR). It is important to identify the severity of PH in patients with VHD to appropriately risk stratify and manage these patients (Magne et al. in JACC Cardiovasc Imaging 8(1):83-99, 2015). In this review, we examine the diagnostic criteria for PH and its pathophysiology. We also focus on the growing evidence supporting the presence of PH secondary to VHD and describe the contemporary surgical and medical therapeutic interventions in this patient population (Fig.1).

Zanubrutinib vs FCR in Fit Treatment-Naive Patients with Chronic Lymphocytic Leukemia: A Matching-Adjusted Indirect Comparison

Background: Fludarabine, cyclophosphamide, and rituximab (FCR) is the standard first-line therapy for fit (physically active, with no major health problems and normal renal function) treatment-naive patients with chronic lymphocytic leukemia (CLL) (Eichhorst et al. Ann Oncol. 2015). Due to the safety profile of FCR, including severe hematotoxicity, and severe infections, treatments with improved efficacy and safety are of interest (Hallek et al. Lancet. 2010; Fischer et al. Blood. 2016). Zanubrutinib, a highly specific and potent small-molecule inhibitor of Bruton tyrosine kinase, has been approved for treatment-naive patients with CLL. However, the comparative efficacy of zanubrutinib vs FCR in patients with CLL has not been investigated. Objective: The objective of this study was to determine the relative treatment effects of zanubrutinib vs FCR in fit treatment-naive patients with CLL. Methods: The CLL10 trial (NCT00769522; Eichhorst et al. Lancet Oncol. 2016) investigated FCR and bendamustine + rituximab (BR), while the SEQUOIA trial compared zanubrutinib to BR (NCT03336333; Tam et al. Lancet Oncol. 2022). Zanubrutinib and FCR were compared by means of an anchored matching-adjusted indirect comparison (MAIC) in which the BR arm of each trial served as a common comparator. Propensity score matching was conducted using patient-level data from SEQUOIA to adjust for differences between trial populations, per National Institute for Health and Care Excellence MAIC methods (Philippo et al. NICE DSU Technical Support Document 18. 2016). Progression-free survival (PFS), as assessed by the independent review committee, was then compared based on the matched patient populations. The selection of matching variables was based on literature review and consultation with clinical experts. As the indirect treatment comparison was anchored on the common comparator arm (BR), patient characteristics that are solely prognostic did not need to be incorporated. The core model included immunoglobulin heavy chain gene mutation, cytogenetic mutations (11q deletion), β2-microglobulin, Binet stage, and age. Geographic region, sex, creatinine clearance, Cumulative Illness Rating Scale (CIRS) score, Eastern Cooperative Oncology Group performance status (ECOG PS), and previous infections were tested in sensitivity analyses. Results: PFS was favorable with zanubrutinib vs FCR, and statistically significant differences were observed in the base case model (hazard ratio [HR], 0.41; 95% CI, 0.20-0.81; p-value=0.01). Sensitivity analyses showed significantly improved PFS with zanubrutinib when adding geographic region (HR, 0.43; 95% CI, 0.21-0.90; p-value=0.03), sex (HR, 0.44; 95% CI, 0.22-0.89; p-value=0.02), ECOG PS (HR, 0.30; 95% CI, 0.14-0.64; p-value<0.01), and previous infections (HR, 0.45; 95% CI, 0.22-0.93; p-value=0.03) to the base case set of matching variables. A sensitivity analysis incorporating CIRS (HR, 0.45; 95% CI, 0.16-1.24; p-value=0.12) showed only numerically favorable PFS with zanubrutinib, owing in part to the low effective sample size (ESS) in the expanded model. Similar results were obtained when creatinine clearance was added to the base case matching set of variables (HR, 0.52; 95% CI, 0.24-1.13; p-value=0.10). Conditional on the number of matching variables, ESS ranged from 174.1 in the core model to 64.05 in the analysis that included the CIRS score. Conclusion:Our findings suggest that zanubrutinib offers clinically meaningful benefits in PFS over FCR in fit treatment-naive patients with CLL. MAICs rely on reporting of relevant patient characteristics in published studies. In the current study, ZAP-70 methylation and TP53 mutation, which were considered treatment effect modifiers, were not reported in CLL10 and were not accounted for in the propensity model.

Broad Superiority of Zanubrutinib (Zanu) Over Bendamustine + Rituximab (BR) Across Multiple High-Risk Factors: Biomarker Subgroup Analysis in the Phase 3 SEQUOIA Study in Patients With Treatment-Naive (TN) Chronic Lymphocytic Leukemia (CLL)/Small Lymphocytic Lymphoma (SLL) without del(17p)

Introduction Zanu is a Bruton tyrosine kinase (BTK) inhibitor with high potency, selectivity, and efficacy and a favorable toxicity profile. In the phase 3 SEQUOIA study (NCT03336333; Tam et al. Lancet Oncol. 2022), zanu treatment demonstrated superior progression-free survival (PFS) in TN patients (pts) with CLL/SLL and without del(17p) (cohort 1) compared with BR treatment (hazard ratio [HR], 0.42; 95% CI, 0.28-0.63; 2-sided P<.0001). We evaluated PFS in both treatment arms in pt subgroups based on various biomarkers, including several known negative prognostic factors for CLL. Methods Details of the SEQUOIA study were described previously (Tam et al. Lancet Oncol. 2022). A total of 479 pts in cohort 1 were randomized to either zanu (n=241) or BR (n=238). Blood (CLL) or bone marrow (SLL) samples collected at screening were used for fluorescence in situ hybridization for chromosome abnormalities, cytogenetic analysis for complex karyotype (CK), next-generation sequencing (NGS) per the European Research Initiative on CLL (ERIC) for immunoglobulin heavy chain variable (IGHV) gene mutations and expressed clones, and ultrasensitive targeted NGS for mutation analysis of 106 genes. For NGS, all pathogenic mutations with variant allele frequency ≥1% were analyzed. The association between biomarkers and PFS was quantified using the log-rank test and HR and summarized by the Kaplan-Meier method. Data cutoff was October 31, 2022. Results Overall, the CLL features analyzed were similarly distributed between both treatment arms (Table). In pts with cytogenetic abnormalities, zanu demonstrated significantly better PFS than BR in those with del(11q) ( P<.001), del(13q) ( P<.001), trisomy 12 ( P<.01), or CKT ≥3 ( P<.01) (Table). Furthermore, zanu treatment conferred a similar PFS benefit to pts with the negative prognostic factor, del(11q) ( P=.05) or intermediate prognostic factor, trisomy 12 ( P=.40), compared to those without these abnormalities (Table). Notably, in the zanu arm, CKT ≥3 was not associated with worse PFS ( P=.18; Table). Pts with either mutated IGHV (mIGHV) or unmutated IGHV (uIGHV) in the zanu arm had significantly better PFS than pts in the BR arm (uIGHV: P<.0001; mIGHV: P<.01; Table); analysis was confirmed by an ERIC-certified provider. Furthermore, PFS was unaffected by IGHV mutational status in pts treated with zanu ( P=.12). In pts with uIGHV, IGHV1-69 was the most prevalent expressed clone in both arms (zanu: 24.6% [31/126]; BR: 30.7% [39/127]). In the present study, pts with uIGHV harboring IGHV1-69 clones showed significantly better PFS with zanu vs BR ( P<.0001). Across all pts, the most frequently mutated genes were ATM (zanu: 11%; BR: 16%), SF3B1 (zanu: 17%; BR: 21%), NOTCH1 (zanu: 20%; BR: 20%), and BRAF (zanu: 11%; BR: 7%; Table). Pts with mutations associated with poor prognosis in CLL had significantly better PFS with zanu than with BR (eg, ATM [ P=.02], BRAF [ P=.01], NOTCH1 [ P<.001], SF3B1 [ P<.001]; Table). Furthermore, zanu-treated pts with or without mutations in ATM ( P=.58), NOTCH1 ( P=.38), and SF3B1 ( P=.39) had similar PFS. Conclusions TN del(17p)-negative pts with CLL/SLL were evenly distributed between zanu and BR arms in SEQUOIA cohort 1 based on the biomarkers analyzed. PFS in zanu-treated pts was superior to that of BR-treated pts in all biomarker subgroups analyzed, including some known negative prognostic markers in CLL such as del11q, CKT≥3, and uIGHV. Importantly, within the zanu-treatment arm, pts with negative prognostic biomarkers showed comparable PFS benefit to pts without those markers. Like with other BTK inhibitors, ibrutinib and acalabrutinib, IGHV mutational status did not affect PFS outcome (Bartosz et al. Hematology in Clinical Practice 2022). However, contrary to what has been reported for TN or relapsed/refractory pts treated with ibrutinib-based regimens, CKT ≥3 was not associated with worse PFS in zanubrutinib-treated patients (Rigolin et al. Blood. 2021; Thompson et al. Cancer. 2015). This study provides further evidence that zanu is a valuable first-line treatment option for pts with CLL/SLL.

Longitudinal Transcriptomic Analysis of CAR-T Cells at Single-Cell Level Allows the Identification of Molecular Mechanisms Promoting Long-Term CAR-T Cell Persistence in MM

Background: CAR-T therapies targeting BCMA are a promising option for Multiple Myeloma (MM) patients. However, a significant number of patients still relapse and the limited persistence of CAR-T cells after infusion has been associated with lack of long-term responses. To shed light on the potential mechanisms driving CAR-T cell persistence we performed longitudinal transcriptomic analysis, at single cell level, of CAR-T cells from a patient enrolled in CARTBCMA-HCB-01 clinical trial (NCT04309981) (Oliver-Caldes A, et al. Lancet Oncol, 2023), that presented detectable CAR-T cells more than one year after CAR-T cell administration. Methodology: Single-cell transcriptomic and TCR analysis was performed on 42.719 CAR-T cells isolated from infusion product (IP) and paired bone marrow (BM) and peripheral blood (PB) samples at 1, 3, 6, 12 and 18 months after infusion (11 samples), using the Chromium Single-Cell Immune Profiling solution from 10x Genomics. Gene Regulatory Network (GRN) analysis was performed using SimiC, a novel machine learning method developed by our group, that infers regulatory dissimilarities form single cell data (Peng J, et al. Commun Biol., 2022). Results: We observed that only 4 out of the 30 patients included in the first pilot academic clinical trial evaluating ARI0002h presented detectable CAR-T cells 12 months after treatment (Oliver-Caldes A, et al. Lancet Oncol, 2023). We performed a deeper analysis of one patient still presenting high levels of CAR-T cells 18 months after treatment that finally ended up relapsing at month 23. scRNA-seq analysis revealed that post-infusion CAR-T cells were mainly composed by effector/effector-memory CD8 + cells, with a polyclonal TCR repertoire. Interestingly, this patient suffered a respiratory tract infection with CMV reactivation during the first month after infusion that was also reflected on CAR-T cells features. Thus, circulating CAR-T cells at M1 expressed high levels of genes involved in IFN response, with an equivalent anti-viral response in CAR-T cells obtained from BM at M3. However, phenotypes of CAR-T cells from both PB and BM samples converged at M6, with a prevailing CAR-T cell population (termed 'CD8 + GZMK') presenting cytotoxic features and expressing key markers related to T cell memory and survival that could explain the prolonged CAR-T cell persistence. However, at M12 we observed changes on CAR-T cell populations, with increased presence of CAR-T cells with more cytotoxic features, enrichment of effector and activation signatures, and expressing lower levels of memory markers ('CD8 + GZMB' cluster), indicating CAR-T cell reactivation. Finally, applying GRNA analysis with SimiC we identified several regulons (FOS and NR4A2), presenting increased activity at M12 that could represent potential drivers of CAR-T reactivation in response to tumor relapse. Further analysis at month 18 are being performed to elucidate mechanisms of tumoral resistance in the presence of CAR-T cells. Conclusions: Our analysis clearly showed that most CAR-T cells after administration presented expression of key markers related to T cell memory and survival without clonal expansion, suggesting that memory induction is crucial for long-term persistence. Interestingly, prior to tumor relapse, we observed reactivation of CAR-T cell with the enrichment of effector and activation signatures, and GRN analysis identified FOS and NR4A2 regulons as potential drivers involved in CAR-T cell reactivation. Overall, scRNA-seq/scTCR-seq coupled with novel machine learning models provides relevant mechanistic insights that could uncover useful targets to be modulated for the development of long-term persistent CAR-T cells.

Long-Term Outcomes from the Phase 3 OCEAN (OP-103) Study: Melflufen and Dexamethasone (Dex) Versus Pomalidomide (Pom) and Dex in Relapsed Refractory Multiple Myeloma (RRMM)

Background/Introduction: Melphalan flufenamide (melflufen) is a first-in-class peptide-drug conjugate that targets aminopeptidases resulting in rapid release of alkylating agents inside tumor cells. Based on the phase 2 HORIZON study and supported by the phase 3, randomized, controlled OCEAN study, melflufen was approved in Europe for use in patients (pts) with triple-class refractory RRMM with ≥3 prior lines of therapy (LoTs) and without prior autologous stem cell transplantation (ASCT) or with a time to progression (TTP) >36 mo after prior ASCT. In the OCEAN study (NCT03151811), melflufen + dex showed superior progression-free survival (PFS) compared with pom + dex (6.8 vs 4.9 mo; hazard ratio [HR]: 0.79; P=0.032); PFS benefit in the melflufen + dex group was mainly driven by pts who had not received prior ASCT. Overall survival (OS) trended in favor of melflufen + dex in pts without prior ASCT and favored pom + dex in pts with prior ASCT (Schjesvold et al. Lancet Haematol. 2022;9:e98). Post-hoc analyses of OCEAN and HORIZON demonstrated that a TTP <36 mo after prior ASCT was a negative prognostic factor for OS with melflufen + dex (Sonneveld et al. Clin Lymphoma Myeloma Leuk. 2023;S2152). Here, we present long-term OS and safety data from the final analysis of the OCEAN study. Methods: Pts with RRMM (2-4 prior LoTs including lenalidomide [len] and a proteasome inhibitor) refractory to len and last LoT were randomized 1:1 (stratified by age, no. of prior LoTs, and International Staging System score) to receive 28-day (d) cycles of melflufen 40 mg intravenously on d1 or pom 4 mg orally (PO) daily on d1 to 21. All pts received dex 40 mg (20 mg for pts ≥75 y) PO on d1, 8, 15, and 22. Pts received therapy until disease progression or unacceptable toxicity. The primary endpoint was PFS, as assessed by independent review committee per IMWG Uniform Response Criteria, and key secondary endpoints were overall response rate, OS, and safety. Results: As of 3 Feb 2023, 495 pts were randomized (246 to melflufen; 249 to pom); median age was 68 y (range, 39-91) and median prior LoTs was 3. In the intent-to-treat (ITT) melflufen and pom populations, median OS was 20.2 mo vs 24.0 mo (HR, 1.09 [95% CI, 0.88-1.35]), at a median follow-up of 40.3 mo and 38.1 mo, respectively. In the target subgroup of the melflufen and pom groups (pts without a prior ASCT or TTP >36 mo after an ASCT), median OS was 23.6 mo vs 19.1 mo (HR, 0.88 [95% CI, 0.67-1.16]); in the non-target population (pts with TTP <36 mo after ASCT), median OS was 15.7 mo vs 27.5 mo (HR, 1.60 [95% CI, 1.15-2.21]), respectively. While any grade hematologic toxicities were more common with melflufen, the occurrence of non-hematologic toxicities was similar in the 2 groups. Grade 3/4 (G3/4) treatment-emergent adverse events (TEAEs) in the safety population (melflufen [n=228] and pom [n=246]) occurred in 90% vs 76% of pts, respectively; most commonly thrombocytopenia (78% vs 13%; occurring with G3/4 hemorrhage in 1% vs 0%), neutropenia (64% vs 50%; occurring with G3/4 infections in 4% vs 7%), anemia (43% vs 19%), and infection and infestations (14% vs 24%). Serious AEs occurred in 43% vs 50% of pts (including pneumonia [6% vs 9%], COVID-19 pneumonia [5% vs 6%], and anemia [4% vs 2%]), and fatal AEs in 14% vs 15% (including COVID-19 pneumonia [4% vs 2%] and pneumonia [2% vs 2%]) in the melflufen and pom groups, respectively. With melflufen and pom, TEAEs led to dose reductions in 52% vs 28% of pts (most frequently thrombocytopenia [32% vs 2%] and neutropenia [12% vs 8%]), and discontinuations in 30% vs 24% of pts, respectively. Deaths occurred in 74% vs 68% of pts in the melflufen and pom groups, with AEs being the primary cause of death ≤30 d after last dose in 8% and 11%, respectively. Conclusion: Long-term results were consistent with those of previous analyses (Schjesvold, et al. Lancet Haematol. 2022;9:e98-e110). While OS trended in favor of pom in the ITT population, OS outcomes continued to be more favorable with melflufen in pts with no prior ASCT or with TTP >36 mo after ASCT. No new safety signals were reported, and AEs were manageable with dose modifications, consistent with previous reports. This long-term follow-up of OCEAN confirms the favorable safety and OS outcomes of melflufen + dex in the target population and supports its continued use as an alternative treatment choice for pts with RRMM who have received ≥2 prior LoT and who have not received ASCT.

ATRA Can Correct Impaired Proplatelet Formation By Regulating HIF-1α/SPHK2/S1P Axis in ITP

Introduction Under normal physiological conditions, the bone marrow (BM) niche is hypoxic, and sustained and consistent oxygen gradients result in stable physiological hypoxia. Hypoxia inducible factor (HIF) is central to mediating the cellular response to hypoxia. Whether hypoxia gradients regulate megakaryocyte (MK) differentiation and maturation and the possible mechanisms of these actions remain unknown. Our previous data showed that impaired proplatelet formation (PPF) contributed to the development of thrombocytopenia in ITP. Recent studies suggest that the S1P signaling pathway in MKs plays a critical role in PPF ( Blood, 2013; J EXP MED, 2012). To further explore the underlying mechanism of impaired PPF in ITP, we found that HIF-1α/SPHK2/S1P axis-mediated cytoskeletal reorganization was defective in the PPF of ITP. All-trans retinoic acid (ATRA), which has been shown to be a promising treatment option for ITP patients in our clinical studies ( Blood, 2021; Lancet Haematology, 2017; Lancet Haematology, 2021), could restore cytoskeletal reorganization and correct impaired PPF. Methods Thirty patients with newly diagnosed ITP and 30 healthy donors were included in our study. The 30 ITP patients received ATRA at a dose of 10 mg orally twice daily for 12 weeks. Bone marrow samples were taken before the first medication (Day 0) and after 12 weeks of ATRA therapy (Day 85). We employed an imaging cytometry platform to perform a comprehensive quantitative analysis of MK distribution and hypoxic status in the bone marrow microenvironment of ITP. Targeted and untargeted metabolomic profiling through metabolomic analysis was performed to explore the relationship between the metabolome and ITP. Results We analyzed the hypoxic status of MKs in the endosteal niche and perivascular niche. In healthy controls, MKs in the perivascular niche exhibited a hypoxic profile, defined by robust HIF-1α expression and strong retention of pimonidazole. MKs in the endosteal region were less hypoxic. These results reflect a spatial hypoxia gradient in the bone marrow microenvironment. In contrast, we found that, irrespective of their location, ITP MKs displayed less hypoxia with no defined spatial gradients. ITP-MKs in the perivascular niche displayed altered cytoskeletal reorganization and impaired proplatelet formation (PPF). Metabolomics data revealed a decreased S1P level in ITP patients. Downregulated sphingosine kinase 2 (SPHK2) in MKs suppressed S1P production. S1P was essential for cytoskeletal reorganization and PPF regulation. In addition, we demonstrated that hypoxia inducible factor-1α (HIF-1α) mediated SPHK2 activation and S1P production. These data suggested that a disrupted hypoxia gradient contributed to impaired PPF via the HIF-1α/SPHK2/S1P axis. We then investigated the effect of ATRA on PPF. The ITP patients received ATRA at a dose of 10 mg orally twice daily for 12 weeks. For patients who received ATRA, S1P levels, HIF-1α and SPHK2 expression levels were increased compared with the pretreatment levels. And ATRA corrected impaired PPF and restored cytoskeletal reorganization by upregulating HIF-1α. Conclusions A disrupted hypoxia gradient contributed to impaired PPF in the ITP bone marrow environment via the HIF-1α/SPHK2/S1P axis. The ATRA-induced correction of impaired PPF is a potential mechanistic explanation for the clinical efficacy of ATRA in ITP.

Disability Adjusted Life-Years (DALYs) As a Measure of the Changing Lifetime Disease Burden in Long-Term Survivors of Allogeneic Hematopoietic Cell Transplantation (HCT)

Background: Although HCT is curative for many hematologic diseases, long-term survivors face a high burden of morbidity and premature mortality compared to the general population. Standard risk measures (prevalence, incidence, hazard, and standardized mortality ratios) do not convey a holistic picture of chronic disease burden because they do not account for degradation in quality of life associated with living with health conditions preceding death. DALY is an established risk measure that accounts for quality of life; it is the sum of years of healthy life lost due to disability (YLD) and years of life lost (YLL) to premature death. YLD weighs the years lived with a health condition by its disability weight (range: 0 for full health, 1 for death). Higher DALY, YLL, YLD indicate worse outcomes. We used microsimulation to estimate DALYs in 5-year survivors of HCT done over four decades, taking into consideration the changing practice of transplantation and the evolving burden of morbidity and mortality over time. Methods: The cohort included 1577 patients who underwent HCT at age ≥18 years (y) at City of Hope in 1976-2011, survived 5y, and were followed to December 31, 2016. Vital status and cause of death (COD) were determined using medical records and the National Death Index Plus. CODs were grouped into eleven broad categories used in a Global Burden of Disease (GBD) study ( Chang et al., Lancet Public Health, 2019). All cause and cause-specific mortality risks by attained age and other risk factors were estimated from deceased patients' data. These risks were applied in microsimulation to project age at death and COD for patients known to be alive. The onset age of COD was estimated for each patient using the age-specific disease incidence rates in GBD (https://www.healthdata.org/gbd). Life expectancy (LE) at time of death was determined from the US Life Tables. YLL was calculated as LE minus age at death. YLD was calculated as age at death minus disease onset age, weighted by disability weights of the COD available at Global Health Data Exchange (https://ghdx.healthdata.org). DALY was computed as YLL+YLD. Microsimulation, performed 1000 times, provided mean YLL, YLD, DALY for each patient. Results: Patient and treatment characteristics evolved over 35 years of HCT era ( Table). Patients who underwent HCT in the earliest era (1976-1989) had characteristics consistent with more favorable post-HCT outcomes compared to patients transplanted in later eras. From 1976-1989 to 2005-2011, there was an increase in patients who were older (median, 28y to 49y), had high relapse risk (26% to 42%), had an unrelated donor (0% to 45%), and developed chronic graft vs host disease (54% to 84%). Conversely, fewer patients received total body irradiation over time (88% to 36%). The proportion of patients transplanted for chronic myeloid leukemia declined from 24% to 5%, and there was a relative increase in patients with myelodysplastic syndrome (4% to 17%). Sex and race/ethnicity remained steady. The most common causes of death were relapse (35%), subsequent malignant neoplasms (22%), cardiovascular disease (10%), diabetes, kidney disease (10%), and infection (8%). Despite more patients with higher risk features receiving HCT in recent eras, median DALY was projected to decline from 19.2y to 15.5y from the earliest to latest HCT era (p<0.0001) ( Figure). YLL also declined from a median of 18.0y to 14.2y across eras (p<0.0001). Although YLD constituted a small portion of DALY, patients were projected to lose a median of 1.6y (1976-1989) to 1.2y (2005-2011) of healthy life due to disease-related disability. The projected age at death for the cohort (median, 67.5y to 73.4y across era) adjusted for rising life expectancy (median, 85y to 88y) suggested that survival after HCT increased in more recent HCT eras: age at death / life expectancy ratio median, 0.79 (1976-1989) to 0.84 (2005-2011) (p<0.0001). Conclusion: Outcomes after HCT have improved during the past four decades in spite of the evolving pattern of higher risk disease and patient characteristics with time. There was notable reduction across all measures (DALY, YLL, YLD), suggesting improvements in both the quantity and quality of survival. Our results highlight the importance and utility of DALY when characterizing health outcomes after HCT, as it provides a more comprehensive assessment of the changing pattern of efficacy associated with transplantation.

Inflammatory Signatures Define a New High-Risk T-Lineage ALL Subtype

T-lineage acute lymphoblastic leukemia (ALL) is an aggressive cancer comprising of diverse subtypes that are challenging to stratify using conventional immunophenotyping and have historically exhibited poor treatment outcomes in response to cytotoxic chemotherapy ( Nguyen, K et al. Leukemia 2008). Early T-cell Precursor ALL (ETP-ALL) represent a distinct subtype that displays a developmental block at the earliest stages of T-cell commitment, accompanied by the aberrant expression of myeloid and stem cell markers ( Coustan-Smith, E. et al. Lancet Oncol. 2009). The classification can be further complicated by T/Myeloid Mixed phenotype acute leukemia (T/My-MPAL), a rare and aggressive malignancy characterized by blasts with both T-cell lymphoid and myeloid markers ( George, B. S. et al. Biomedicines 2022). Accurate clinical diagnosis of T-lineage ALL is hindered by phenotypic variations among its subsets. Nevertheless, the precise diagnosis holds critical importance as drug sensitivity in preclinical models of T-lineage ALL is closely linked to the differentiation state, such as the sensitivity of ETP-ALL to the BCL-2 inhibitor venetoclax ( Chonghaile, T. N. et al. Nat Cancer 2021). This highlights the critical need to identify consistent phenotypic traits associated with unique therapeutic vulnerabilities to effectively tailor therapy to individual patients. To gain insights into subset-specific therapeutic vulnerabilities, we performed an integrative multiomic analysis of bone marrow (BM) samples (n=21) from newly diagnosed T-lineage ALL patients, including T-cell ALL (T-ALL), ETP-ALL, and T/My-MPAL. Leveraging cellular indexing of transcriptomes and epitopes in conjunction with T-cell receptor sequencing, we identified a distinct subset of patients spanning all three subtypes characterized by the high frequency of leukemic cells that most closely resemble healthy hematopoietic stem and progenitor cells (HSPC) by Symphony ( Kang, J. B. et al. Nat Commun 2021) query mapping, indicative of an earlier stage of differentiation arrest ( Figure 1A bottom). Pathway analysis revealed an inflammatory signature in patients with high frequencies of stem-like cells suggestive of an inflammatory microenvironment akin to high-risk AML ( Lasry, A. et al. Nat Cancer 2023), which we refer to as inflammatory T-lineage ALL. Next, we mined our multiomic data to generate a comprehensive geneset for inflammatory T-lineage ALL scoring in single cells and bulk samples ( Figure 1A top). Interestingly, Olink cytokine array analysis uncovered upregulation of production of cytokines IL-1β, IL-18, and/or OSM in inflammatory T-lineage ALL samples. Thus, this comprehensive multiomic analysis identified a subset of T-lineage ALL patients characterized by early differentiation arrest and inflammatory signatures. To explore the relationship between inflammatory T-lineage status and clinical response, we utilized the ssGSEA algorithm to score activity of our inflammatory signature in bulk RNA-sequencing data from the TARGET T-lineage ALL dataset (n=265, 74/265 inflammatory) ( Liu, Y. et al. Nat Genet 2017) and the Tran et al. study (n=27, 17/27 inflammatory) ( Tran, T. H. et al. Blood Adv 2022). We found a significant association between high inflammatory ssGSEA score and elevated MRD. To uncover the inflammatory T-lineage ALL mutational landscape, we performed whole exome sequencing on our study cohort, and compiled the reported mutations in the TARGET dataset. Interestingly, inflammatory T-lineage ALL patients, as predicted by inflammatory signature activity, had a significantly higher prevalence of mutations affecting cytokine signaling ( NRAS, JAK3), chromatin remodeling genes ( EZH2), and the WT1 oncogene. These analyses demonstrate a significant relationship between inflammatory status, genomic lesions, and MRD. Finally, we demonstrate that inflammatory, but not non-inflammatory, T-lineage ALL cells are significantly more sensitive to BCL-2 inhibitor venetoclax ex vivo (Figure 1B).Overall, our study has identified a new subtype of T-lineage ALL defined by an early arrest in T-cell development, inflammatory signaling, a unique mutational landscape, sensitivity to venetoclax, and an association with adverse clinical parameters.

Analysis of Ventricular Arrhythmias and Sudden Death with Acalabrutinib from 5 Prospective Clinical Trials

Background: The approval of ibrutinib, the first covalent Bruton tyrosine kinase inhibitor (BTKi), provided an effective non-chemotherapy option for the treatment of B-cell malignancies, such as chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL). While generally well tolerated, ibrutinib demonstrated significant toxicities in clinical studies, particularly cardiovascular adverse events such as hypertension, atrial fibrillation, and ventricular arrhythmias (VAs). Post hoc analysis of the phase 3 FLAIR trial in patients with previously untreated CLL reported an unexplained sudden or cardiac death rate of 0.5 patients per 100 patient-years with ibrutinib plus rituximab (Hillmen et al. Lancet Oncol. 2023). Acalabrutinib is a next-generation, more selective, irreversible BTKi approved to treat CLL/SLL and relapsed/refractory mantle cell lymphoma. Herein, the rates of nonfatal and fatal VAs and sudden deaths (SDs) from 5 acalabrutinib clinical trials of patients with CLL were analyzed. Methods: Incidence and relative risk of nonfatal and fatal VAs and SDs were analyzed using pooled data from 5 prospective acalabrutinib clinical trials (nonrandomized trials: CL-001 [acalabrutinib monotherapy], CL-003 [acalabrutinib + obinutuzumab]; randomized trials: CL-006 [acalabrutinib vs ibrutinib], CL-007 [acalabrutinib ± obinutuzumab vs chlorambucil + obinutuzumab], CL-309 [acalabrutinib vs idelalisib + rituximab OR bendamustine + rituximab]). Acalabrutinib-containing arms were pooled as a single group; comparators, which were standard-of-care therapies, were pooled as a separate group. Events were identified by comprehensive data extraction of terms capturing nonfatal and fatal VAs and SDs (terms are defined in Table 1 footnote). Nonfatal events were analyzed with and without the inclusion of premature ventricular contractions (PVCs). A subset of fatal events also was analyzed after adjudication through manual clinical review by the sponsor's medical, clinical, and safety leadership. Results: In total, 1299 patients received acalabrutinib (cumulative exposure, 4568.4 patient-years) and 585 received standard-of-care therapies (comparator group) (941.3 patient-years). Considering only fatal VAs and SDs, 5 (0.4%) and 3 (0.5%) patients had an event in the acalabrutinib and comparator groups, respectively, via comprehensive data extraction ( Table 1). Based on exposure-adjusted event rates per 100 patient-years, fatal VAs and SDs were numerically less frequent for acalabrutinib versus comparators; however, relative risks did not statistically favor either group. This trend persisted when events were adjudicated through manual clinical review. After adjudication, 2 of 5 patients in the acalabrutinib group and 2 of 3 patients in the comparator group were determined to have fatal VA or cardiac-related SD. Nonfatal VAs were reported in 11 (0.8%) patients in the acalabrutinib group and 3 (0.5%) patients in the comparator group. When PVCs were excluded, 2 patients in each group (acalabrutinib, 0.2%; comparator, 0.3%) had nonfatal VAs. These nonfatal VAs consisted of ventricular fibrillation (acalabrutinib, n=2 [grades 2 and 4]; comparator, n=1 [grade 4]) and unspecified VA (comparator, n=1 [grade 1]). Although the acalabrutinib group had a lower exposure-adjusted event rate per 100 patient-years than the comparator group, relative risk did not favor either group. With both fatal and nonfatal VAs (with or without PVCs), time to event was notably longer with acalabrutinib than with comparators ( Table 1). A sensitivity analysis excluding the ibrutinib-treated patients from the comparator cohort yielded overall similar results (data not shown). Conclusions: This pooled analysis of prospective acalabrutinib clinical trials demonstrates that the risk of VA and SD with acalabrutinib is low and similar to that of standard-of-care therapies when adjusted for exposure. The current analysis with more than 1200 patients treated with acalabrutinib points to favorable safety outcomes and no specific trend with SDs and VAs. Additional analyses with larger cohorts from the clinical development program and all postmarketing sources will continue to further characterize the safety profile of acalabrutinib.

Pre- and Post-Treatment Immune Contexture Correlates with Long Term Response in Large B Cell Lymphoma Patients Treated with Axicabtagene Ciloleucel (axi-cel)

Background: Axi-cel is a US and EU-approved autologous anti-CD19 chimeric antigen receptor (CAR) T cell therapy for pts with relapsed/refractory large B cell lymphoma after 1 prior therapy as a result of ZUMA-7 (NCT. In ZUMA-1 (NCT02348216), the pivotal study which initially led to the approval of axi-cel in the 3L+ setting, the objective response rate was 83% (58% complete response rate; Locke et al. Lancet Oncol. 2019) and at 5 years follow up an OS rate of 42.6%, 5 years durable response and 5 years disease specific survival at 51% ( Neelapu et al. Blood 2023). T cell-related biology (Immunosign 21; Immunoscore®IC) measured pretreatment in the tumor microenvironment (TME) was associated with response to axi-cel ( Scholler et al., 2022). Increased density of activated PD-1+LAG-3+/−TIM-3−CD8+ T cells, measurable pretreatment, facilitates clinical response in pts post-axi-cel. This expanded analysis characterized the impact of axi-cel treatment on the TME immune contexture and examined associations between immune cell subsets and relapse. Methods: Baseline and post-infusion tumor biopsy samples were analyzed by multiplex immunohistochemistry ( Brightplex®). Four panels were developed and applied to assess T cell infiltration (CD3 CD8 FOXP3 TIM3 PD1 LAG3 TOX), regulatory T cell subtyping (CD3 CD8 GATA3 TBET RORg BCL6), T cell activation & exhaustion (CD3 CD8 TIM3 LAG3 PD1 GZMB KI67), macrophage (CD68 CD64 CD163 CD204 CD206 PDL1) and MDSC (CD3 CD11B CD68 CD14 CD15 LOX1 S100A9) subsets. Transcriptomic analysis was performed using nCounter® Pan Cancer panel. The association between T cell subtypes and macrophages cell subset density, and probability to relapse was evaluated. T test values were based on Brightplex analysis. Results: 34 tumor biopsies (16 at baseline, 18 post-infusion) from 26 pts were analyzed including 11 relapsed (6 CR/5 PR) and 15 durable response (15 CR). The baseline and post-infusion TME comprised all major macrophages, MDSC and T cell subsets, with diverse distribution across samples analyzed. Low proinflammatory M1 macrophage density was observed at baseline and post-infusion across patients. In relapsed patients, baseline tumors were enriched with a significantly higher proportion of protumoral M2 macrophage (p<0.0001). A shift of M1-M2 polarization was observed between baseline and post-infusion (Baseline 33%M1M2 / 65%M2, post infusion 76%M1M2 / 21%M2, p<0,0001) across the cohort. In contrast, in relapsed patients, M-MDSC cell density was significantly increased post treatment (p=0.0420). In addition, Innate lymphocyte cells subtype 2 (ILC2) cell density, previously described to be positively correlated with improved overall survival in CRC ( Huang et al. 2021 Cancers), was significantly decreased in relapsed patients post infusion (p=0.019).Post-infusion, durable response was associated with a significant increase in pan-Macrophages (p=0.0360) and cytotoxic T cell subset densities: CD8 naïve T cells (p=0.016); Activated Cytotoxic T cell (GZMB+)(p=0.0203); PD1+TIM3+LAG3- cytotoxic T cell (p=0.0117); CD8 regulatory T cell (FOXP3+)(p=0.012). Regarding T helper lineage, ongoing response was associated with a significant increase of: CD4 naïve T cells (p=0.021); T helper Th2 (p=0.020); cytotoxic T lymphocyte TC2 and TC1/TC2 cell densities (respectively p=0.0052 and 0.011).Additional characterization of the immune contexture and correlative analysis of cell subsets will be presented. Conclusion: These results suggest that baseline proportions of protumor M2 macrophages may predict disease relapse after axi-cel treatment. Following infusion, relapse is also associated with spatial enrichment of M-MDSC, whereas durable response is related to increases in T cell subpopulation densities inclusive of cytotoxic T cells, T helper, TC1/TC2 ratios and innate T-subpopulations. These findings suggest that axi-cel treatment drastically impacts tumor immune mobilization, infiltration and contexture, which correlates with long term response.

Polatuzumab Vedotin, Zanubrutinib and Rituximab Achieved Rapid and Deep Response in Previously Untreated Frail and Elderly Diffuse Large B-Cell Lymphoma Patients

Introduction: The incidence of diffuse large B-cell lymphoma (DLBCL) increases with age, while first-line treatment for frail and elderly DLBCL is a debating topic. R-miniCHOP, a reduced intensity regimen of R-CHOP, is currently the standard of care for this population, yet with limitation due to toxicities (Frédéric Peyrade et al., Lancet Oncol, 2011). Chemo-free regimens combining Bruton kinase inhibitors, rituximab and lenalidomide showed fewer toxicities, but compromised on efficacy in elderly DLBCL in multiple previous reports (Pengpeng Xu et al., Lancet Healthy Longev, 2022; Haiyan Yang et al., ASH 2022). First-line treatment balancing efficacy with safety is urgently needed for frail and elderly DLBCL patients. Since polatuzumab vedotin was approved in China for first-line DLBCL this year and reportedly had synergistic effect with rituximab in cell line research (Natsumi Kawasaki et al., Br J Haematol, 2022), we conducted a study of polatuzumab vedotin plus zanubrutinib and rituximab (ZPR) in previously untreated frail and elderly DLBCL to assess the efficacy and safety. Here we present the characteristics of 12 enrolled patients and preliminary results. Methods: Previously untreated DLBCL patients aged over 70 years old, or aged between 60 to 69 with ECOG score 2 to 4 were enrolled in this study. All patients received a triplet regimen of ZPR. Twenty-one days was a cycle. Zanubrutinib was given 160mg twice a day orally from day 1 to day 21. Rituximab was given 375mg/m 2 intravenously on day 1. Polatuzumab vedotin was administered at a dosage of 1.8mg per kilogram intravenously on day 1. Patients would receive 6 cycles of ZPR regimen if assessed partial response (PR) or complete response (CR) after 3 cycles. If assessed CR after 6 cycles of ZPR, patients would receive zanubrutinib 160mg twice a day alone for another 6 cycles. Whole body 18F-FDG PET/CT scan or contrast-enhanced CT scan of cervical, thoracic, abdominal and pelvic parts was scheduled every 3 cycles. Treatment response was assessed by 2014 Lugano response criteria. Adverse events were graded by CTCAE 5.0. The primary endpoint is overall response rate (ORR) after 6 cycles of ZPR. The secondary endpoints are CR rate after 6 cycles of ZPR, and 2-year progression-free survival (PFS). Other endpoint is safety (Figure 1). Results: Twelve patients were enrolled in our study until Jul 24, 2023. Median age was 75 (range: 67-87). Six patients were female and six patients were male. Nine (75%) patients were classified non-GCB type according to cell of origin by Hans algorithm. Nine (75%) patients were assessed stage Ⅲ to Ⅳ by Ann Arbor staging system. Nine (75%) patients had an international prognostic index (IPI) score of 2 or more. One (11%) patient had double-hit risk factor in 9 patients with gene rearrangement results. Six (75%) patients had MYD88 L265P mutation in 8 patients with sequencing results. After a median follow up of 2.1 months, four patients had PET/CT assessment results after 3 cycles of ZPR, and four (100%) of them achieved CR. The ORR after 3 cycles of ZPR regimen was 100% (Figure 2).Grade 3 to 4 adverse events occurred in two patients, that one patient had grade 3 aspartate aminotransferase increase and alanine aminotransferase increase, and the other patient had grade 3 pneumonitis. Grade 1 to 2 adverse events included neutrophil count decrease (16%), dizziness (8%), and epistaxis (8%). All patients recovered from adverse events under best supportive care. There was no discontinuation of ZPR regimen due to severe adverse events. Conclusion: Based on these results in our study, ZPR regimen showed rapid and deep response in previously untreated frail and elderly DLBCL patients with manageable safety profiles. We registered and initiated a phase 2b trial (NCT05940064) to further evaluate the efficacy and safety of ZPR regimen as first-line treatment for this population. Ongoing treatment and follow up of these twelve patients will also be further updated.

Acrue: A Phase 2, Open-Label Study of Acalabrutinib in Combination with Rituximab in Elderly and/or Frail Patients with Treatment-Naive Diffuse Large B-Cell Lymphoma

Background and Significance: Patients aged ≥80 years with diffuse large B-cell lymphoma (DLBCL) account for approximately 18% of DLBCL cases in the United States (Morrison et al. Future Oncol. 2018). Standard-of-care chemoimmunotherapy (CIT) regimens such as rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone (R-CHOP), including reduced doses of CHOP (R-miniCHOP), are not well tolerated in elderly and/or frail patients, leading to high treatment-related mortality rates (Boslooper et al. Leuk Lymphoma. 2014; Freudenberger et al. Ann Hematol. 2021; Peyrade et al. Lancet Oncol. 2011). Therefore, safe, effective, and convenient treatment alternatives to CIT are needed. Acalabrutinib is a highly selective, second-generation Bruton tyrosine kinase inhibitor approved for the treatment of patients with relapsed/refractory mantle cell lymphoma and in patients with chronic lymphocytic leukemia. Acalabrutinib monotherapy has demonstrated encouraging activity in patients with DLBCL in phase 1b (NCT02112526) and phase 2 (NCT04002947) studies (Strati et al. Haematologica. 2021; Roschewski et al. Blood. 2021). However, neither of these studies were conducted specifically in a population of elderly and/or frail patients. Study Design and Methods: ACRUE (NCT05952024) is a phase 2, open-label, single-arm, global, multicenter study of acalabrutinib plus rituximab in elderly and/or frail patients with treatment-naive (TN) DLBCL who are unable to tolerate standard CIT ( Figure). Eligible patients are aged ≥80 years or aged 65-79 years with impairment in ≥1 activity of daily living (ADL) or instrumental ADL (IADL), or with cardiac/renal/liver function impairment or other comorbidities that render them unable to tolerate CIT. Furthermore, patients must have histologically confirmed CD20-positiveDLBCL (not otherwise specified), according to WHO 2016 classification, an Ann Arbor classification of stage II, III, or IV disease, an Eastern Cooperative Oncology Group performance status of 0-2, and ≥1 measurable lesion. Patients with active central nervous system involvement by lymphoma, leptomeningeal disease, or spinal cord compression, or significant cardiovascular disease within 6 months of screening are excluded. Target enrollment is 80 patients. Patients will receive acalabrutinib plus rituximab combination for 8 cycles (acalabrutinib 100 mg orally twice daily beginning on day 1 of cycles 1-8; rituximab 375 mg/m 2 intravenously on C1D15, then 1400 mg subcutaneously, on day 1 of cycles 2-8; cycles 1-8 are 21 days/cycle), followed by acalabrutinib monotherapy until disease progression, unacceptable toxicity, or 24 months (cycles 9-28 are 28 days/cycle). The primary endpoint is the percentage of patients with grade 3-4 treatment-emergent adverse events. The key secondary endpoint is investigator-assessed objective response rate per Lugano 2014 criteria. Other secondary endpoints include progression-free survival, event-free survival, overall survival, duration of response, and Timed Up and Go test. Exploratory biomarker analyses may include genomic characterization of baseline samples to evaluate predictors of survival, and of progression samples to evaluate genomic resistance mechanisms. Descriptive safety and efficacy data will be summarized. If patients discontinue treatment prior to completing 28 cycles of therapy, they will be followed every 12 weeks after their last treatment dose for up to 2 years. Patients who are still on treatment at the end of the study and are deriving clinical benefit may receive continued treatment, with assessments reverting to standard of care. Enrollment is planned to begin Q4 of 2023 in approximately 40 sites from North and South America and Asia. Summary: This study will help determine if acalabrutinib plus rituximab combination therapy is tolerable and effective in elderly and/or frail patients with TN DLBCL who are unable to tolerate CIT.

Predictors of Outcomes with Venetoclax-Based Treatment in Patients with Progressive Disease or Intolerance after Covalent BTK Inhibitors

Background: Venetoclax (Ven) is a standard of care for the treatment of patients (pts) with CLL/SLL. In the relapsed setting, there is very limited data on the efficacy of Ven after covalent BTK inhibitors ([cBTKi], ibrutinib, acalabrutinib, or zanubrutinib), and there is no prospective trial data on the feasibility of a Ven-based fixed-duration therapy after cBTKi failure. Recently, novel treatment options like pirtobrutinib, a non-covalent BTKi, have shown high efficacy after cBTKi, prompting scrutiny of Ven-based therapies after cBTKi failure, including Ven monotherapy and combination treatment with anti-CD20 antibodies. In this study, we aim to evaluate: 1) The efficacy of Ven-based therapy after cBTKi, 2) Predictors of the efficacy of Ven in this setting, and 3) The feasibility of fixed-duration therapy with Ven after BTKi. Methods: In this single-institution, retrospective study, we used our institutional CLL registry to identify patients with a CLL/SLL diagnosis who received Ven (with or without anti-CD20 antibodies) for at least two months after prior treatment with a cBTKi. Patient-level clinical data were extracted from electronic medical records. Progressive disease (PD) was defined by the treating physician based on clinical, laboratory, and/or imaging data. Progression-free survival (PFS) and Overall survival (OS) were calculated using the Kaplan-Maier estimates. Results: We identified 80 pts who started Ven between 1/2016-12/2022 after prior treatment with a cBTKi. Median age was 62 (range: 40-80), and 53% were male. FISH abnormalities included: 58% del17p, 25% del11q, 48% del 13q, and 19% with trisomy 12. The median prior lines of treatment were 2 (range: 1-7). Prior first cBTKis were ibrutinib (92.5%), acalabrutinib (6.5%), and zanubrutinib (1.5%). A total of 7 pts (9%) received more than one prior cBTKi (1 ibrutinib, 6 acalabrutinib). Prior cBTKi was used as first-line treatment in 21 pts (26%), and 51 (64%) pts received prior treatment with chemotherapy. The median duration of cBTKi treatment was 25 months (range 1-92). Reasons for cBTKi discontinuation were PD (n=56, 70%) and intolerance (n=24, 30%). Most pts started Ven-based therapy immediately after cBTKi (n=53, 66%), although 21 (26%) received one interim line, and 6 (7.5%) received more than one interim line. Ven was used either as monotherapy (n=45, 56%) or with anti-CD20 antibodies (n=35, 44%). The median time on Ven was 11 months (2-55 months). At the time of analysis, 75% of pts had stopped Ven, with the most common reason for discontinuation being PD (31, 53%), followed by treatment completion (19, 32%). Median PFS after starting Ven was 29 months (95% CI: 17-44) for the entire cohort. Median PFS was significantly inferior in pts who experienced PD on prior cBTKi compared with those who were intolerant to prior cBTKi (17 months vs. 55 months, p=0.002). Inferior PFS was also observed in patients with del17p (7 months vs. 26 months without del17p, p=0.003) and in pts who received Ven monotherapy vs. combination therapy with anti-CD20 antibodies (21 months vs. not reached, p=0.01). There was no difference in PFS based on prior treatment with chemotherapy or for pts who received treatments between cBTKi and Ven. In multivariable analysis, having del17p (HR 3.04, 95%CI [1.38-6.65]), Ven monotherapy (HR 3.19, 95% CI [1.52-6.68]) and PD on prior cBTKi (HR 4.29, 95% [1.50-12.29]) were associated with inferior PFS. With a median of 28 months of follow-up, 26 pts (32%) stopped Ven and did not experience PD. This included 23% of pts with PD after prior cBTKi and 54% of pts intolerant to prior cBTKi (p=0.007). Conclusion: Venetoclax-based therapy after cBTKi provides disease control, but not to the extent reported by the only prospective trial in this setting (Jones, Lancet Oncol, 2018). Inferior PFS was observed in pts who had PD after prior cBTKi (vs. being intolerant), pts who received Ven monotherapy (vs. combined with anti-CD20 antibodies), and those with del17p. While this study may be limited in assessing the feasibility of fixed-duration Ven therapy after cBTKi, our results indicate that such an approach may only be feasible in a subset of pts who stopped cBTKi because of intolerance and not PD.

Updated Results from a Matching-Adjusted Indirect Comparison of Efficacy Outcomes for Isatuximab Plus Pomalidomide and Dexamethasone Versus Daratumumab Plus Pomalidomide and Dexamethasone in Patients with Relapsed and/or Refractory Multiple Myeloma

Introduction: Findings from a previously conducted matching-adjusted indirect comparison (MAIC) between isatuximab plus pomalidomide and dexamethasone (IsaPd) and daratumumab plus pomalidomide and dexamethasone (DaraPd) among relapsed and/or refractory multiple myeloma (RRMM) patients receiving at least two prior lines of therapy (LoT) including lenalidomide and a proteasome inhibitor (PI) demonstrated a significantly better overall survival (OS) and a positive trend for progression-free survival (PFS), overall response rate (ORR), and very good partial response or better (≥VGPR) rates with IsaPd than that with DaraPd, with a similar safety profile for both treatments [Leleu X et al, Value in Health 2022;25(1):S39]. The present study used longer follow-up data from the phase 3 ICARIA trial (NCT02990338) to update the previous MAIC, evaluating the comparative efficacy of IsaPd versus DaraPd. Methods: A MAIC was performed with the latest available individual patient-level data (IPD) from the ICARIA trial (IsaPd arm, n = 154; Pd arm, n = 153), data cutoff of March 14, 2022, and aggregate data from study publications of the phase 3 APOLLO trial (DaraPd arm, n = 151; Pd arm, n = 153, NCT03180736) [Dimopoulos MA, et al. Blood 2022;140 (Suppl 1):7272-7274; Dimopoulos MA, et al. Lancet Oncol. 2021;22(6):801-812; Dimopoulos MA, et al. Blood 2020;136(Suppl 1):5-6]. To match patients with APOLLO, patients in ICARIA who did not meet the inclusion criteria of APOLLO were excluded. In APOLLO, 11% patients received only one prior LoT. As patients with one prior LoT were not enrolled in ICARIA, it was not feasible to match on this factor; however, the treatment effect of DaraPd versus Pd on PFS among these patients was similar to patients in APOLLO with ≥2 prior LoTs. Therefore, this difference in study populations is unlikely to bias the MAIC. Remaining patients in ICARIA were then weighted using standardized MAIC weights (mean value = 1.0) generated using the method of moments, with active treatment and control arms matched separately to ensure any residual imbalances between active and control arms in APOLLO were replicated in the matched ICARIA sample. Anchored MAICs for the hazard ratio (HR) for OS and PFS, odds ratio (OR) for ORR, and ≥VGPR for IsaPd versus DaraPd were calculated using the Bucher method [Bucher HC, et al. J Clin Epidemiol. 1997;50(6):683-691]. Results: MAIC weighting was able to match all characteristics included in the matching algorithm, with standardized mean difference (SMDs) <0.0001 after weighting. The effective sample size after weighting was 28.1 for IsaPd and 43.1 for Pd. Based on an anchored MAIC, OS was significantly better with IsaPd versus DaraPd (HR [95% CI]: 0.49 [0.24-0.98]; p = 0.04) (Figure 1). A numerical trend in favor of IsaPd versus DaraPd was observed for PFS, ORR, and ≥VGPR, although the differences were statistically non-significant (Table 1). Conclusions: This MAIC analysis demonstrated a significant benefit in OS and a positive trend in the improvement of PFS, ORR, and ≥VGPR in favor of IsaPd compared with DaraPd. These findings were consistent with the previous MAIC with a longer follow-up data (present analysis used a maximum follow-up of 4.5 years of OS data from both trials, whereas the previous analysis used a follow-up of 3 years from ICARIA and unknown from APOLLO), which highlights the therapeutic potential of IsaPd in RRMM, particularly for patients who become refractory to lenalidomide and a PI.

Tafasitamab for the Treatment of Relapsed/Refractory (R/R) Diffuse Large B-Cell Lymphoma (DLBCL) in the US Real-World Setting

Introduction: Tafasitamab (tafa) is a CD19-targeting immunotherapy indicated in combination with lenalidomide (len) for the treatment of adult patients with R/R DLBCL ineligible for autologous stem cell transplantation (ASCT). The 2020 FDA accelerated approval of tafa in this setting was based on findings from L-MIND, a multicenter, open-label, single-arm, phase 2 trial (Salles G, et al. Lancet Oncol. 2020;21:978-988). Since this approval, there has been a paucity of real-world studies (RWS) evaluating the outcomes of patients with R/R DLBCL who received tafa in the community setting. We conducted this RWS to describe the patient characteristics, treatment patterns, and outcomes of patients who received tafa for R/R DLBCL across practice settings in the US. Methods: A retrospective, multisite, physician-abstracted medical chart review RWS was conducted in US adults who initiated tafa (with or without len) on or after Oct 21, 2020 for R/R DLBCL outside of the clinical trial setting. Patients were required to have at least 4 months of follow-up after tafa initiation unless the patient died during this period. Participating physicians from Cardinal Health's Oncology Provider Extended Network-approximately 83% from community oncology practices-abstracted data from the medical records of patients into electronic case report forms. Data were summarized using descriptive statistics for all patients and for subgroups based on whether tafa was received as second-line (2L) or third-line (3L) therapy for R/R DLBCL. Whereas some patients received tafa as fourth-line (4L) or fifth-line (5L) therapy, data for these patients were not analyzed separately due to small numbers. Results: A total of 181 patients were included in this study, with a median follow-up time of 6.5 (range, 0.9-27.4) months since initiating tafa. Key patient characteristics, treatment patterns, and outcomes are summarized in Table 1. At initiation with tafa, the majority of patients had an Eastern Cooperative Oncology Group performance status of 0-2 (98%), Ann Arbor stage of III-IV (93%), and a revised International Prognostic Index score of 3-5 (75%). The majority of patients (72%) received tafa as 2L therapy for R/R DLBCL, whereas 24% received tafa as 3L, 3% as 4L, and 2% as 5L. The median time from initial diagnosis of DLBCL to initiation of tafa was 20 (interquartile range [IQR], 12-35) months. Prior to tafa, 12% had undergone ASCT and 3% had received chimeric antigen receptor T-cell therapy. The starting dose of concomitantly administered len varied: 68 (38%) received 25 mg; 43 (24%) received 20 mg; 29 (16%) received 15 mg; 31 (17%) received 10 mg; 2 (1%) received 5 mg; and 8 (4%) patients did not receive concomitant len. Len dose reductions were experienced during treatment by 19% of patients. Among the 60 patients who discontinued tafa, reasons for discontinuation included progression confirmed by scan (50%), progression defined clinically (17%), toxicity (15%), patient/caregiver request (3%), complete response (2%), and other reasons (13%). The real-world overall response rate for tafa was 76% (95% confidence interval [CI], 69-82%), with a real-world complete response rate of 18% (95% CI, 13-24%). The real-world progression-free survival probability at 6 months post tafa initiation was 0.8 (95% CI, 0.7-0.8). At the time of data collection, most patients were still alive (80%), among whom 84% were still receiving tafa. Conclusions: Findings from this real-world analysis support the clinical benefit of tafa when used in early lines of treatment for R/R DLBCL, as demonstrated in L-MIND. Importantly, the patient population included in this study was racially and ethnically diverse, with nearly one-third of patients from typically underrepresented racial groups and approximately one-sixth of Hispanic ethnicity. Further, this population consisted of patients treated predominantly in a community oncology setting, which is the treatment setting for the majority of patients with DLBCL in the US. Most patients were still on tafa at the time of data collection and follow-up was limited in duration; the median time to complete response in L-MIND (Duell J, et al. Haematologica. 2021;106:2417-2426) was longer than the median follow-up time in this study. Therefore, longer follow-up of these patients is warranted to better understand long-term outcomes of tafa among this diverse patient population.

Development of the Treatment Preference in Myelodysplasia Questionnaire (TPMQ) for Clinicians (mTPMQ), Carers (cTPMQ), and Patients (pTPMQ)

Objective: Myelodysplastic syndrome (MDS) is a disease of the hematopoietic stem cells and leads to an increased risk of developing acute myeloid leukemia (AML).[1] The injectable hypomethylating agents (HMA) azacitdine and decibtabine have been shown to be superior to best supportive care in improving cytopenias in intermediate to high-risk patients.[2, 3] These injectable HMAs regimens are commonly administered in a healthcare facility, with patients traveling to the facility on consecutive days. The recent availability of an oral form of decitabine, ASTX 727 (a fixed-dose combination of decitabine and the cytidine deaminase inhibitor cedazuridine), may significantly impact patient, carer, and clinician choices. To assess preference for an injectable vs. orally administered HMA, three questionnaires were developed based on qualitative input from clinicians, patients, and carers: the Treatment Preference in Myelodysplasia Questionnaire (TPMQ) for clinicians (mTPMQ), carers (cTPMQ), and patients (pTPMQ). Methods: This was a non-interventional, cross-sectional qualitative interview study consisting of interviews with patients with MDS who were eligible for or were being/had been treated with injectable azacitidine, their carers and their treating clinicians. Data were collected from qualitative mixed method interviews composed of concept elicitation and cognitive debriefing. Two sites were identified to enrol participants. During concept elicitation, participants were asked about MDS and treatment options to best understand the constructs of interest relative to their specific circumstance and preferences. Cognitive debriefing occurred after participants reviewed a draft of the questionnaire. Based on the interviews, the questionnaires were revised to ensure the constructs of importance were captured and the questionnaires, response options, and instructions were all well understood. Results: 15 participants completed interviews (5 clinicians for the mTPMQ, 5 patients for the pTPMQ, and 5 carers for the cTPMQ). Concepts identified as important included across groups included: easier to be compliant, less travel time, less time in the clinic, less painful, costs and convenience (Table 1). The mTPMQ underwent 3 revisions. Overall, clinicians indicated the directions, items and response options were understandable and clear. Comments that significantly modified the questionnaire concerned splitting the last question from 1 to 2 questions, depending on which treatment the patient would be receiving. Patient participants thought the pTPMQ captured concepts of interest and was easy to understand. Modifications were made to the questionnaire either as a direct result of patient participant comments (e.g., changed skip directions to “turn to page X, question X”), study team discussions of the interviewer's comments and notes and making items consistent between questionnaires. Carer participants thought the cTPMQ captured the constructs of interest and the questions were clear and the questionnaire was easy to answer. Modifications were made to the questionnaire as a direct result of carer participant comments (e.g., changed “We spend less time in the clinic” to “It means less time in the clinic for the patient”), study team discussions of the interviewer's comments and notes (e.g., added “It makes it easier for the patient to continue treatment”) and making items consistent between questionnaires. Conclusions: The findings of the participant interviews suggest the concepts assessed in the assessments are complete. Respondents could respond to items and talk about all aspects assessed in the questionnaires in meaningful ways and were able to comprehend the instructions and response options on a categorical scale. The TPMQs are based on direct input from the population of interest and assess the constructs of importance to each group. 1. Bell, J.A., et al., Clin Lymphoma Myeloma Leuk, 2018. 18(4): p. e157-e166. 2. Kantarjian, H., et al., Cancer, 2006. 106(8): p. 1794-803. 3. Fenaux, P., et al., Lancet Oncol, 2009. 10(3): p. 223-32.

Senescent Immune Signature in Frail and Vulnerable Newly Diagnosed Myeloma Patients: A Cohort Analysis from the UK-MRA Myeloma XIV Fitness Study

Introduction Physical frailty is a key factor driving treatment tolerability and outcome in multiple myeloma (MM). There is a close interplay between physiological aging, frailty and immune dysfunction, with the presence of a malignant disease overlay adding an additional dimension to the host response biology. The UKMRA Myeloma XIV FiTNEss study is the first phase 3 randomised clinical trial to explore the utilisation of frailty assessment as a tool to guide and optimise treatment decision making and deliverability. Here we report on the relationship between physical frailty and T cell dysfunction in a cohort of patients within this landmark study. Methods Multiparameter flow cytometry was used to assess the baseline T cell phenotype in the peripheral blood of 49 participants with NDMM receiving treatment within FiTNEss. Patients were assessed for frailty using the IMWG frailty score and additionally assessed for clinical vulnerability using the UKMRA MRP score (Cook et al, Lancet Haematol. 2019). The IMWG frailty score combines activities of daily living assessment and co-morbidity burden with age to generate a validated, prognostic frailty score. The UKMRA MRP is a risk profile which uses routinely collected clinical data including age, WHO performance status, ISS stage and CRP to define prognostic risk categories with the high-risk category encompassing frailer individuals. Normal T cell subsets were assessed based on CD45RA and CD28 phenotype while dysfunctional subsets (exhausted and senescent) were defined using CD28 and KLRG1. Results The median age of participants was 79 (range 68-90) and 55% were male. The distribution by IMWG was fit 18%, unfit 33%, frail 49% and by MRP was low risk 31%, intermediate risk 28%, high risk 38%. Absolute lymphocyte count (ALC) decreased with increasing frailty (p=0.02), with no patients classified as fit by IMWG or low risk by MRP criteria having a lymphocyte count below the lower limit of normal. No relationship was seen between ALC and age. The ratio remained stable across the IMWG cohorts, suggesting a global reduction in lymphocyte subsets. An accumulation of CD8 TEMRA cells was seen in the MRP high risk cohort (p=0.0085). No differences were seen between cohorts based on the IMWG frailty score. The accumulation of CD8 TEMRA cells indicates a loss of T cell differentiation plasticity and is associated with a cytotoxic and pro-inflammatory immune response. CD4 dysfunctional subsets were comparable across the cohorts. CD8 dysfunctional subsets were most marked in the frail and high-risk cohorts. The MRP high-risk cohort had a skewed ratio of exhausted to senescent cells (p=0.02), with a reduction in CD8 exhausted populations compared to the fit and unfit cohorts (p=0.0477) and a trend towards increased senescent cell burden (p=0.07). This was echoed within the IMWG frail cohort where elevated numbers of senescent CD8 cells were identified (p=0.02). No relationship was seen between CD8 senescent burden and age, CRP, ISS or WHO PS when each variable was assessed independently, suggesting that the presence of this immune population is not directly related to disease burden or physiological aging alone, but reflects a more complex interplay between host response biology and disease characteristics. Principal component analysis demonstrated a high degree of immunological heterogeneity within the MRP low and intermediate risks cohorts, while the high-risk cohort had a more uniform immunological profile (Figure 1A). Significant overlap between a proportion of the non-high-risk cohorts and high-risk cohorts was seen and requires further exploration. A multi-variable correlation analysis identified correlations between the CD4:8 ratio and CD8 cell senescent burden, MRP and ISS, highlighting the important interplay between immunological dysfunction, clinical vulnerability and disease burden (Figure 1B). Conclusion The T cell immunological landscape of the most vulnerable patient populations with newly diagnosed MM is characterised by an accumulation of senescent CD8 T cells which cannot be attributed to age or disease burden alone. Immune senescence is known to be associated with a chronic inflammatory state which may drive the acquisition of the physiological changes, such as sarcopenia, which characterise a frailty phenotype. This provides an opportunity for targeted intervention to address the biological drivers of disease related frailty.

Real-Life Experience with Lenalidomide Plus Anti-CD20 Antibodies for the Treatment of Patients (pts) with Relapsed/Refractory (R/R) Indolent Non-Hodgkin Lymphoma (iNHL)

Introduction: The combination of lenalidomide and anti-CD20 antibodies (Ab) is an accepted treatment for R/R iNHL and often used as comparator in registration-directed clinical trials. In pts with R/R marginal zone lymphoma (MZL) or follicular lymphoma (FL) lenalidomide plus rituximab (R2) produced a median PFS of 27.6 months and 5-year OS 83.2% (Leonard, ASH 2022). Similarly, lenalidomide and obinutuzumab (RO) led to high PFS and OS rates in pts with R/R FL (2-year PFS 65% and OS 87%, Morschhauser, Lancet Haematol. 2019). Whether these results are reproducible in clinical practice is unknown. Methods: We retrospectively analyzed our institutional experience in patients with R/R FL or MZL treated at MSK with R2 or RO identified on our electronic lymphoma database. Patients with previous history of histological transformation (HT) or who received R2/RO in combination with other agents were excluded. Planned lenalidomide starting dose was 20 mg orally daily for 21/28 days for twelve 28-day cycles; dose adjustments were considered in case of impaired renal function per standard practice. Rituximab was administered at 375 mg/m2 weekly for 4 weeks, then every 28 days for five 28-day cycles; and obinutuzumab at 1000 mg on D1, 8, and 15 of C1, then on D1 of each 28-day cycle for 6 cycles. Response was assessed according to the 2014 Lugano criteria; for splenic MZL and extranodal MZL patients, the ESMO guidelines were adopted to assess disease response (Zucca, Annals of Onc. 2020). PFS was defined as the time from treatment initiation to disease progression or death, and OS as the time from treatment initiation to death from any cause. Patients undergoing autologous (auto) or allogeneic (allo) stem cell transplantation (SCT) were censored at that time. Results: 85 pts were treated between June 2013 and December 2022. Median age was 65 years (range 39-94); 71 patients (83.5%) had FL and 14 (16.5%) had MZL, and 18 patients (21%) had bulky disease (>7cm). The median number of prior therapies was 2 (range 1-8), 39 pts (46%) had ≥3 prior treatments, and 44 (53%) were refractory to their last regimen; 86% received R2 and 14% RO (Table). At the time of this analysis, pts had received a median of 9 cycles and 8 (10%) received more than 12 cycles. Eight (9%) pts are on therapy, 35 (41%) completed the planned treatment, 17 (20%) discontinued due to progression, 4 (5%) due to adverse events (AEs), 5 (6%) because they underwent auto- or allo-SCT, and 7 (8%) by medical decision, while in 9 cases (11%), treatment was prematurely discontinued in pts who achieved complete response (CR) after 6 cycles. Lenalidomide dose reduction was required in 47 pts (53%) due to non-hematologic AE (20 pts), cytopenia (12), impaired renal function (6), or other reasons (9). AEs of any grade occurred in all but one pt. The most frequent were neutropenia (45%), fatigue (47%), thrombocytopenia (33%), constipation (32%), rash (25%), and anemia (22%). Five patients (6%) developed a second cancer, including myelodysplastic syndrome (1), mucoepidermoid carcinoma (1), and non-melanoma skin cancers (3). The median duration of follow-up is 30.8 months. End of treatment disease response was evaluable in 76 patients; the remaining 8 were still receiving treatment at time of data cutoff and 1 discontinued R2 due to grade 3 colitis before the first disease assessment. The overall response rate (ORR) was 66%, with 55% achieving CR. There were no differences in response rates between pts receiving R2/RO in 2nd line or ≥3rd line, or between pts treated with R2 and RO. The median PFS was 21 months, and the 2-year PFS rate was 44.3% (95% CI: 32.7-60.0%). Seventeen pts died, 10 due to lymphoma and 7 due to other causes. HT occurred in 6 pts. The median OS was not reached, and the 2-year OS rate was 84.4% (95% CI: 75.9-92.6%) (Figure). Conclusion: This is the first real-life study of combined lenalidomide and anti-CD20 Ab for pts with R/R iNHL. Efficacy results were comparable to those reported in clinical trials and we observed no new safety signals. Our findings reaffirm the role of lenalidomide plus anti-CD20 Ab in the management of R/R iNHL and help define the benchmark for registration-directed clinical trials that utilize R2/RO as the control arm.

The HLA-B Leader MM Genotype Has a Negative Impact on Outcomes in HLA-B One Antigen Mismatched and HLA-B Matched Cord Blood Transplantation without Anti-Thymocyte Globulin: Analysis of the Japanese Society for Transplantation and Cellular Therapy

Background: The dimorphism at position -21 of exon 1 in HLA-B gives rise to leader peptides featuring either methionine (M) or threonine (T) as the second residue in the processed leader peptide (HLA-B leader). The cell surface abundance of HLA-E is positively correlated with the copy number of −21M HLA-B. In hematopoietic cell transplantation (HCT), the MM genotype in the recipients is associated with worse outcomes after HLA-matched and mismatched unrelated bone marrow and peripheral blood stem cell transplantation compared with TT (Kanaya M, et al. ASH Annual Meeting 2021, Petersdorf EW et al. Lancet Haematol. 2020 and Blood. 2020). Eurocord/EBMT study revealed HLA-B leader mismatch in HLA-B mismatched combination is associated with relapse and non-relapse mortality (NRM) risks after cord blood transplantation (CBT) (Petersdorf EW et al. Haematologica 2021). Notably, approximately 70% of patients in the literature administrated anti-thymocyte globulin (ATG) for graft-versus-host disease (GVHD) prophylaxis. The impacts of HLA-B leader dimorphism on the outcomes of CBT still needed to be elucidated more. Particularly, we were interested in the significance of HLA-B leaders in CBT without an ATG, then we conducted a retrospective study to elucidate it. Methods: Clinical data of 10,569 patients who underwent single-unit CBT for acute myeloid leukemia (AML, n=5,518), myelodysplastic syndrome (MDS, n=1,483), acute lymphoblastic leukemia (ALL, n=2,020), and lymphoma (n=1,548) between 2008 - 2021 were provided by the Japanese Data Center for Hematopoietic Cell Transplantation. HLA-A, -B, -C, and -DRB1 typing was performed at least at the antigen level. The leader genotype is defined based on the two HLA-B allotypes. All patients were not used ATG for GVHD prophylaxis. Study outcomes were overall survival (OS), cumulative incidences of relapse, NRM, and grade II-IV acute GVHD (aGVHD). Multivariable Cox regression analysis including covariables; disease risk index (DRI), myeloablative/reduced-intensity conditioning, presence of total body irradiation as a conditioning regimen, short-term methotrexate or mycophenolate mofetil for GVHD prophylaxis, recipient cytomegalovirus serostatus. All statistical analyses were performed with EZR. Results: The median patient's age was 53 (range 0-88) years. The numbers of patients with HLA-B leader TT, MT, and MM genotype were 7,639 (72.3 %), 2,707 (25.6 %), and 223 (2.1 %), respectively. To classify HLA-B match/mismatch status, we stratify “HLA-B one locus mismatched (GVH direction)” and HLA-B matched (no GVH direction mismatch)“. First, we focused on HLA-B one antigen mismatched (HLA 5/6 antigen matched, n = 1172) setting as shown in the previous literature. Recipient MM genotype showed significantly worse OS and relapse in the multivariate analysis compared to MT or TT genotype (Tx) (Figure) (OS: hazard ratio [HR] 0.543 [95% CI, 0.311 - 0.947]; p = 0.032, relapse: [HR] 2.863 [95% CI, 1.330 - 6.165]; p = 0.007). Recipient MM didn't relate to relapse and grade II-IV aGVHD. Next, we explored the impacts of HLA-B leaders in HLA-B antigen-matched CBT (3/6 - 6/6 matched, n = 3995). Donor MM genotype demonstrated a negative impact for OS and NRM, not relapse and aGVHD, compared to Tx donor (Figure) (OS: [HR] 0.738 [95% CI, 0.542 - 1.000]; p 0.050, NRM: [HR] 1.695 [95% CI, 1.097 - 2.621]; p = 0.018). Especially, OS and NRM of donor MM genotype were significantly inferior to Tx in HLA 6/6 matched (n = 914) CBT (OS: [HR] 0.311 [95% CI, 0.187 - 0.518]; p < 0.001, NRM: [HR] 3.216 [95% CI, 1.931 - 5.356]; p = 0.034). Conclusions: ATG would affect immune dynamics strongly in CBT via in-vivo T cell depletion, yet the current study demonstrated the HLA-B leader was still a significant risk factor in CBT without ATG for the first time. Recipient MM genotype in HLA-B one locus mismatched (HLA 5/6 matched) and donor MM genotype in HLA-B matched were the risk factors for worse CBT outcomes. We concluded MM genotype donors would be avoided to decrease NRM in HLA-B matched CBT. The precise mechanism of negative impact in CBT outcomes in patients or donor MM genotype remains to be clarified, however, we assume higher level HLA-E expression may be crucial in terms of NKG2A inhibitory signaling to T cells/NK cells and T-cell direct recognition for peptide/HLA-E complex in CBT.

Long-Term Outcomes after Unrelated Marrow Transplantation for Aplastic Anemia with Optimized Cyclophosphamide Dose (BMT CTN 0301)

Background: Seventy-nine patients (aged ≤65 years) with aplastic anemia received HLA-matched or single locus mismatched unrelated donor marrow transplantation (BMT) on a clinical trial ( BMT CTN 0301; NCT00326417) between 02/2006 and 12/2013 ( Lancet Haematol. 2015, 2:e367-75). The median ages of patients enrolled on the cyclophosphamide (Cy) 50 mg/kg and 100 mg/kg strata were 25 and 18 years, respectively, and 80% were Caucasians. That trial determined the minimum dose of Cy required for engraftment when added to total body irradiation (TBI 2 Gy), anti-thymocyte globulin (rabbit-ATG 9 mg/kg) and fludarabine (120 mg/m 2). Graft versus host disease (GVHD) prophylaxis was cyclosporine and methotrexate. Although Cy at 50 mg/kg (n=38) and 100 mg/kg (n=41) were both effective for day 100 and 1-year outcomes, the computed optimum Cy dose by Bayesian method was 50 mg/kg. Early results showed a 1-year incidence of graft failure after Cy 50 mg/kg and 100 mg/kg of 12% and 15%, respectively. The corresponding probabilities of 1-year survival were 92% and 76%. Methods: Thirty-seven patients who received Cy at 50 mg/kg and 31 patients who received Cy at 100 mg/kg were alive ≥1 year after BMT. These patients now have median follow ups of 7 and 8.7 years, respectively. The incidence of graft failure and chronic GVHD was calculated using the cumulative incidence estimator and the probability of survival using the Kaplan-Meier estimator. Results: In the trial cohort, 31 of 38 patients (82%) who received Cy 50 mg/kg were HLA-matched to their donor at HLA-A, B, C and DRB1, compared to 27 of 41 (66%) receiving Cy 100 mg/kg. Table 1 shows outcomes of the 38 and 41 patients who received Cy at 50 mg/kg and Cy at 100 mg/kg. With Cy 50 mg/kg there was 1 late graft failure at 4 years (cumulative total 5). Five deaths occurred at 1.5, 1.8, 4.2, 5.2 and 12.2 years after BMT (cumulative total 6). All chronic GVHD occurred within 2 years. Late toxicities (n=8; 22%) included liver cirrhosis (n=1, 1.25 years), myocardial infarction (n=1, 3.8 years), congestive heart failure (n=1, 8.7 years), acute renal failure requiring dialysis (n=1, 3.8 years), avascular necrosis (n=1, 9 years), gonadal dysfunction in pre-pubertal children at BMT (n=2, 1.4 and 3.9 years), and secondary cancer (n=1, pancreatic adenocarcinoma 3.1 years). The 8-year survival among the 38 patients receiving Cy 50 mg/kg was 85% despite late toxicities, including death, being more common beyond the first year after BMT. With Cy 100 mg/kg, there were no late graft failures. There was 1 death at 8.6 years (in a patient with underlying dyskeratosis congenita (cumulative total 11). One patient was diagnosed with chronic GVHD at 7 years. Late toxicities (n=6; 19%) included pulmonary (n=2, ARDS 8 years and restrictive airway disease 1.9 years), chronic renal failure requiring dialysis (n=1, 15 years), avascular necrosis (n=1, 1.5 years), gonadal dysfunction (n=1, 2.6 years), and secondary cancer (n=1, myelodysplastic syndrome 8.9 years). The 8-year survival among the 41 patients receiving Cy 100 mg/kg was 76% and all except 1 death occurred within the 1 st year after BMT. Causes of death after the first year of BMT are shown in Table 2. Conclusion: Eight-year survival ≥75% and sustained engraftment confirm both Cy at 50 mg/kg and Cy at 100 mg/kg with TBI 2 Gy, ATG and fludarabine are effective conditioning for unrelated BMT for aplastic anemia but life-long surveillance for late complications is required. Chronic GVHD was common and proved fatal in several patients despite most donor-recipient pairs who received Cy at 50 mg/kg being HLA-matched at the allele-level.