Failure Of Therapy Research Articles

Comparison of outcomes following intravenous magnesium compared with intravenous labetalol and oral nifedipine in 355 pregnant Han Chinese women with preeclampsia.

As per the American College of Obstetricians and Gynecologists in 2013, magnesium sulfate is the gold standard for the management of preeclampsia, but it has a short action time that does not provide stable maintenance of blood pressure. Labetalol is currently recommended as first-line treatment by the national UK guidance. This study included 355 pregnant Han Chinese women with preeclampsia and aimed to compare outcomes following intravenous magnesium compared with intravenous labetalol and oral nifedipine. Women received 4 g intravenous magnesium sulfate followed by the maintenance dose of 1 g/h intravenous magnesium sulfate (MS cohort, n = 104) or intravenous labetalol (LB cohort, n = 115), or oral nifedipine (NF cohort, n = 136). Therapy success: systolic blood pressure ~140 mm Hg and diastolic blood pressure ~90 mm Hg, therapy failure: persistent systolic blood pressure ≥ 160 or diastolic blood pressure ≥ 110 mm Hg after maximum dosage of therapy (EL). Women of all cohorts successfully decreased systolic and diastolic blood pressures at EL as compared to them before therapy conditions (P < .001, for all). At EL, systolic and diastolic blood pressures of women of the LB cohort decreased more than those of women of the MS and NF cohorts (P < .05, for all). Therapy was more successful in women of the LB cohort than those of the NF cohort (107 [93%] vs 112 [82%], P = .0132). More numbers of women were reduced blood pressure after 1 day of therapy from the LB cohort than those of the NF (75 [65%] vs 21 [15%]) and MS (75 [65%] vs 35 [34%]) cohorts (P < .0001 for both). Labetalol-induced tachycardia, bradycardia, and intracranial hemorrhage in pregnant women and respiratory distress syndrome and hypoglycemia in neonates. Intravenous labetalol provides proper reduction of blood pressure in Han Chinese women with preeclampsia but has the risk of undesirable maternal and neonatal adverse effects (Level of Evidence: IV; Technical Efficacy: Stage 4).

Discovery of New Anti-Cancer Agents against Patient-Derived Sorafenib-Resistant Papillary Thyroid Cancer.

Thyroid cancer is the most well-known type of endocrine cancer that is easily treatable and can be completely cured in most cases. Nonetheless, anti-cancer drug-resistant metastasis or recurrence may occur and lead to the failure of cancer therapy, which eventually leads to the death of a patient with cancer. This study aimed to detect novel thyroid cancer target candidates based on validating and identifying one of many anti-cancer drug-resistant targets in patient-derived sorafenib-resistant papillary thyroid cancer (PTC). We focused on targeting the sarco/endoplasmic reticulum calcium ATPase (SERCA) in patient-derived sorafenib-resistant PTC cells compared with patient-derived sorafenib-sensitive PTC cells. We discovered novel SERCA inhibitors (candidates 33 and 36) by virtual screening. These candidates are novel SERCA inhibitors that lead to remarkable tumor shrinkage in a xenograft tumor model of sorafenib-resistant patient-derived PTC cells. These results are clinically valuable for the progression of novel combinatorial strategies that facultatively and efficiently target extremely malignant cancer cells, such as anti-cancer drug-resistant PTC cells.

The role of irrigation fluid in transurethral resection of the prostate outcomes and surgeon performance.

Transurethral resection of the prostate (TURP) is the gold-standard for the treatment of benign prostate enlargement (BPE) associated with lower urinary tract symptoms (LUTS), after failure of conservative therapy. At present, only resection-rate (grams of prostate resected over time) is regarded as an efficiency parameter to evaluate the skill of the operator and to assess the outcome of the procedure. Five surgeons performed TURP using a Gyrus-type bipolar system in 123 patients with BPE/LUTS who came to our observation from June 2016 to December 2019. The amount of irrigation fluid used during the procedure was registered and correlated to the operating time, resection-rate, prostate adenoma weight, post-operative bladder irrigation time, intraoperative bleeding and days of catheterization. We found an inverse correlation between the amount of irrigation fluid used during TURP and the resection-rate recorded for all operators, according to Spearman's Correlation (r = -0.78, p = 0.002); a direct correlation was also found between the amount of irrigation fluid and the adenoma weight. Finally, we also found a direct correlation with intraoperative bleeding and the duration and amount of bladder irrigation during and after TURP. The amount of irrigation fluid used is proposed as a reliable parameter to estimate the efficiency of the endoscopic procedure as well to assess the skill of the operator and shortterm results. The observed data encourage the possibility of applying this new efficiency indicator to all endoscopic maneuvers.

Incidence of subsequent surgical decompression following minimally invasive approaches to treat lumbar spinal stenosis: A retrospective review.

Surgical decompression is the definitive treatment for managing symptomatic lumbar spinal stenosis; however, select patients are poor surgical candidates. Consequently, minimally invasive procedures have gained popularity, but there exists the potential for failure of therapy necessitating eventual surgical decompression. To evaluate the incidence and characteristics of patients who require surgical decompression following minimally invasive procedures to treat lumbar spinal stenosis. Retrospective review. Patients who underwent minimally invasive procedures for lumbar spinal stenosis (Percutaneous Image-guided Lumbar Decompression [PILD] or interspinous spacer device [ISD]) and progressed to subsequent surgical decompression within 5 years. The primary outcome was the rate of surgical decompression within 5 years following the minimally invasive approach. Secondary outcomes included demographic and comorbid factors associated with increased odds of requiring subsequent surgery. Patient data were collected using the PearlDiver-Mariner database. The rate of subsequent decompression was described as a percentage while univariable and multivariable regression analysis was used for the analysis of predictors. A total of 5278 patients were included, of which 3222 (61.04%) underwent PILD, 1959 (37.12%) underwent ISD placement, and 97 (1.84%) had claims for both procedures. Overall, the incidence of subsequent surgical decompression within 5 years was 6.56% (346 of 5278 patients). Variables associated with a significantly greater odds ratio (OR) [95% confidence interval (CI)] of requiring subsequent surgical decompression included male gender and a prior history of surgical decompression by 1.42 ([1.14, 1.77], p = 0.002) and 2.10 times ([1.39, 3.17], p < 0.001), respectively. In contrast, age 65 years and above, a diagnosis of obesity, and a Charlson Comorbidity Index score of three or greater were associated with a significantly reduced OR [95% CI] by 0.64 ([0.50, 0.81], p < 0.001), 0.62 ([0.48, 0.81], p < 0.001), and 0.71 times ([0.56, 0.91], p = 0.007), respectively. Minimally invasive procedures may provide an additional option to treat symptomatic lumbar spinal stenosis in patients who are poor surgical candidates or who do not desire open decompression; however, there still exists a subset of patients who will require subsequent surgical decompression. Factors such as gender and prior surgical decompression increase the likelihood of subsequent surgery, while older age, obesity, and a higher Charlson Comorbidity Index score reduce it. These findings aid in selecting suitable surgical candidates for better outcomes in the elderly population with lumbar spinal stenosis.

Real-World Effectiveness and Safety of SDZ ETN, an Etanercept Biosimilar, in Patients with Rheumatic Diseases: Final Results from Multi-Country COMPACT Study.

COMPACT, a non-interventional study, evaluated the persistence, effectiveness, safety and patient-reported outcomes (PROs) in patients with rheumatoid arthritis (RA), axial-spondyloarthritis (axSpA) or psoriatic arthritis (PsA) treated with SDZETN (etanercept [ETN] biosimilar) in Europe and Canada. Patients (aged ≥ 18years) who have been treated with SDZETN were categorised on the basis of prior treatment status (groupsA-D): patients in clinical remission or with low disease activity under treatment with reference ETN or biosimilar ETN and switched to SDZETN; patients who received non-ETN targeted therapies and switched to SDZETN; biologic-naïve patients who started SDZETN after conventional therapy failure; or disease-modifying anti-rheumatic drug (DMARD)-naïve patients with RA considered suitable for treatment initiation with a biologic and started on treatment with SDZETN. The primary endpoint was drug persistence, defined as time from study enrolment until discontinuation of SDZETN treatment. Of the 1466 patients recruited, 844 (57.6%) had RA, 334 (22.8%) had axSpA and 288 (19.6%) had PsA. Patients had an ongoing SDZETN treatment at the time of enrolment for an observed average of 138days (range 1-841); 22.7% of patients discontinued SDZETN through 12months of study observation. Overall, all the patients receiving SDZETN showed good treatment persistence at 12months with discontinuation rates of 15.2%, 25.7% and 27.8% in groupsA, B and C, respectively. Across all patient groups, no major differences were observed in the disease activity and PRO scores between baseline and month12. Injection-site reactions were low across the treatment groups. These results support the effectiveness and safety of SDZETN treatment in patients with RA, axSpA or PsA in real-life conditions. The treatment persistence rates observed were consistent with previously published reports of patients treated with reference or other biosimilar ETN. No new safety signals were identified.

EPCO-37. DISSECTING GBM EVOLUTION FOLLOWING STANDARD-OF-CARE BY LARGE-SCALE LONGITUDINAL SINGLE NUCLEUS RNA-SEQUENCING

Abstract The heterogeneity within tumors has long been associated with therapy failure and disease progression. Recent advances in single-cell RNA-sequencing technologies have enabled us to dissect the cellular diversity in glioblastoma (GBM). However, how these cellular programs change longitudinally under therapy remains poorly understood. To address this question, we collected and profiled a large-scale longitudinal cohort of 59 matched IDH-wildtype GBM sample pairs provided by 7 centers worldwide by single-nucleus RNA-sequencing (snRNA-seq) and whole-exome/whole-genome sequencing. The majority of patients (51 patients) in this cohort received standard-of-care (temozolomide and radiation) following initial tumor resection. Leveraging this large-scale snRNS-seq dataset of 457,442 cells, we detected novel cellular states and associations between malignant and tumor microenvironment (TME) cells, and then performed longitudinal analyses. The recurrent samples showed significantly lower malignant cell fraction (p=0.002) and reciprocal increase in proportions of glio-neuronal TME cell types (oligodendrocytes, neurons and astrocytes). The TME composition, malignant cell state proportions and baseline expression programs were retained at recurrence more than expected by chance, but overall were not well conserved between primary and recurrent samples. A subset of pairs (12/59 pairs) enriched with glio-neuronal TME at recurrence showed a significant transcriptomic shift and was associated with better clinical course (p=0.02). The tumors that acquired radiation-related small deletion phenotype underwent a transition towards MES/Hypoxia phenotype (0/9 at primary, 6/9 at recurrence, p=0.02). We defined pairs as likely responders or non-responders to treatment based on the MGMT methylation status of the primary tumor sample. This uncovered diverging evolutionary trajectories in cellular programs between the two groups. Strikingly, the changes in malignant state frequency and baseline malignant expression profile were strongly associated with specific changes in the TME composition. Our findings based on high-resolution longitudinal snRNA-seq analyses highlight the diverse evolutionary trajectories in GBM that are shaped by TME changes and treatments.

Inhibition of EIF2α Dephosphorylation Decreases Cell Viability and Synergizes with Standard-of-Care Chemotherapeutics in Head and Neck Squamous Cell Carcinoma.

Drug resistance is a common cause of therapy failure in head and neck squamous cell carcinoma (HNSCC). One approach to tackling it is by targeting fundamental cellular processes, such as translation. The eukaryotic translation initiation factor 2α (EIF2α) is a key player in canonical translation initiation and integrates diverse stress signals; when phosphorylated, it curbs global protein synthesis. This study evaluates EIF2α expression and phosphorylation in HNSCC. A small-molecule inhibitor of EIF2α dephosphorylation, salubrinal, was tested in vitro, followed by viability assays, flow cytometry, and immunoblot analyses. Patient-derived 3D tumor spheres (PD3DS) were cultured with salubrinal and their viability assessed. Lastly, salubrinal was evaluated with standard-of-care chemotherapeutics. Our analysis of RNA and proteomics data shows elevated EIF2α expression in HNSCC. Immunohistochemical staining reveals increasing EIF2α abundance from premalignant lesions to invasive and metastatic carcinoma. In immunoblots from intraoperative samples, EIF2α expression and steady-state phosphorylation are higher in HNSCC than in neighboring normal tissue. Inhibition of EIF2α dephosphorylation decreases HNSCC cell viability and clonogenic survival and impairs the G1/S transition. Salubrinal also decreases the viability of PD3DS and acts synergistically with cisplatin, 5-fluorouracil, bleomycin, and proteasome inhibitors. Our results indicate that pharmacological inhibition of EIF2α dephosphorylation is a potential therapeutic strategy for HNSCC.

Individuals with rotator cuff tears unsuccessfully treated with exercise therapy have less inferiorly oriented net muscle forces during scapular plane abduction

Exercise therapy for individuals with rotator cuff tears fails in approximately 25.0 % of cases. One reason for failure of exercise therapy may be the inability to strengthen and balance the muscle forces crossing the glenohumeral joint that act to center the humeral head on the glenoid. The objective of the current study was to compare the magnitude and orientation of the net muscle force pre- and post-exercise therapy between subjects successfully and unsuccessfully (e.g. eventually underwent surgery) treated with a 12-week individualized exercise therapy program. Twelve computational musculoskeletal models (n = 6 successful, n = 6 unsuccessful) were developed in OpenSim (v4.0) that incorporated subject specific tear characteristics, muscle peak isometric force, in-vivo kinematics and bony morphology. The models were driven with experimental kinematics and the magnitude and orientation of the net muscle force was determined during scapular plane abduction at pre- and post-exercise therapy timepoints. Subjects unsuccessfully treated had less inferiorly oriented net muscle forces pre- and post-exercise therapy compared to subjects successfully treated (p = 0.039 & 0.045, respectively). No differences were observed in the magnitude of the net muscle force (p > 0.05). The current study developed novel computational musculoskeletal models with subject specific inputs capable of distinguishing between subjects successfully and unsuccessfully treated with exercise therapy. A less inferiorly oriented net muscle force in subjects unsuccessfully treated may increase the risk of superior migration leading to impingement. Adjustments to exercise therapy programs may be warranted to avoid surgery in subjects at risk of unsuccessful treatment.

Risk Factors for Radiotherapy Failure in the Treatment of Spinal Metastases.

Retrospective cohort study. To build a predictive model for risk factors for failure of radiation therapy, hypothesizing a higher SINS would correlate with failure. Patients with spinal metastasis being treated with radiation at a tertiary care academic center between September 2014 and October 2018 were identified. The primary outcome measure was radiation therapy failure as defined by persistent pain, need for re-irradiation, or surgical intervention. Risk factors were primary tumor type, Karnofsky and ECOG scores, time to treatment, biologically effective dose (BED) calculations using α/β ratio = 10, and radiation modality. A logistic regression was used to construct a prediction model for radiation therapy failure. One hundred and seventy patients were included. Median follow up was 91.5days. Forty-three patients failed radiation therapy. Of those patients, 10 required repeat radiation and 7 underwent surgery. Thirty-six patients reported no pain relief, including some that required re-irradiation and surgery. Total SINS score for those who failed reduction therapy was <7 for 27 patients (62.8%), between 7-12 for 14 patients (32.6%), and >12 for 2 patients (4.6%). In the final prediction model, BED (OR .451 for BED > 43 compared to BED ≤ 43; P = .174), Karnofksy score (OR .736 for every 10 unit increase in Karnofksy score; P = .008), and gender (OR 2.147 for male compared to female; P = .053) are associated with risk of radiation failure (AUC .695). A statistically significant association between SINS score and radiation therapy failure was not found. In the multivariable model, BED ≤ 43, lower Karnofksy score, and male gender are predictive for radiotherapy failure. SINS score was among the candidate risk factors included in multivariable model building procedure, but it was not selected in the final model. Prognostic level III.

Optimization of a Three-Dimensional Culturing Method for Assessing the Impact of Cisplatin on Notch Signaling in Head and Neck Squamous Cell Carcinoma (HNSCC).

Head and neck squamous cell carcinoma (HNSCC) is a prevalent cancer type, with cisplatin being a primary treatment approach. However, drug resistance and therapy failure pose a significant challenge, affecting nearly 50% of patients over time. This research had two aims: (1) to optimize a 3D cell-culture method for assessing the interplay between tumor cells and cancer-associated fibroblasts (CAFs) in vitro; and (2) to study how cisplatin impacts the Notch pathway, particularly considering the role of CAFs. Using our optimized "3D sheet model" approach, we tested two HNSCC cell lines with different cisplatin sensitivities and moderate, non-mutated NOTCH1 and -3 expressions. Combining cisplatin with a γ-secretase inhibitor (crenigacestat) increased sensitivity and induced cell death in the less sensitive cell line, while cisplatin alone was more effective in the moderately sensitive line and sensitivity decreased with the Notch inhibitor. Cisplatin boosted the expression of core Notch signaling proteins in 3D monocultures of both lines, which was counteracted by crenigacestat. In contrast, the presence of patient-derived CAFs mitigated effects and protected both cell lines from cisplatin toxicity. Elevated NOTCH1 and NOTCH3 protein levels were consistently correlated with reduced cisplatin sensitivity and increased cell survival. Additionally, the Notch ligand JAG2 had additional, protective effects reducing cell death from cisplatin exposure. In summary, we observed an inverse relationship between NOTCH1 and NOTCH3 levels and cisplatin responsiveness, overall protective effects by CAFs, and a potential link between JAG2 expression with tumor cell survival.

Somatic Mutations in Cancer-Related Genes Were Observed More Frequently in AYA CML Patients Compared to Elderly at Diagnosis, Whereas the Frequency Was Markedly Higher in Elderly Patients during TKI Treatment

CONCLUSIONS Several works showed that patients with chronic myeloid leukemia (CML) diagnosed at the age of 18 to 39 years (adolescent and young adults, AYAs), had a worse outcome of tyrosine kinase inhibitors (TKI) therapy compared to elderly patients. Only limited data on the molecular background differing AYA from the other age groups are available. To characterize the somatic mutation landscape of AYA CML patients in chronic phase at the time of diagnosis and follow-up in comparison to pediatric and elderly patients, and to follow their clonal evolution. Ph-positive acute lymphoblastic leukemia (Ph+ ALL) patients were included for comparison. Samples from the time of diagnosis (CML children N=18; AYA N=69; elderly N=70; Ph+ ALL children N=32, AYA N=13, elderly N=39) and TKI treatment follow-up (CML AYA N=69, elderly N=70) were analyzed with custom panel (22 whole genes, 40 selected exons). To follow the clonal evolution vital cells available from 10 CML patients were FACS sorted into populations of neutrophils, monocytes, T- and B-lymphocytes. Genomic BCR::ABL1 (g BCR::ABL1) and mutations were quantified using ddPCR in the sorted cells. At the time of diagnosis, a slightly higher frequency of somatic mutations was detected in AYA CML patients (28%, 19/69) compared to elderly (20%, 14/70) and children (11%, 2/18). The similar trend with markedly higher differences was observed in Ph+ ALL patients (62% 8/13 of AYA, 31% 12/39 of elderly, 22% 7/32 of pediatric). The most frequently mutated genes in AYA and elderly CML patients were identically ASXL1, DNMT3A, and TET2. Other mutated genes in AYA were FBXW7, IDH2, IKZF1 PHF6 and KDM6A, SETD2, SF3B1 in elderly. Considering the treatment response, a higher number of mutations at diagnosis were found in the AYA patients that responded optimally in follow-up (21%, 7/33) than in elderly 4% (1/26) and a slightly higher frequency of mutations in AYA patients who later failed on TKI (35%, 8/23) than in elderly 30% (13/43). In contrary, the frequency of acquired mutations or persisted mutations from diagnosis detected during TKI therapy was higher in elderly that failed on TKI therapy (67%, 29/43) than in AYA (52%, 12/23). The treatment failure was associated with de novo mutations in ABL1, RUNX1, TET2, ASXL1 in AYA and elderly CML patients, and in addition in IKZF1, and in DNMT3A, WT1, JAK2, respectively. Sorted cells were analyzed from the time of optimal response in 7 CML patients (+ diagnostic sample of 2 patients) with mutated ASXL1 in 6 patients, FBXW7 in 1 patient, DNMT3A in 1 patient and IDH2 in 1 patient. Samples from the time of therapy failure were available for sorting of 3 patients (+ diagnostic sample of 1 patient). All mutations were detected in g BCR::ABL1 positive neutrophiles and monocytes. The g BCR::ABL1 levels matched exactly with the levels of mutations except for 1 patient with ASXL1-mutated subclone (VAF 5%), which was found in g BCR::ABL1 positive cells. Similar data were found in patients with therapy failure. However, in one patient with secondary failure, the ASXL1 mutation detected at diagnosis disappeared. In all 10 CML patients, g BCR::ABL1 and mutations were not detected in T- and B- lymphocytes under the therapy. In diagnostic samples some g BCR::ABL1 and mutations positive T- and B- lymphocytes were found, but the levels were 3-4 log lower compared to levels in neutrophils and monocytes, which likely correspond to the contamination (purity check was done after sorting) than to the presence of a very small subset of physiological lymphoid cells. Of all age groups, AYAs had the highest number of mutations detected at the time of diagnosis with differences in the spectrum between CML and Ph+ ALL. A markedly higher de novo acquisition of mutations was observed in elderly CML patients during TKI treatment. Moreover, therapy failures were observed more frequently in elderly than in AYA CML patients. Clonal analysis of 10 CML patients outlined that myeloid, but not lymphoid stem/progenitor cells were mutated. Supported by MZCR NU21-07-00225

TP53 Aberrant B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) Are Uniquely Sensitive to a Combination of Pyrimidine De Novo Synthesis Blockade and Replication Checkpoint Inhibition

Background Unlike most solid tumors, the incidence of TP53 aberrations in ALL is only 3% at diagnosis. However, in relapsed ALL this number increases to 12%. More importantly, at relapse, loss of p53 function is associated with therapy failure, with an overall survival of only 20%. Hence, there is a clear clinical need for more effective strategies to treat TP53 aberrant relapsed ALL. Owing to their high proliferative state, leukemic blasts show a high dependency on pyrimidines for DNA synthesis. A lack of pyrimidines leads to replication stress, causing activation of p53 and the DNA damage response pathway. Results We used CRISPR/Cas9 to generate TP53 deficient variants of the TP53 proficient BCP-ALL cell lines Nalm6 and RCH-ACV. We used these cell lines in a CRISPR screens and high throughput drug screen, and identified a strong synergy between the targeting of pyrimidine synthesis and DNA checkpoint kinase ATR and its downstream effectors Chk1 and Wee1 in TP53 deficient ALL. To validate these results, we tested the efficacy of a checkpoint inhibition together with Orludodstat (BAY-2402234), an inhibitor of Dihydroorotate Dehydrogenase (DHODH), a key enzyme in de novo pyrimidine biosynthesis. This clinical grade inhibitor was evaluated in either wild type or TP53 deficient BCP-ALL and T-ALL cell line models and patient derived xenografts (PDX). A strong apoptotic response was observed when Orludodstat was combined with the ATR inhibitor Berzosertib, most notably in TP53 deficient ALL. This p53 dependency was confirmed using ex vivo cultures of patient derived xenografts (PDX) of diagnosis and relapse pairs, where each diagnostic sample was wild type for TP53, and the relapse carried a TP53 aberration. While little or no synergy was observed in the diagnostic samples, a strong efficacy of the combination was observed in the matched TP53 deficient relapsed samples (Figure 1 A). Preliminary results from i n vivo experiments using p53 deficient patient derived xenografts indicate that the combination of Orludodstat and Berzosertib is well tolerated, while significantly delaying leukemic growth (Figure 1 B). Conclusion Together, our findings suggest that the combined targeting of de novo pyrimidine synthesis and ATR signaling may present a specific and non-genotoxic vulnerability for TP53 aberrant ALL.

Myelodysplastic Syndrome Hematopoietic Stem Cells Can Undergo Differentiation Trajectory Rewiring to Escape Venetoclax Vulnerability

Myelodysplastic syndromes (MDS) are incurable neoplasms that arise from early hematopoietic stem cells (HSCs). The standard of care for MDS is hypomethylating agent (HMA) therapy. However, the disease invariably fails these agents, resulting in fatal cytopenias or progression to acute myeloid leukemia (AML) with a dismal prognosis. We previously showed that the cellular hierarchy of MDS HSCs predicts the mechanisms of progression after HMA failure and can guide the design or choice of specific therapeutic approaches targeting these cells (Ganan-Gomez et al., Nature Medicine 2022). MDS HSCs in one of two differentiation states, long-term HSCs or lymphoid-primed multipotent progenitors, have a “common myeloid progenitor (CMP) pattern” or “granulocytic-monocytic progenitor (GMP) pattern” of differentiation. HSCs in these 2 differentiation states persist throughout the disease course and, driven by the recurrent activation of BCL2- or NF-κB-mediated survival pathways, respectively, expand at disease progression. In our preclinical studies, pharmacologically inhibiting these pathways depleted MDS HSCs and reduced tumor burden, which suggests that only patients with “CMP pattern” MDS may benefit from venetoclax-based therapies. Accordingly, patients with “CMP pattern” MDS, whose disease progressed after frontline HMA failure, enrolled in clinical trials of venetoclax-based therapy (NCT04160052, NCT04550442, or NCT04655755; n=28: 12 “CMP pattern” MDS and 16 “GMP pattern” MDS) had a shorter cumulative time to complete remission (P=0.012) and a longer relapse-free survival duration (14.0 months vs 5.2 months) than those with “GMP pattern” MDS did. Consistent with the hypothesis that venetoclax-based therapy selectively targets different HSC populations after HMA therapy failure, bone marrow (BM) HSC frequencies were significantly decreased at the time of remission after venetoclax-based therapy in “CMP pattern” MDS patients, but not in those with “GMP pattern” MDS. These results explain why “GMP pattern” MDS are resistant to venetoclax therapy. However, 8 of the 12 patients with “CMP pattern” MDS relapsed during venetoclax treatment and/or progressed to AML after an initial remission. In 2 patients with “CMP pattern” MDS, sequential flow cytometry and next generation sequencing analyses showed that the HSC hierarchy switched to “GMP pattern” MDS before venetoclax failure. This immunophenotypic hierarchical change was associated with the detection or expansion of STAG2- or RUNX1-mutant clones with isolated trisomy 8. To evaluate whether the immunophenotypic switch resulted from the distinct differentiation potential of early HSCs or from the aberrant expression of cell surface markers, we performed sequential single-cell RNA sequencing analyses of BM mononuclear cells isolated from the 2 “CMP pattern” MDS patients whose disease changed hierarchy before venetoclax failure. Our analyses revealed that after undergoing significant depletion during disease remission, HSCs expanded at therapy failure. Differential expression analysis showed that the expansion of STAG2- or RUNX1-mutant clones in the HSC compartment not only rewired MDS HSCs' differentiation state towards a granulo-monocytic-biased trajectory but also changed HSCs' survival dependency from BCL2-mediated anti-apoptotic pathways to TNFa-induced pro-survival NF-κB signaling, thus enabling HSCs to evade the cytotoxic effects of venetoclax (Fig. 1). The anti-apoptotic gene MCL1, a direct transcriptional target of NF-κB, was also significantly upregulated in HSCs at the time of progression, which suggests that the combination of venetoclax with an MCL1 inhibitor can overcome secondary failure to venetoclax. Together, these results reveal a novel mechanism of venetoclax resistance. HSCs under venetoclax therapy undergo survival pressure, which results in the expansion or acquisition of clones carrying specific mutations that change HSCs' survival dependencies (Fig. 2). Our study suggests that MDS patients receiving venetoclax-based therapy should be monitored at the molecular level and enrolled in clinical trials of other targeted agents before their disease undergoes clonal evolution and HSC hierarchical rewiring.

Two-Year Follow-up Results of C-CAR066, a Novel Anti-CD20 Chimeric Antigen Receptor Cell Therapy (CAR-T) in Relapsed or Refractory (r/r) Large B-Cell Lymphoma (LBCL) Patients after Failure of CD19 CAR-T Therapy

Background: Disease recurrence or progression represents a significant challenge in r/r LBCL treated with CAR-T. In a large analysis of r/r LBCL pts who receive further therapy after CD19 CAR-T treatment failure, the overall response rate (ORR) is 39% (CR 20%; PR 19%) and the median post-treatment-OS and post-treatment-EFS are 8.5 months and 1.9 months respectively. C-CAR066 is a novel 2nd generation CAR-T therapy targeting CD20 antigen. We previously reported that C-CAR066 had shown a favorable safety profile and promising efficacy in pts with r/r LBCL following failure to CD19 CAR-T therapy. At a median follow-up of 4.2 months, the ORR was 100%, with 70.0% (7/10) achieving complete response (CR) (Liang et al. ASCO 2021. #2508). Here we present the updated results to include more pts (n=14) and a longer follow-up period. Methods: This first-in-human (FIH) study is an open-label, dose-finding, phase 1 investigator-initiated trial (IIT) to determine the safety and efficacy of C-CAR066 in r/r LBCL (including DLBCL, tFL and PMBCL) after previous CD19 CAR T-cell treatment failure. This study was conducted at two clinical sites in China. C-CAR066 was administered at doses of 2.0x10 6 CAR-T cells/kg and 3.0x10 6 CAR-T cells/kg after a 3-day conditioning chemotherapy. The primary objective was to assess the safety and tolerability of C-CAR066. Cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) were graded according to ASTCT 2019 criteria. The secondary objectives were to evaluate C-CAR066 efficacy and pharmacokinetics (PK). Response was assessed per Lugano 2014 criteria. The study follow-up period was 2 years. Results: Between Oct 16, 2019, and Aug 17, 2021, a total of 14 pts received C-CAR066. 11 pts (78.6%) were DLBCL, 3 pts (21.4%) were tFL. The median age was 54.5 years (range, 37-67) with 2 pts (14.3%) ≥65 years. 12 pts (85.7%) were in Ann Arbor Stage III/IV. 7 pts (50.0%) were double-expressor lymphoma. Median number of prior lines of therapy was 5 (range, 2-7). All pts had prior CAR-T therapy, which included CD19 CAR-T (12 pts), CD19/CD22 bispecific CAR-T (1 pts), and CD19/CD79b bispecific CAR-T (1 pts). 12 pts responded to the prior CAR-T therapy (2 CR and 10 PR), and the median duration of response (DOR) was 1.5 months. Median time from prior CAR-T to C-CAR066 infusion were 5.45 months (range, 3.4-14.2). 7 pts (50.0%) received bridging therapy. As of Jan 15, 2023, 12/14 pts (85.7%) experienced CRS, all were grade 1/2, except for 1 pt dosed at 3.0×10 6 CAR- T cells/kg experienced a grade ≥3 CRS. This pt recovered after receiving corticosteroids and tocilizumab. The median time to CRS onset was 5.5 days (range, 2-15), and the median time to resolution was 4.0 days (range, 1-15). No pts experienced ICANS. Grade ≥3 neutropenia, anemia, thrombocytopenia, and infection were reported in 92.9%, 50.0%, 21.4% and 14.3% of pts, respectively. No new safety signals were observed in this updated 2-year follow-up. Investigator assessed ORR was 92.9%, with 57.1% CR. Median time to first response and CR were 1.0 month (range, 0.9-2.8) and 2.5 month (range, 1.0-2.8), respectively. With the median follow-up of 19.5 months, median PFS and DOR were 9.4 months and 8.3 months, respectively. 5 pts remained in CR 12 months after infusion, and 4 of them remained in CR after 24 months. 6 deaths occurred due to progressive disease. Median post-treatment-OS had not been reached at 24 months. 13 of 14 pts had assessable PK and pharmacodynamic data (one pt dropped out before day 28). The median T max was 11 days (range, 10-23), the median C max was 398,996 copies/µg gDNA (range, 51,667-1,286,932), and the median AUC 0-28day of 4,437,474 copies/µg∙day (range, 431,534-17,842,217), the median T last was 59 days (21~571). We have observed that C-CAR066 can effectively expand and eliminate CD19+/CD20+ B cells in patients. The median time to B cell depletion was 5.5 days (range, 4-11). B cell recovery was observed in 6 (46.2%) pts from 53 to 366 days after the C-CAR066 infusion, 3 of them remained in CR by the data cutoff date. Conclusions: Longer term follow-up demonstrated that C-CAR066 can produce a deep and durable response with a favorable safety profile in pts with r/r LBCL who failed prior CD19 CAR-T therapy.

Matching-Adjusted Indirect Comparison (MAIC) of Brexucabtagene Autoleucel (Brexu-cel) and Pirtobrutinib in Patients with Relapsed/Refractory (R/R) Mantle Cell Lymphoma (MCL) Previously Treated with a Covalent Bruton Tyrosine Kinase Inhibitor (cBTKi)

Introduction: Patients with MCL often require multiple lines of therapy and exhibit a generally unfavorable prognosis, particularly following failure of cBTKi therapy. Therapies such as chimeric antigen receptor (CAR) T-cell therapies and non-covalent BTKi therapies have shown promise in improving outcomes. Brexu-cel (formerly known as KTE-X19) is the only CAR T-cell therapy approved in the US for R/R MCL based on results from a phase 2 multicenter, single-arm trial (ZUMA-2; NCT02601313) in patients with R/R MCL who had 1-5 prior therapies, including a BTKi. Pirtobrutinib, a highly selective, non-covalent BTKi, was recently approved in the US for treatment of R/R MCL after at least two lines of systemic therapy including a BTKi, based on ongoing results from a multicenter phase 1/2 study (BRUIN; NCT03740529). In the absence of a direct head-to-head trial comparing brexu-cel and pirtobrutinib, we performed an unanchored MAIC to estimate the relative treatment effects of these therapies in the post-BTKi setting for R/R MCL. Methods: Individual patient-level data were available for ZUMA-2 (N=68; 35.8 months median follow-up as of July 24, 2021 data cut-off) whereas study-level data were available for BRUIN (N=90 in the BTKi pre-treated cohort; 23.5 months median follow-up as of July 29, 2022 data cut-off). Longer-term overall survival (OS) data for ZUMA-2 from the July 23, 2022 data cut-off (46.1 months median follow-up) were available and used. Logistic propensity score models were used to weight the brexu-cel infused population from ZUMA-2 to match the aggregate baseline characteristics of the BTKi pre-treated cohort in BRUIN. These baseline characteristics represented clinically relevant prognostic factors which were pre-specified based on input from clinical experts and data availability. The base-case model incorporated the top five most pertinent factors, which were reported in at least 50% of patients in both trials: blastoid morphology, MCL International Prognostic Index, number of prior lines of therapy, disease stage, and prior autologous stem cell transplantation. A sensitivity analysis additionally incorporated TP53 mutation (missing in &amp;gt;50% of patients in ZUMA-2 and BRUIN) and Ki-67 proliferation index (missing in &amp;gt;50% of patients in BRUIN). Several prognostic factors (response to prior BTKi therapy, response to last therapy, and duration on prior BTKi therapy) could not be considered due to not being reported for BRUIN. The effective sample size was calculated as a measure of the degree of precision after weighting, reflecting the extent of overlap in the distribution of covariates between the trial populations. Relative treatment effects were expressed as odds ratios or hazard ratios with 95% confidence intervals. Outcomes included objective response (ORR), complete response (CR), duration of response (DOR), progression-free survival (PFS), and overall survival (OS). Results: In the base-case MAIC, a significant difference in the odds of ORR and CR in favor of brexu-cel versus pirtobrutinib was observed (Table 1). Similarly, brexu-cel demonstrated a significant improvement in PFS (Table 2). OS and DOR point estimates were in favor of brexu-cel when compared to pirtobrutinib, although not statistically significant. These findings were consistent with unadjusted naïve analyses as well as the sensitivity analyses including adjustment for TP53 mutation and Ki-67 proliferation index. Conclusions: While acknowledging the inherent limitations of an unanchored indirect comparison, our findings suggest that brexu-cel offers clinically and statistically significant efficacy benefits in terms of ORR, CR, and PFS compared to pirtobrutinib in patients with R/R MCL after prior cBTKi therapy. Both treatments were not statistically different in terms of OS and DOR although the estimated hazard ratios indicated a favorable trend for brexu-cel; however, given the relatively shorter follow-up and the high degree of censoring in BRUIN, an updated analysis incorporating longer follow-up data with more events from BRUIN could provide more reliable results. Although efforts were made to ensure a robust approach to the selection of prognostic factors for inclusion in the model, the possibility of some residual confounding variables cannot be completely ruled out. Nevertheless, these results provide valuable insights that may help inform treatment decisions for this population.

Outcomes of Patients with Newly Diagnosed Multiple Myeloma Experiencing Treatment Failure after Upfront Quadruplet Therapy and Autologous Stem Cell Transplantationgayathri Ravi 1, Susan Bal 1, Kelly Godby 1, Smith Giri 1, Clare Ubersax 1, Abigail Tucker 1, Luciano J Costa 1

Background The combination of a proteasome inhibitor (PI) an immunomodulatory agent (IMiD), an anti-CD38 monoclonal antibody (anti-CD38MoAb) and dexamethasone, i.e. quadruplet therapy (QUAD) leads to highest rate of complete responses (CR) and prolonged progression-free survival (PFS) in newly diagnosed multiple myeloma (NDMM) in the context of autologous stem cell transplantation (ASCT). While there are treatment failures reported after QUADs and they may be exquisitely challenging to treat the published literature in this setting is scarce. Methods We included all patients treated with QUADs between January 2016 and May 2023 with intention to proceed with ASCT to identify treatment failures and report outcomes. Treatment failures were classified into primary failure (PF) defined as lack of objective response after at least 4 QUAD cycles or progression during or within 60 days of completing QUAD therapy; early progression (EP), defined as disease progression 2-12 months of completing QUAD therapy, and late progression (LP) defined as disease progression &amp;gt; 12 months from completing QUAD therapy. For each of these groups we analyzed disease features, treatment refractoriness (as patients in EP and LP may have varying refractoriness depending on maintenance therapy received), response to subsequent therapy and outcomes- Overall response rates(ORR), Progression free survival(PFS) and Overall Survival(OS). Results We identified 248 sequential patients treated with QUADs, including 191 (77%) treated with daratumumab, bortezomib, lenalidomide and dexamethasone (D-VRd), and 57 (23%) treated with daratumumab, carfilzomib, lenalidomide and dexamethasone (D-KRd). Median follow up for all patients is 20.4 months (IQR 11.3-34.0) from onset of QUAD therapy. We found 11 patients with PF, 9 with EP and 11 with LP (Table) for a total of 31 treatment failures. Patients with QUAD failures were more likely to have ISS-3, R-ISS3 and 2+ high-risk chromosome abnormalities than the overall QUAD population. Median follow up from QUAD failure is 5.2 months for PF, 11.2 months for EP and 10.7 months for LP. All 31 patients with QUAD failure received subsequent therapy and 28 patients have &amp;gt; 1 month follow up enable assessment of response to subsequent therapy (Table). Cytotoxic chemotherapy was frequently used in patients with PF (60%) whereas anti-CD38 monoclonal Ab containing regimen was commonly used with increasing time from initial QUAD therapy reaching 89% in patients with LP. ORR to next line of therapy was 20% for PF, 33% for EP and 67% for LP. Of the 28 patients, 13(46%) participated in subsequent treatment clinical trial and 16(57%) received subsequent T-cell redirecting therapy either bispecific antibody or CAR-T cell therapy . Median PFS on next line of therapy was 2.4 months for PF, 3.7 months for EP and 7.4 months for LP. Median OS from QUAD failure was only 6.3 months for PF and 11.5 months for EP. No deaths (0/11) observed in LP group (Figure). Conclusions Multiple myeloma patients with delayed progression (&amp;gt;12 months) from end of QUADs have reasonable response to subsequent therapy. Despite access to experimental therapies including T-cell redirected therapies, patients with earlier failure of QUAD therapy (0-12 months) have poor response rates to subsequent treatments and significantly shorter survival. These findings highlight the urgent need for better predictive models to identify patients with QUAD failure and preemptively deploy treatments with novel mechanisms of action.

ATM Germline Pathogenic Variants Affect Treatment Outcomes in Children with Acute Lymphoblastic Leukemia/Lymphoma and Ataxia Telangiectasia

Introduction Ataxia telangiectasia (A-T) is a multisystem disorder caused by biallelic germline pathogenic variants (PV) in the ATM gene. An important feature of A-T is an increased predisposition to cancer with a reported incidence of 25%, primarily attributed to hematological malignancies. Patients with A-T and cancer are usually excluded from therapeutic clinical trials, limited information thus exists concerning their treatment outcomes and toxicity profiles and consequently, optimal management strategies are unclear and an unmet need. In this multinational study, we aimed to investigate the characteristics and outcomes of leukemia and lymphoma in a large cohort of children with A-T and to determine risk factors which impact treatment outcome in order to generate consensus and data-based prospective treatment recommendations. Methods This study of patients aged ≤25 years with A-T and hematological malignancies was conducted through the International BFM Study Group. Patient data were collected from medical records, including specific patient comorbidities. Each reported ATM PV identified in the cohort was classified as null (resulting in complete loss of ATM activity) or hypomorphic (allowing residual ATM activity) according to the expected functional activity of the ATM protein and published functional studies. Patients with reported ATM PV were then classified as Group A (two null PV) or Group B (at least one hypomorphic PV). Results We report 202 pediatric and adolescent/young adult patients with A-T and hematological malignancies from 25 countries. The cohort included 82 patients with ALL/LBL (41%), predominantly (85%) of T-cell lineage, 91 with mature B-cell lymphomas (45%), 21 with Hodgkin lymphoma (10%) and 8 with other hematological malignancies (4%) (Fig 1). Of 111 patients with classifiable germline ATM variants, 82 (74%) were classified as Group A and 29 as Group B (26%). The distribution of patients with Group A and Group B germline ATM PV differed considerably between tumor types with 44% of the patients with lymphoblastic leukemia/lymphoma classified as Group B vs. 5% of those with mature B cell lymphomas ( p&amp;lt;.001). In total, 185 patients (92%) treated with curative intent were included in the outcome analyses, 135 (73%) of whom were treated with attenuated therapy regimens. Four-year OS and EFS for the entire cohort were 50.8% (95% CI 43.6-59.1) and 47.9% (95% CI 40.8-56.2), respectively. Surprisingly, cure rates of patients with A-T and malignancy did not appear to improve significantly with therapy modernization over the last four decades with 4-year EFS rates of 41.6% (95% CI 26.6-65), 49.6% (95% CI 38.8-63.4) and 48.0% (95% CI 37.4-61.7) for those treated before 2000, between 2000-2009 and since 2010, respectively ( p=.54). The major cause of treatment failure for the entire cohort was treatment-related mortality (TRM) with a 4-year cumulative incidence of 32.8% (95% CI 19.5-32.4), followed by progressive cancer in 14.5% (95% CI 10-19.8) and second malignancy in 4.9% (95% CI 13.1-85.8) (Fig 2). We identified factors that were significantly associated with survival for this unique patient population. Older age had a significantly deleterious effect upon survival with 4-year EFS rates of 69.1% (95%CI 55.9-85.4), 45.3% (95% CI 34.5-59.4), 39.8% (95% CI 25-63.2), and 33.7% (95% CI 20.8-54.6) for children aged ≤5 years, 5-10 years, 10-15 years and ≥15 years, respectively ( p=.003). The type of germline ATM PV also had a significant impact: 4-year EFS for patients with Group B ATM PV was 78.7% (95% CI 63.7-97.2) vs. 39.4% (95% CI 29-53.3) for Group A ( p&amp;lt;.001). Group A PV were associated with an increased TRM (OR 9.3; 95% CI 1.6-180.1; p=.042) and decreased EFS (HR .371 95% CI 16.6-82.6; p=.009). Conclusions We demonstrate in this first comprehensive international study that leukemia and lymphoma in children with A-T are curable. While the standard treatment stratification system for patients with hematological malignancies without A-T focuses upon cancer relapse/progression as the main cause of treatment failure, TRM was the main cause of therapy failure in patients with A-T and was strongly associated with the underlying germline ATM variant type. This study further fulfills an unmet need for international collaboration and provides a platform for data-based guidelines for a novel risk stratification system and optimal therapy selection for this unique patient population.

A Novel Online Decision Support Tool to Determine Practice Patterns and Concordance with Experts in the Treatment of Patients with Immune Thrombocytopenia

Background: Immune thrombocytopenia (ITP) is a complex disease involving dysregulation of the immune system with inadequate platelet production and increased platelet destruction. When treatment is warranted, corticosteroids are frequently used as first-line therapy. Steroid-refractory patients eventually require alternative treatments, of which there are several approved and well-studied options. However, these second-line treatments have not been rigorously compared in clinical trials. To assist healthcare professionals (HCPs) in managing patients with ITP, we developed an online interactive decision support tool to provide expert recommendations for treatment. Here we report an analysis of practice patterns among HCPs and experts for ITP cases after initial therapy failure and the impact of expert recommendations on HCP decisions. Methods: An online tool was developed with 4 experts providing recommendations on management of 332 unique patient cases based on patient- and disease-specific factors that the experts considered relevant to making treatment decisions. HCPs entered patient case details including age, treatment setting, platelet count, current presentation, and presence of risk factors for bleeding, along with their intended management approach, before being shown the expert recommendations for their specific scenario. Results: Between October 2022 and July 2023, 311 patient cases were entered by 256 HCPs, including 194 (62.4%) cases of treatment-exposed patients (Table). Of the treatment-exposed cases, 72.1% were submitted by physicians, and 23.2% were submitted by HCPs in North America. There was strong alignment between HCP treatment selection and at least 1 expert recommendation (69%; P = .0001). There was consensus among experts for 94.4% of cases with patients ≥21 years of age who were treatment exposed. In these cases, experts recommended thrombopoietin receptor agonists (TPO-RA) for 83.5% of cases in contrast to HCPs recommending them in 38.2% of cases. Alternatives for TPO-RA chosen by HCPs in cases with patients ≥21 years of age who were treatment exposed included rituximab (29.6%), immunoglobulin (23.5%), fostamatinib (12.5%), splenectomy (9.8%), observation (8.6%), or unsure (16%). There was consensus among experts for 72% of cases with patients &amp;lt;21 years of age who were treatment exposed. For cases with patients &amp;lt;21 of age who were treatment exposed, HCPs chose immunoglobulin (28.6%), observation (28.6%), rituximab (14.3%), splenectomy (4.7%), or unsure (23.8%). In 47.6% of cases where HCPs indicated an alternative selection, they indicated that expert recommendations changed their treatment choice. HCPs indicated there were barriers to implementation in 7% of indicated cases. Conclusion: Based on our analysis of an online treatment decision tool, practice patterns for the management of patients with ITP aligned between HCPs and at least 1 tool expert in 69% of treatment-experienced cases. For previously treated patients, HCPs were less likely to choose a TPO-RA compared with tool experts, although very few identified barriers to implementation. Although specific treatment utilization patterns differed between HCPs and experts based on data from this tool, HCPs appeared amenable to practice changes based on expert recommendations. Online decision support tools can offer opportunities for HCPs to receive education and expert guidance in choosing optimal treatment regimens for ITP, a condition where treatment guidelines may not address every unique patient scenario.

Patterns and Safety of Glucocorticosteroids Use Following CD19 CAR-T Cell Therapy for B-Cell Lymphoma

Introduction: CAR T-cell therapy is a well-established therapy for relapsed and/or refractory B cell non-Hodgkin's lymphomas (R/R B-NHL). However, CAR-T cell therapy may cause significant toxicities such as CRS and ICANS. Glucocorticosteroids play a pivotal role in managing these toxicities but can also suppress T cell function and may impede the persistence and effectiveness of CAR-T cells. The impact of glucocorticosteroids has resulted in conflicting conclusions in the literature. Objective: This single-center, retrospective, observational study aimed to compare the outcomes of patients with R/R B-NHL who received steroids to those who did not after CAR-T cell therapy. Methods: Patients' records were reviewed to identify any steroid administration within 30 days of CAR-T cell infusion. Patients receiving steroids within 7 days before CAR-T infusion were excluded to focus on CAR-T impact without confounding baseline steroid use. Information on the type of steroids, indication, timing and duration was collected. Steroids were expressed as dexamethasone equivalent. Steroids-associated variables were analyzed as both continuous and categorical variables, using median as the threshold. Cox regression models and Kaplan-Meier analysis was employed for PFS and OS estimation using steroids as a time-dependent covariate and a 30-day landmark analysis, with events as disease progression or death for PFS and death only for OS. Results: 292 patients with R/R B-NHL received commercial CAR-T cell therapy (48% axi-cel, 24% tisa-cel, 22% liso-cel, and 6% Brexu-cel), primarily for large B cell cell lymphoma (85%), mantle cell (11%) or follicular (4%) lymphoma. Only 49 of the 292 patients (17%) received steroids between the day of infusion (day 0) to 30 days after. The median cumulative dexamethasone-equivalent dose at 30 days was 60 mg (range: 1-1982), the median day from CAR-T cells infusion to steroids initiation was 7 days (range 0-26) and the median duration of corticosteroid use was 3 days (range, 1-27). Patients receiving steroids had a significantly higher number of lines of prior therapies (p=0.036), more stage 3-4 disease (p=0.027), and elevated pre-lymphodepletion LDH levels(p=0.004). However, there were no other significant differences, including CD19 CAR-T product type, between the groups. The rates of grade 2 or higher CRS and ICANS were documented in 42% and 18%, respectively. ICANS (47%), CRS (14%), or both (21%) were the leading indications for steroid administration. Other indications for administration of steroids were progression of disease (n=4), adrenal insufficency (n=3), graft vs host disease (n=1), and premedication with intravenous immune globulin (n=1). With a median follow-up of 20 months, median PFS and OS were 6.8 months (95% CI 6-11) and 27 months (95% CI 18-not reached), respectively. The use, duration, and cumulative dose of steroids, whether analyzed as continuous or categorial variables in univariable Cox regression models, were not associated with PFS and OS in a 30 landmark analysis ( Figure). The analysis of the adminstration of steroids as a time-dependent covariate did not show significant differences for either PFS or OS. Importantly, patients exposed to steroids were also not at increased risk for bloodstream infections (p=0.68). Conclusion: In this largest analysis to date on the impact of steroids on CAR-T outcomes, steroid exposure following CAR-T cell infusion was not associated with a greater likelihood of CAR-T cell therapy failure. Our results deviate from previous observations that associate using steroids with shorter PFS and OS (Starti et al, Blood 2021, Terao et al, JTCT 2023). The discrepancy is likely due to our study's exclusion of steroid use prior to CAR-T and statistical analysis. Overall, our findings suggest that steroid administration for CAR-T cell therapy toxicity management is safe and does not appear to have a negative impact on efficacy.

Acquired Venetoclax Resistance in an In Vivo Model of B-Cell Precursor Acute Lymphoblastic Leukemia Is Characterized By Altered Functions of Apoptosis Regulators

Deregulation of cell death pathways such as apoptosis is a hallmark of cancer and contributes to leukemogenesis and treatment failure in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Apoptosis is controlled by different pro- and anti-apoptotic molecules either inducing or counteracting cell death signaling. Inhibition of anti-apoptotic molecules of BCL-2 family cell death inhibitors has been developed as therapeutic strategy. Venetoclax (VEN) binds selectively to BCL-2 resulting in apoptosis induction and has shown clinical activity in hematologic malignancies, but primary and secondary resistance have also been described. In this study, we aimed to identify resistance mechanisms acquired during VEN treatment leading to therapy failure of BCL-2 inhibition. In order to mimic a clinical situation with consecutive treatment cycles, we employed a patient-derived human BCP-ALL xenograft mouse model with subsequent courses of VEN administration to ALL bearing recipients in vivo. First, we estimated VEN-sensitivities in a series of BCP-ALL PDX samples (N=30) by ex vivo titration of individual VEN-EC 50 values and identified a range of heterogeneous VEN-activities. A VEN-sensitive (low VEN-EC50) PDX-ALL sample with a pro-B ALL phenotype carrying a KMT2A:: MLL1 gene fusion was selected and transplanted onto immunodeficient NOD/SCID recipient mice. Upon leukemia manifestation (presence of 5% human ALL cells in peripheral blood), mice were either treated with VEN (100 mg/kg, two times five days) or vehicle control. Recipients were sacrificed upon onset of leukemia-related morbidity and ALL cells isolated from spleens were transplanted onto secondary recipients, which were again treated with VEN or vehicle. ALL cells isolated from secondary recipients were retransferred onto tertiary mice and again exposed to in vivo VEN-treatment providing a total of three subsequent transplantation and in vivo VEN-therapy cycles. Assessing VEN sensitivities of the ALL cells initially transplanted and isolated after VEN-treatment cycles by using an ex vivo cytochrome c release assay showed increasing VEN-resistance. Accordingly, comparison of VEN to control in vivo treated cells showed significantly decreased sensitivities over treatment cycles. Importantly, also the time periods between treatment and leukemia re-occurrence in mice indicating decreasing VEN-sensitivity became shorter with every additional in vivo therapy course. Paired RNA- seq analysis revealed that transcript levels of the pro-apoptotic molecules BAX and BIM were significantly downregulated in VEN-exposed/resistant ALL as compared to vehicle control treated cells. Basal BH3 profiling analysis showed impaired apoptotic priming (significantly lower cytochrome c release after BIM and PUMA peptide exposure) and decreased dependence on anti-apoptotic BCL-2 and MCL-1 indicating a shift from a pro-apoptotic to a pro-survival state. This shift was also reflected in an e x vivo drug treatment assay showing decreased sensitivity to the MCL-1 inhibitor S63845 and the BCL-XL inhibitor A-1331852 in ALL cells from VEN- treated mice compared to control- treated mice (S63845 EC 50 1.5 vs. 2.2 µM, A-1331852 EC 50 9.3 vs. 15.3 µM). In line with the low BAX levels in ALL cells with acquired VEN-resistance, PDX BCP-ALL samples with intrinsic VEN resistance also showed lower BAX protein expression. Taken together, acquired VEN-resistance was recapitulated in a co-clinical trial model of BCP-ALL with repeated in vivo treatment cycles showing lower drug sensitivities along with increasingly reduced in vivo anti-ALL activity of VEN. Characterization of acquired VEN-resistance revealed decreased functional dependency on anti-apoptotic proteins and downregulation of pro-apoptotic BIM and BAX, thus pointing to an imbalance of pro- and anti-apoptotic molecules, which can be potentially targeted by directed compounds bypassing resistance to specific BCL-2 inhibition.