ALBERT

Description: Background Chronic lymphocytic leukemia (CLL) is a common B-cell lymphoproliferative neoplasm. Although not required for the diagnosis of CLL, important prognostic information is obtained with fluorescence in-situ hybridization (FISH) testing for chromosomal aberrations including del(11q), trisomy 12, del(13q), and del(17p). Abnormalities involving TP53, the ATM gene, and cases constituting complex cytogenetics (3 or more abnormalities), among others, have been shown to impact patient survival and response to traditional chemoimmunotherapy (CIT). Cytogenetic microarray analysis (CMA) is a laboratory technique used to detect unbalanced chromosomal abnormalities. CMA allows for comparison of hundreds of discrete loci within a chromosome and has been validated in CLL, with greater sensitivity in detecting genomic alterations compared to FISH. The acquisition of new genomic alterations is termed "clonal evolution" and has been shown to have a detrimental effect on survival in patients with CLL (Shanafelt TD, et al. J Clin Oncol. 2006). CIT results in a higher proportion of complete responses (CR) and eradication of CLL clones compared to targeted therapies. While targeted therapies may successfully suppress CLL clones, a CR is rare with single-agent treatment (Byrd JC, et al. Blood. 2015). CMA may be a valuable tool in monitoring clonal evolution throughout a patient's disease course and may have a role in evaluation of patients on suppressive therapies due to the relatively high risk of clonal emergence. Methods This was a single-center retrospective study. Patients with pathologically-confirmed CLL were analyzed utilizing data obtained from the electronic medical record. Data was extracted from the CMA reports and clinic documentation. To be included in the study, at least two serial CMA evaluations were required, separated by at least four weeks. In the interval between CMA results, subsequent systemic treatment was recorded, if any. Treatments were categorized as ibrutinib/idelalisib ("I"), any cytotoxic chemotherapy ("C"), and no treatment/observation ("N"). Change in the number of abnormalities detected by CMA was calculated for each interval and evaluated as both continuous and categorical (increase/no change/decrease). Patients with over three abnormalities were deemed to have complex disease. Genomic DNA of bone marrow or peripheral blood was extracted using Qiagen's Gentra Puregene kit. Chromosome microarray assay was performed using ThermoFisher Affymetrix CytoScan HD according to the manufacturer's protocol. Copy number and allele analysis was performed using Affymetrix Chromosome Analysis Suite using the default settings. Results There were 34 patients who met inclusion criteria, each with 2-4 CMA results, for a total of 81 CMA results. Forty-seven treatment intervals were analyzed. The median age was 61 years, and 64% of patients were male. Based on the first CMA result, the median number of detected abnormalities was 2 and mean was 2.6; 13 patients (38%) had complex disease. Complexity was not associated with a clinically significant change in the number of chromosomal abnormalities regardless of what treatment was received, if any (p=0.14). Overall, 18% of patients (6/34) had a decrease in the number of abnormalities detected by CMA, while 21% (7/34) had an increase. Analyzing the data by treatment group, the mean change in number of abnormalities was -0.5, -0.6 and +0.3, in groups I, C and N, respectively (p=0.26) (Figure 1). Among the patients in group I, 23% (3/13) had a decrease in the number of abnormalities, while 23% demonstrated an increase. For those in group C, 25% (2/8) and 25% experienced increase and decrease, respectively. For patients in group N, 14% (3/21) had an increase and 0% had a decrease in CMA abnormalities. Among the two patients who experienced Richter's transformation, the complexity of their chromosomal abnormalities increased over time and preceded clinical emergence of transformation. Discussion CMA can sensitively identify the emergence or clearance of CLL clones and help detect clonal evolution throughout periods of treatment or observation. Though a relatively small number of patients had serial CMA data for evaluation, these data suggest that CMA should be prospectively analyzed in patients with CLL and may be used as a dynamic clinical tool to potentially modify treatments in patients with sub-clinical evidence of treatment failure. Figure 1. Figure 1. Disclosures Barta: Janssen: Membership on an entity's Board of Directors or advisory committees; Merck, Takeda, Celgene, Seattle Genetics, Bayer: Research Funding.

Description: Whether tumor-reactive T cells can infiltrate into the tumor to execute effector function is essential for controlling tumor growth. CD103 is an integrin protein (αE) that binds integrin β7 to form the heterodimeric integrin complex αEβ7. CD103 is important for T cell retention in peripheral tissues by interacting with E-cadherin and a promising prognosis biomarker for assessment of tumor-reactive T cells infiltrating in the tumor from various types of cancer, such as lung cancer, ovarian cancer and cervical cancers. However, CD103 is not expressed on the surface of circulating peripheral blood T cells that are genetically modified to express a chimeric antigen receptor (CAR) for adoptive T cell therapy. Whether CD103 expression on the surface of tumor-reactive CAR T cells is functionally important for their anti-tumor activity has not been previously determined. Using a preclinical model of human lymphoma expressing E-cadherin, we demonstrate that engineering of CD19-specific human CAR T cells with CD103 significantly improves their therapeutic effects on eliminating pre-established human lymphoma in immune deficient NSG mice (NOD.scid.Il2Rγcnull). We synthesized a codon optimized CD19-specific CAR containing 4-1BB and CD3zeta intracellular signaling domains (named CD19-BBz-CAR), cloned it into lentiviral vector and infected human T cells. As expected, the resultant human CD19-BBz-CAR T cells possessed potent capacity to cure human B cell leukemia in NSG mice that had been intravenously inoculated with Raji leukemic/lymphoma cells. Notably, while approximately 10% of non-CAR T cells produced high levels of CD103 from these NSG mice, CD19-BBz-CAR T cells failed to upregulate CD103, suggesting that the expression of CD19-BBz-CAR inhibits the induction of CD103 in vivo. Ex vivo assay confirmed that CD19-BBz-CAR caused dose-dependent decrease of CD103 expression in human T cells cultured in the presence of TGF-β1. This effect was mediated by the expression of costimulatory molecule 41BB, which is known essential for sustaining CD19-BBz-CAR T cells in vivo. To circumvent the repression effect of 41BB on induction of CD103, we incorporated the gene encoding integrin αE into the CAR structure to generate CD103-CD19-BBz-CAR T cells. Intriguingly, as compared to conventional CD19-BBz-CAR T cells, CD103-CD19-BBz-CAR T cells expressed high levels of CD62L and CD45RA, which resemble less differentiated T cells, produced higher levels of IL-2, which is crucial for promoting T cell expansion and function, and underwent greater expansion in cultures. Upon adoptive transfer into NSG mice that had subcutaneous human Raji lymphoma, CD103-engineering of CD19-BBz-CAR T cells dramatically decreased the distal metastasis of lymphoma, increased the infiltration of CAR T cells into the solid lymphoma, and improved the in vivo persistence of tumor-reactive CAR T cells. As a result, transfer of CD103-CD19-BBz-CAR T cells significantly increased overall survival rate of lymphoma mice compared to conventional CD19-BBz-CAR T cells (40% versus 10%, p

Description: More than 70% of Epstein–Barr virus (EBV)-negative Hodgkin lymphoma (HL) cases display inactivation of TNFAIP3 (A20), a ubiquitin-editing protein that regulates nonproteolytic protein ubiquitination, indicating the significance of protein ubiquitination in HL pathogenesis. However, the precise mechanistic roles of A20 and the ubiquitination system remain largely unknown in this disease. Here, we performed high-throughput CRISPR screening using a ubiquitin regulator-focused single-guide RNA library in HL lines carrying either wild-type or mutant A20. Our CRISPR screening highlights the essential oncogenic role of the linear ubiquitin chain assembly complex (LUBAC) in HL lines, which overlaps with A20 inactivation status. Mechanistically, LUBAC promotes IKK/NF-κB activity and NEMO linear ubiquitination in A20 mutant HL cells, which is required for prosurvival genes and immunosuppressive molecule expression. As a tumor suppressor, A20 directly inhibits IKK activation and HL cell survival via its C-terminal linear-ubiquitin binding ZF7. Clinically, LUBAC activity is consistently elevated in most primary HL cases, and this is correlated with high NF-κB activity and low A20 expression. To further understand the complete mechanism of NF-κB activation in A20 mutant HL, we performed a specifically designed CD83-based NF-κB CRISPR screen which led us to identify TAK1 kinase as a major mediator for NF-κB activation in cells dependent on LUBAC, where the LUBAC-A20 axis regulates TAK1 and IKK complex formation. Finally, TAK1 inhibitor Takinib shows promising activity against HL in vitro and in a xenograft mouse model. Altogether, these findings provide strong support that targeting LUBAC or TAK1 could be attractive therapeutic strategies in A20 mutant HL.