Purpose: Since CD7 may represent a potent target for T-lymphoblastic leukemia/lymphoma (T-ALL/LBL) immunotherapy, this study aimed to investigate safety and efficacy of autologous CD7-chimeric antigen receptor (CAR) T cells in relapsed and refractory (R/R) T-ALL/LBL patients, as well as its manufacturing feasibility. Experimental Design: Preclinical phase was conducted in NPG{trade mark, serif} mice injected with Luc+ GFP+CCRF-CEM cells. Open label phase I clinical trial (NCT04004637) enrolled patients with R/R CD7-positive T-ALL/LBL who received autologous CD7-CAR T cells infusion. Primary endpoint was safety, secondary endpoints included efficacy, pharmacokinetic and pharmacodynamic parameters. Results: CD7 blockade strategy was developed using tandem CD7 nanobody VHH6 coupled with an ER/Golgi-retention motif peptide to intracellularly fasten CD7 molecules. In preclinical phase CD7 blockade CAR T-cells prevented fratricide and exerted potent cytolytic activity, significantly relieving leukemia progression and prolonged the median survival of mice. In clinical phase, the complete remission (CR) rate was 87.5% (7/8) three months after CAR T cells infusion; one leukemia patient achieved minimal residual disease negative CR and one lymphoma patient achieved CR for more than 12 months. Majority of patients (87.5%) only had grade 1 or 2 cytokine release syndrome with no T-cell hypoplasia or any neurological toxicities observed. The median maximum concentration of CAR T cells was 857.2 cells/µL at approximately 12 days and remained detectable up to 270 days. Conclusions: Autologous nanobody-derived fratricide-resistant CD7-CAR T cells demonstrated a promising and durable antitumor response in R/R T-ALL/LBL with tolerable toxicity, warranting further studies in highly aggressive CD7-positive malignancies.

The role of excision repair cross-complementation group 6-like (ERCC6L) has been reported in several cancers, but little is known about its expression and function in laryngeal squamous cell carcinoma (LSCC). In this study, the expression of ERCC6L in LSCC was determined by immunohistochemistry and its correlation with prognostic factors was analyzed. Furthermore, cytological functional validation elucidated the role and underlying mechanisms of ERCC6L dysregulation in LSCC. Our data revealed that ERCC6L expression was elevated in LSCC and it’s correlated with TNM stage. In addition, ERCC6L knockdown LSCC cells showed decreased proliferation and migration, increased apoptosis, and reactive oxygen species (ROS). Mechanically, overexpression of ERCC6L promoted nuclear translocation of FOXM1 to facilitate direct binding to the KIF4A promoter and upregulated KIF4A expression. Furthermore, KIF4A knockdown attenuated the role of ERCC6L overexpression in promoting proliferation, migration, and tumorigenesis of LSCC cells. In summary, ERCC6L promoted the binding of FOXM1 and KIF4A in LSCC cells to drive their progression, which may be a promising target for precision therapy in this disease.

e19574 Background: Lymphoma is common cancer worldwide, a large group of lymphoid hematopoietic malignancies including Non-Hodgkin lymphoma (NHL) and Hodgkin lymphoma (HL) two major type. Lymphoma includes more than 30 unique subtypes, originating from B, T and NK cells. Currently, diagnosis and classification of lymphoma is based on excisional lymph node biopsy through immunohistochemistry and in situ hybridization. However, invasive biopsies have significant limitations and carry procedural risks to patients, and it cannot account for spatial inter- and intra-tumor heterogeneity due to sampling from only one location in a single tumor lesion. In the past few years, an emerging class of methods named Liquid biopsy can potentially improve on these limitations. Methods: 135 untreated stage I-IV lymphoma patients and 399 healthy individuals were enrolled in this study. 8ml peripheral blood was collected from each participant after enrollment. Cell-free DNA (cfDNA) was extracted and subjected to shallow whole-genome sequencing (sWGS), and partly plasma was used to quantify the expression of 7 tumor serum protein markers. We developed a newly blood-based assay, which integrated the newly next-generation technology of sWGS of cfDNA and protein marker (CA125), with artificial intelligence (AI) technology. Though our multi-dimensional assay, the cancer risk score (CRS) of each sample were calculated, and was used for lymphoma early diagnosis. Results: After compared each protein marker between lymphoma and healthy groups, only CA125 could be used to screen the lymphoma with a sensitivity of 25.9% at extremely high specificity 98.0% and an area under the curve (AUC) of 69.5%. As for the genomic feature of cfDNA, the characteristics of copy number aberrations (CNA) and fragment size (FS) were significantly different between lymphoma and healthy groups (p value < 0.0001 by student’s t-test). Both FS and CNA demonstrated improved sensitivity (36.3% and 67.4% respectively) compared to CA125. When the three signatures were incorporated into the CRS model, it achieved the best performance allowing 95 lymphoma cases to be identified with a sensitivity of 70.4% at 98.0% specificity, and an AUC of 88.0%. In stage I/II and III/IV lymphoma cases, the sensitivity was 40.0% and 85.7% respectively. For each lymphoma subtype, the lymphoma which from B-cell had higher sensitivity than that from NK/T cell (71.0% vs 64.3%), especially in HL (91.7%), all originated from B cell. Conclusions: We newly find the traditional protein marker CA125 can be used for lymphoma screening, which was integrated with genomic feature CNA and FS of cfDNA as a multidimensional assay for early detection of lymphoma with sufficient accuracy. The performance of our efficient and non-invasive assay for detecting lymphoma is 70.4% sensitivity at extremely high specificity 98.0%, especially for 40.0% sensitivity for early patients (stage I/II) screening.

e19547 Background: Lymphomas represent a diverse group of diseases that arise from a clonal proliferation of lymphocytes. Periodic imaging is the current standard of care for clinical evaluation of treatment response for lymphoma patients, but It bears obvious shortcomings. Therefore, more accurate method is needed to evaluate treatment effect of Lymphoma. Methods: We present a real-time blood-based treatment response monitoring assay SeekInClarity to assess tumor load and response to varied treatment protocols in patients with lymphoma. A novel multidimensional molecular tumor burden (MTB) model was utilized. Copy number aberrations (CNAs) and fragment size (FS) patterns across the genome from sWGS data and levels of 7 PTMs (AFP, CEA, CA153, CA125, CA199, CYFRA21-1, CA724) are exploited to establish the MTB model. Patients with lymphoma were radiological assessed at baseline and reassessed regularly after treatment. The patients also had 10 ml venous blood samples collected for SeekInClarity at baseline and every two treatment cycles. Results: At baseline, 64.5% (80/124) patients were tested positive by SeekInClarity, implying the high MTB. In particular, genome-wide or focal CNA patterns were observed in 45.2% patients, and FS profile abnormality was witnessed from 42.5% patients, indicating both features are fundamental and ubiquitous surrogates of tumor burden. Elevated level of PTMs was also reported from 23.4% patients. All patients received various combination therapies with chemotherapies or immunotherapies as the backbone, and evaluated efficacy by clinical imaging. MTB evaluations of patients who underwent the follow-up SeekinClarity were compared with clinical imaging results. For the 44 patients who had negative MTB at baseline, 33 of them were performed following SeekinClarity and kept negative after treatment, and their imaging evaluations all showed remarkably curative effect. Furthermore, in the 80 patients with positive MTB, 57 patients were performed subsequent SeekinClarity tests, and a concordance of 81.6% was achieved with clinical evaluation. In addition, there was an 18.8% discordance between imaging evaluation and SeekinClarity. For these who had clinical partial response but unchanged or higher MTB, more tests and clinical evaluations are needed to assure the true clinical outcomes. Conclusions: This proof-of-concept study demonstrated SeekInClarity, a non-invasive, multidimensional multi-omics blood-based assay, can promptly evaluate the treatment response of patients with lymphoma. By implementing this assay, the dynamic change of MTB can help physicians to make well-informed decisions on the upcoming therapeutic strategies for each patient in conjunction with imaging. This study is still ongoing, and more cases and more time points are included to demonstrate the clinical performance of the assay.

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