Citations

Abstract Background: Chimeric Antigen Receptor (CAR) T cell therapy is arguably one of the most significant breakthroughs in cancer treatment. There are currently five FDA-approved products that are commercially available. However, despite their success, these CAR T-cell therapies cannot induce long-term durable responses in approximately 50% of leukemia or lymphoma-treated patients. Similarly, the results of CAR T-cells in solid tumors have been somewhat disappointing. Therefore, there is an urgent need to design and develop novel CAR T cells with improved efficacy in hematologic malignancies and solid tumors. ROR1 is a carcinoembryonic antigen expressed in different cancers and is associated with tumor stemness, proliferation, metastatic transformation, and treatment resistance. In this project, we optimize an anti-ROR1 CAR using a humanized single-chain variable fragment (scFv) with second (2G) or third-generation (3G) costimulatory domains. Methods: Several optimization steps in silico were performed using a selected scFv binding domain that targets ROR1. Those included codon optimizations, positional arrangement of heavy-light chains, evaluation of the ideal length of linkers based on tridimensional modeling of the docking between the antibody-like paratope with the target antigen (Figure 1A). After this initial scFv optimization process, we constructed a lentiviral vector that encodes CARs using the selected scFv linked to a transmembrane domain CD28 and different signaling endodomains for 2G and 3G variants (CD28, 41BB, ICOS, OX40), each linked to the T cell receptor CD3z domain. The cytotoxic activity of these constructs was evaluated using an in vitro rechallenge luciferase assay in ROR1 expressing JeKo-1 cells and ROR1(negative) controls. Results: The 2G 41BB-z construct with V H-V L scFv orientation and a long linker (V H-L-V L) showed optimal cytotoxicity with a CAR expression level in T cells of 36% (Range 28-49% for other constructs, Figure 1B-C). The V H-L-V L 41BB-z construct was evaluated comparatively using a rechallenge cytotoxic assay with 3G constructs that expressed CD28, ICOS, or OX40 signaling domains using JeKo-1 and ROR1(negative) target cells as controls. All the tested constructs showed specific ROR1 medicated cytotoxicity. CD28-41BB-z and ICOS-41BB-z showed the lowest cytotoxicity levels during the Day 1 of the repetitive rechallenge. However, the cytotoxicity levels of those constructs gradually increased during the 7 days of rechallenge and were closed to the levels induced by the 2G- 41BB-z construct (>80% of cytotoxicity). There were no significant differences in CAR T cells subsets generated by the different constructs during the 7 days of rechallenge with a predominance of effector memory phenotype (CCR7-, CD45RA-) and no difference in PD1 expression. Conclusions: Our results demonstrate that optimization of the CAR constructs enhances T-cell effector function and cytotoxicity against ROR1+ target cells. In previous studies, 3G CARs have shown longer persistence of the transduced T cells in peripheral blood, sustained and regulated cellular activation, improved solid tumor infiltration, and positive modulation of the tumor microenvironment. Our preclinical in vitro optimization demonstrates strategies to generate 3G constructs with a progressive and modulated cytotoxic profile that may confer benefits when tested in vivo in terms of enhanced persistence and lower adverse events profile. Additional experiments in vivo will be presented during the meeting to corroborate our findings. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Nonalcoholic fatty liver disease (NAFLD) is a global health problem that develops through unclear molecular mechanisms. The P2X7 purinergic receptor (P2RX7) is an ATP‐gated ion channel that belongs to the P2XR family. Thus far, studies on P2RX7 in NAFLD have been largely contradictory. Integrating experiments and modeling, we elucidate the dynamic processes of lipid droplet fusion and degradation following regulation of P2RX7. We show that activation of P2RX7 can activate the AMPK/ULK1 pathway to promote autophagosome generation and lysosomal degradation of autophagosomes. Inhibiting P2RX7 has the opposite effect. Notably, we find that lipid droplets become larger by the fusion of dysfunctional lysosomes but cannot be degraded by them following P2RX7 inhibition. Our study provides evidence that P2RX7 activation improves NAFLD by promoting lipophagy.

Abstract Usually used in emergency settings, bedside sonographic measurement of optic nerve sheath diameter can aid in diagnosing elevated intracranial pressure. We report a case of a 26-year-old male hospitalized for CAR T-cell therapy with Axicabtagene Ciloleucel for treatment of relapsed diffuse large B-cell lymphoma, who developed progressive symptoms of immune effector cell-associated neurotoxicity syndrome. Fundoscopic examination suggested the presence of blurred optic disc margins. Bedside ocular ultrasound revealed wide optic nerve sheath diameters and bulging optic discs bilaterally. The patient had a ventriculostomy placed for monitoring and received treatment with steroids and mannitol, as well as tocilizumab. After 7 days in the ICU, the patient recovered with no evidence of long-term neurological deficits.

Abstract Ibrutinib-based therapies are costly and require continuous administration. We hypothesized combining BTK inhibition with anti-CD20 monoclonal antibodies would yield deep remissions allowing discontinuation. We enrolled 32 therapy-naïve CLL patients to receive ibrutinib plus obinutuzumab, followed by single-agent ibrutinib. Patients could discontinue ibrutinib after 36 months with sustained complete response (CR). We evaluated treatment safety, efficacy, and outcomes after ibrutinib discontinuation. The overall response rate was 100%, 28% achieved a CR, and 12.5% achieved bone marrow undetectable minimal residual disease. At a three-year median follow-up, 91% remain in remission with 100% overall survival. Five patients in sustained CR stopped ibrutinib and have not progressed. Eight non-CR patients discontinued for other reasons, with only two progressing. The treatment was safe, with a lower IRR rate. All patients responded to treatment with longer time-to-progression after discontinuation of ibrutinib. Our data support the evaluation of ibrutinib discontinuation strategies in more extensive clinical trials.

Background: The role of the critical shoulder angle (CSA) as a risk factor for rotator cuff tear (RCT) remains controversial. Studies on the association between the CSA and RCT show considerable differences in design, and this could be responsible for the variation in study results. Purpose: To (1) describe the reliability of CSA measurement and (2) evaluate the results of the studies reporting the association between the CSA and RCT using meta-analytical techniques to explore potential sources of variation of study results. Study Design: Systematic review and meta-analysis; Level of evidence, 4. Methods: MEDLINE, EMBASE, and CINAHL electronic databases were searched through June 30, 2019. Case-control and cross-sectional studies reporting the association of the CSA and RCT were selected. The weighted mean difference in the CSA was estimated using a random-effects model. Prediction interval was computed to better express uncertainties in the effect estimate. Metaregression and subgroup analyses were performed to explore potential sources of heterogeneity. Results: A total of 14 studies, including 1154 cases and 1271 controls, were identified. Of these studies, 79% (11/14) assessed the reliability of the CSA measurement, demonstrating an excellent intraobserver (range, 0.91-0.99) and interobserver (range, 0.87-0.99) reliability. Compared with controls, cases with RCT showed larger measurements of the CSA (3.3° [95% CI, 2.3°- 4.4°]). However, there was a high heterogeneity ( I 2 = 93%), and the 95% prediction interval (-0.4° to 7.1°) included no difference in the CSA. Results of the metaregression analysis showed a significant association of several methodological aspects with the heterogeneity. The difference in the CSA tended to be larger when only full-thickness tears were included, when no specific defined criterion for assessing radiographic viewing perspective was used, in studies with smaller sample sizes, and in studies at higher risk of bias. Conclusion: While the CSA can be reliably measured, the difference in the CSA between cases and controls varied from very large to modest or almost no difference. Several determinants of heterogeneity were determined. Owing to this heterogeneity, it is difficult to gain an insight into the strength and exact nature of the association between the CSA and RCT with the current evidence.

Background Many hormones display distinct circadian rhythms, driven by central regulators, hormonal bioavailability, and half-life. A set of 11-oxygenated C19 steroids (11-oxyandrogens) and pregnenolone sulfate (PregS) are elevated in congenital adrenal hyperplasia and other disorders, but their circadian patterns have not been characterized. Participants and methods Peripheral blood was collected every 2 h over 24 h from healthy volunteer men (10 young, 18–30 years, and 10 older, 60–80 years). We used mass spectrometry to quantify 15 steroids, including androstenedione (A4), testosterone (T), 11β-hydroxy- and 11-ketotestosterone (11OHT, 11KT),11β-hydroxy- and 11-ketoandrostenedione (11OHA4, 11KA4), and 4 ∆5-steroid sulfates. Diurnal models including mesor (rhythm adjusted median), peak, and nadir concentrations, acrophase, and amplitude were computed. Results 11OHA4 followed a rhythm similar to cortisol: acrophase 8:00 h, nadir 21:00 h and were similar in young and old men. 11KT had similar diurnal patterns, but the peak was lower in older than in young men, as was the case for A4. All four steroid sulfates were higher in young vs older men. PregS and 17-hydroxypregnenolone sulfate (17OHPregS) showed sustained elevations between 8:00 and 18:00 h, and nadirs around midnight, while DHEAS and AdiolS displayed minimal diurnal variations. All 4 11-oxyandrogens correlated tightly with cortisol (r from 0.54 for 11OHT to 0.81 for 11OHA4, P < 0.0001 for all), but very weakly with T, supporting their adrenal origin and ACTH governance. Conclusions 11-Oxyandrogens, PregS, and 17OHPregS display distinct circadian and age variations, which should be accounted for when used as clinical biomarkers.

Abstract Background: Amplification of EGFR and its active mutant EGFRvIII are common in glioblastoma (GB). While EGFR and EGFRvIII play critical roles in pathogenesis, targeted therapy with EGFR-tyrosine kinase inhibitors (TKIs) or antibodies has shown limited efficacy. To improve the likelihood of effectiveness, we targeted adult patients with recurrent GB enriched for simultaneous EGFR amplification and EGFRvIII mutation, with osimertinib/bevacizumab at doses described for non-small cell lung cancer (NSCLC). Methods: We retrospectively explored whether previously described EGFRvIII mutation in association with EGFR gene amplification could predict response to osimertinib/bevacizumab combination in a subset of 15 patients treated at recurrence. The resistance pattern in a subgroup of subjects is described using a commercial NGS panel in liquid biopsy.Results: There were ten males (66.7%), and the median patient’s age was 56 years (range 38-70 years). After their initial diagnosis, 12 patients underwent partial (26.7%) or total resection (53.3%). Subsequently, all cases received IMRT and concurrent and adjuvant temozolomide (TMZ; the median number of cycles 9, range 6-12). The median follow-up after recurrence was 17.1 months (95% CI 12.3-22.6). All patients received osimertinib/bevacizumab as a second-line intervention with a median progression-free survival (PFS) of 5.1 months (95% CI 2.8-7.3) and overall survival (OS) of 9.0 months (95% CI 3.9-14.0). The PFS6 was 46.7%, and the overall response rate (ORR) was 13.3%. After exposure to the osimertinib/bevacizumab combination, the main secondary alterations were MET amplification, STAT3, IGF1R, PTEN, and PDGFR.Conclusions: While the osimertinib/bevacizumab combination was marginally effective in most GB patients with simultaneous EGFR amplification plus EGFRvIII mutation, a subgroup experienced a long-lasting meaningful benefit. The findings of this brief cohort justify the continuation of the research in a clinical trial. The pattern of resistance after exposure to osimertinib/bevacizumab includes known mechanisms in the regulation of EGFR, findings that contribute to the understanding and targeting in a stepwise rational this pathway.

Abstract Richter's transformation (RT) is a clinically challenging and aggressive transformation of CLL (Chronic lymphocytic leukemia) to lymphoma. Immune-checkpoint therapy has improved outcomes in selective lymphoma types. Nearly 40% of RT patients respond to anti-PD1, while the underlying CLL do not show any response, and 10% of CLL patients progress post-anti-PD1 treatment. Previous studies mostly focused on T-cell responses elicited by anti-PD1, however, intrinsic changes in CLL and RT B-cells induced by the treatment remains elusive. We hypothesized that pre-existing active transcriptional circuitry and distinct B-cell subsets in the CLL and RT B-cells may determine the response to anti-PD1 treatment. Peripheral blood mononuclear cells (PBMCs) from 3 patients who received pembrolizumab(anti-PD1) as a single agent. Samples were obtained at baseline and 3 months post-treatment initiation. Among these patients, 2 are progressors while 1 is a responder. We performed single-cell RNAseq (10X Genomics) on B-cells enriched from PBMCs derived from 3 samples (1 Responder: RS2, 2 progressors: CLL14 and CLL17) before and after treatment using chromium single cell 3' gene expression library. A total of ~10000 cells were captured with an average detection of 1000 genes/sample. To delineate the transcriptional changes upon the anti-PD1 treatment, we first integrated the single-cell data from 6 samples (before and after Pembro) and performed unsupervised clustering analysis using UMAP (Uniform Manifold Approximation and projection). We identified 8 different clusters based on highly expressed marker genes. Cluster 2(C2) was increased in the responder as compared to the progressors post-pem treatment. To determine the activated pathways in each cluster, we performed gene-set enrichment analysis (GSEA) using the Enrichr method. This analysis revealed upregulation of distinct pathways related to IFNG/MYC(C1), IFNG(C2), ER stress(C3), B-cell dependent T cell activation related pathway(C4), MYC targets/TNFα(C5,6,7) indicating intra-tumor transcriptional heterogeneity. To decipher the intratumor cell differentiation changes, we performed pseudotime trajectory analysis using Monocle 2 coupled with B cell developmental stage markers. We identified 7 different states, with distinct changes of state in responder sample RS2 (State 4,5, 6, and 7 were increased in RS2 post-treatment) while CLL samples(progressors) did not have distinct changes. Integration with B cell development revealed that aggressive B cells with CD38 expression were highly induced upon anti-PD1 in responder RS2 while regulatory (CD1d) and memory B cells (CD27) remained the same, suggesting PD1 induced intrinsic changes contributing to the disease response. We identified distinct transcriptional states in B cells within responders and progressors which may govern the intra-tumor and inter-tumor heterogeneity to the anti-PD1 response. Citation Format: Prajish Sundaram Iyer, Lu Yang, Yang Zhi Zhang, Hyo Jin Kim, Charla Secreto, Sutapa Sinha, Neil Kay, Stephen Ansell, Wei Ding, Lili Wang. Tumor intrinsic changes determine immune-checkpoint response in Richter's transformation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1612.

Background: Trastuzumab (Herceptin) is the key systemic therapy for HER2-positive breast cancer. However, the initial response rate is limited to approximately 50% in patients. Moreover, most patients, especially at an advanced stage, eventually develop acquired resistance. Understanding the mechanisms of trastuzumab resistance is crucial for achieving better treatment outcome in this group of patients. Methods: A trastuzumab-resistant (TR) cell line was developed using the BT474 HER2-positive breast cancer cell line. Whole-transcriptome expression array was performed and the TR-related gene NDUFA4L2 was identified by differential expression analysis between BT474 and BT474-TR. Mitochondrial localization of NDUFA4L2 was confirmed by immunofluorescence and western blotting using mitochondrial fractionation. Mitochondrial function and energy metabolism were evaluated using Seahorse, ATP production, and lactate production assays, and cellular reactive oxygen species (ROS) levels were determined using DCFDA. NDUFA4L2 expression in patients was evaluated by immunohistochemistry, and relapse-free survival was analyzed using the Kaplan–Meier method. Results: NDUFA4L2 was highly expressed in the TR HER2-positive breast cancer cell line. High expression level of NDUFA4L2 was associated with shorter relapse-free intervals in trastuzumab-treated HER2-positive breast cancer patients. Overexpression of NDUFA4L2 enhanced Warburg effects, enhanced aerobic glycolysis, reduced oxygen consumption, and lowered ROS production. Mechanistically, overexpression of NDUFA4L2 facilitated mitochondrial relocalization of HER2 and suppressed ROS production, thus rendering cancer cells more resistant to trastuzumab treatment. Conclusions: We identified NDUFA4L2 as a new biomarker and potential therapeutic target for TR HER2-positive breast cancer.