B2M Genes Research Articles

Generation of GGTA1-/-β2M-/-CIITA-/- Pigs Using CRISPR/Cas9 Technology to Alleviate Xenogeneic Immune Reactions.

Xenogeneic organ transplantation has been proposed as a potential approach to fundamentally solve organ shortage problem. Xenogeneic immune responses across species is one of the major obstacles for clinic application of xeno-organ transplantation. The generation of glycoprotein galactosyltransferase α 1, 3 (GGTA1) knockout pigs has greatly contributed to the reduction of hyperacute xenograft rejection. However, severe xenograft rejection can still be induced by xenoimmune responses to the porcine major histocompatibility complex antigens swine leukocyte antigen class I and class II. We simultaneously depleted GGTA1, β2-microglobulin (β2M), and major histocompatibility complex class II transactivator (CIITA) genes using clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins technology in Bamma pig fibroblast cells, which were further used to generate GGTA1β2MCIITA triple knockout (GBC-3KO) pigs by nuclear transfer. The genotype of GBC-3KO pigs was confirmed by polymerase chain reaction and Sanger sequencing, and the loss of expression of α-1,3-galactose, SLA-I, and SLA-II was demonstrated by flow cytometric analysis using fluorescent-conjugated lectin from bandeiraea simplicifolia, anti-β2-microglobulin, and swine leukocyte antigen class II DR antibodies. Furthermore, mixed lymphocyte reaction assay revealed that peripheral blood mononuclear cells from GBC-3KO pigs were significantly less effective than (WT) pig peripheral blood mononuclear cells in inducing human CD3CD4 and CD3CD8 T-cell activation and proliferation. In addition, GBC-3KO pig skin grafts showed a significantly prolonged survival in immunocompetent C57BL/6 mice, when compared with wild-type pig skin grafts. Taken together, these results demonstrate that elimination of GGTA1, β2M, and CIITA genes in pigs can effectively alleviate xenogeneic immune responses and prolong pig organ survival in xenogenesis. We believe that this work will facilitate future research in xenotransplantation.

A versatile oscillating-flow microfluidic PCR system utilizing a thermal gradient for nucleic acid analysis.

We report the development of a versatile system based on the oscillating-flow methodology in a thermal gradient system for nucleic acid analysis. Analysis of DNA and RNA samples were performed in the device, without additional temperature control and complexity. The technique reported in this study eliminates the need for predetermined fluidic channels for thermocycles, and complexity involved with additional incubation steps required for RNA amplification. A microfluidic device was fabricated using rapid prototyping by simply sandwiching dual side adhesive Kapton tape and a polydimethylsiloxane spacer between glass microscope slides. Amplification of the 181-bp segment of a viral phage DNA (ΦX174) and B2M gene in human RNA samples was demonstrated using the system. The developed system enables simultaneous acquisition of amplification and melt curves, eliminating the need for postprocessing. A direct comparison between the oscillating-flow system and a commercial real-time polymerase chain reaction (PCR) instrument showed complete agreement in PCR data and improved sample-to-result time by eliminating an additional 30 min melt curve step required in commercial PCR systems.

Identification of Hub Genes and Potential Molecular Mechanisms in Patients with HBV-Associated Acute Liver Failure.

Hepatitis B virus (HBV) infection is a major cause of acute liver failure (ALF) in China, and mortality rates are high among patients who do not receive a matched liver transplant. This study aimed to determine potential mechanisms involved in HBV-ALF pathogenesis. Gene expression profiles under access numbers GSE38941 and GSE14668 were downloaded from the Gene Expression Omnibus database, including cohorts of HBV-ALF liver tissue and normal samples. Differentially expressed genes (DEGs) with false discovery rates (FDR) <0.05 and |log2(fold change)| >1 as thresholds were screened using the Limma package. Gene modules associated with stable disease were mined using weighed gene co-expression network analysis (WGCNA). A co-expression network was constructed and DEGs were analyzed using gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. A gene-based network was constructed to explore major factors associated with disease progression. We identified 2238 overlapping DEGs as crucial gene cohorts in ALF development. Based on a WGCNA algorithm, 10 modules (modules 1-10) were obtained that ranged from 75 to 1078 genes per module. Cyclin-dependent kinase 1 (CDK1), cyclin B1 (CCNB1), and cell-division cycle protein 20 (CDC20) hub genes were screened using the co-expression network. Furthermore, 17 GO terms and 6 KEGG pathways were identified, such as cell division, immune response process, and antigen processing and presentation. Two overlapping signaling pathways that are crucial factors in HBV-ALF were screened using the Comprehensive Toxicogenomics Database (CTD). Several candidate genes including HLA-E, B2M, HLA-DPA1, and SYK were associated with HBV-ALF progression. Natural killer cell-mediated cytotoxicity and antigen presentation contributed to the progression of HBV-ALF. The HLA-E, B2M, HLA-DPA1, and SYK genes play critical roles in the pathogenesis and development of HBV-ALF.

First Report on Ex Vivo Delivery of Paracrine Active Human Mesenchymal Stromal Cells to Liver Grafts During Machine Perfusion.

Machine perfusion is rapidly becoming the standard for graft preservation and provides new opportunities for organ salvage, reconditioning, and repair. Based on their immune-modulatory and regenerating properties,1 arguable mesenchymal stromal cells (MSCs) are the most promising cell therapy for graft repair on the pump, though proof of concept is still lacking.2 Here, we show the feasibility of delivering clinically relevant numbers of human MSCs (hMSCs; 5–10 × 106/kg) during 30-minute hypothermic oxygenated machine perfusion in porcine liver grafts (n = 8; Figure 1). Full methods are described in Supplemental Materials and Methods (SDC, https://links.lww.com/TP/B821). To track the biodistribution of hMSC, cells were genetically labeled with click beetle red luciferase.3 As shown in Figure 2, bioluminescent imaging of both the anterior and posterior side of the liver showed a wide range and patchy distribution of hMSC which retain throughout the liver after 30 minutes of perfusion. No significant difference between arterial (n = 4) or venous infusion (n = 4) was observed. Histological and RNA-expression analysis confirmed the delivery of the hMSC throughout the liver grafts. About 104–105 hMSC were estimated to be still present per kilogram of liver graft. Importantly, the hMSC retained their paracrine activity after infusion. Using both Luminex and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, increasing levels of human-specific interleukin-6 and interleukin-8 released in porcine blood were shown during 4-hour normothermic perfusion. In conclusion, this is the first report showing an effective delivery of hMSC in a liver machine perfusion model. Evidence of paracrine activity of hMSC after delivery indicates regenerative and immune-modulatory effects during normothermic graft perfusion, though this needs further research in a transplantation model.FIGURE 1.: Graphical abstract of the procedure. The timeline indicates the details of each time point. Pig livers are procured after a warm ischemia period of 30 min and infused with 5 × 106–107 click beetle red luciferase (CBred2)-labeled human mesenchymal stromal cell (hMSC) either via the portal vein or hepatic artery. The distribution of hMSC is visualized by bioluminescence imaging (BLI) immediately (time point 0) after infusion using an IVIS Imaging System 200. The liver is then placed on a purpose-made perfusion device in which it is perfused with cold (10°C) oxygenated University of Wisconsin (UW)-organ preservation fluid. After 30 min of perfusion, the distribution of the hMSC is again imaged using BLI. After imaging, the livers were perfused for 4 h with their own, heparinized, full blood at normothermic temperature (37°C) by ex vivo machine perfusion after which biopsies were collected for further analysis. The BLI signal is quantified, presence of hMSC was assessed in biopsies taken from left and right periphery and center of the liver using human-specific antibodies, proteomic and genetic analysis. HOPE, hypothermic oxygenated machine perfusion; qPCR, quantitative polymerase chain reaction.FIGURE 2.: Effective delivery of paracrine active human mesenchymal stromal cells (hMSCs) during normothermic hepatic machine perfusion. Live imaging of the livers (n = 8) infused with click beetle red luciferase (CBred2)-transduced hMSC in the hepatic artery (n = 4; A, B, E, F) or portal vein (n = 4; C, D, G, H) using the IVIS Imaging System 200, directly after infusion (directly after infusion, A, B, C, D) and after 30 min of perfusion (E, F, G, H). Images are representative images showing bright luciferin-based luminescence at the posterior (A, C, E, G) and anterior side (B, D, F, H) of the liver, demonstrating whole organ coverage of the infused hMSC. The presence of hMSC was confirmed by immunohistochemical staining for copepod green fluorescent protein (copGFP) (I arrows; ×40 objective) at end of perfusion, whereas no staining was observed in liver biopsies before infusion (J). Representative pictures are shown. Human-specific B2M gene expression was determined by nested quantitative polymerase chain reaction (qPCR) to confirm the presence of hMSC in the pig liver at the genetic level (M). Shown are the raw cycle threshold (Ct) values of B2M expression of 3 pigs. B2M expression was measured in the pig samples that were perfused with hMSC, (pig 1–3) and in nonperfused pig livers (n = 3). Luminex-based analysis using human-specific cytokines showed that interleukin (IL)-6 (O) and IL-8 (P) was secreted in the liver perfusate in 2 of the 4 pigs that were infused via the hepatic artery (filled symbols). In pig livers that were not infused with hMSC, no IL-6 or IL-8 was detected (open symbols). Human-specific IL-6 and IL-8 protein release was confirmed in the same 2 pig livers by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis (data not shown). nd, not detectable.

Genetic and epigenetic analysis of the beta-2-microglobulin gene in microsatellite instable colorectal cancer.

One of the most common mechanisms of immune evasion in MSI colorectal cancers (CRCs) is loss of HLA class I expression due to mutations in B2M gene which can become a negative predictor for checkpoint blockade therapy. The aim of this study was the determination of prevalence of B2M somatic mutations in MSI CRC patients and relationship between B2M mutations and lymphocytes infiltration and other clinicopathological features as well as detection of methylation changes in B2M promoter region which can be another mechanism of immune escape. In our study, 37 MSI-H and 5 MSI-L patients were selected for screening of B2M mutational and methylation status. The characterization of patients was based on standard histopathological diagnosis and TNM classification; BRAF, KRAS mutations, tumor-infiltrating lymphocytes and peritumoral lymphoid reaction were also determined. MSI analysis was performed using fragment analysis. B2M mutations were identified by Sanger sequencing, and methylation of CpG islands in promoter region was detected by methylation-specific PCR. Heterozygous mutations in the B2M gene were detected in five MSI-H patients (13.5%), while the mutation c.45_48delTTCT was determined in four patients and mutation c.276delC was found in two patients. One of these five patients was compound heterozygote harboring both mutations. Methylation of the promoter region of the B2M gene was observed in one patient with MSI-H colorectal cancer. Detection of genetic and epigenetic changes in B2M gene could be important in personalized therapy for CRC patients as these changes may be one of the mechanisms of secondary resistance of MSI positive tumors to immunotherapy.

B2M Gene Expression Reflects an Immunologically Active Tumor Microenvironment in DLBCL

Introduction Interactions between tumor cells and the immune system play a critical role in regulating tumor development. Immune-based therapies have shown variable responses in diffuse large B-cell lymphoma (DLBCL) which suggests that the immune cell composition of the tumor microenvironment (TME) influences response to these agents. However, the key determinants of the immune TME are poorly understood. We have recently shown that indolent lymphomas can be stratified as immunologically 'hot' or 'cold' (Tobin et al J. Clin Oncol, in press) and sought to test for this in DLBCL. Also, given that beta-2-microglobulin (B2M) has a key role in antigen presentation and its loss is a frequent immune evasion mechanism in DLBCL, we determined the effect of B2M expression on the nature and magnitude of immune infiltration in the tumor microenvironment of DLBCL. Methods Ninety-seven de novo systemic DLBCL FFPE biopsies underwent targeted exon re-sequencing of B2M (Illumina) in addition to quantitative gene expression of Β2M, immune effector (CD4, CD8, CD56, CD137), immunosuppressive macrophage markers (CD68, CD163) and immune checkpoints (TIM3, LAG3, PD1 and PDL1) (Nanostring Technologies). B2M promoter methylation by mass array, immunohistochemistry for the above markers and high throughput T-cell receptor β sequencing (Adaptive Biotechnologies) were performed on a subset of cases. Genomic findings were validated in a whole exome and transcriptome cohort of approximately 1000 DLBCL samples (Reddy et al Cell 2017). Results Β2MMut were detected in 14/97 (14.4%) samples and had lower Β2M gene expression compared to Β2MWT (p = 0.0094) and all of these showed B2M protein loss. However, 29/40 (72%) of B2MWT samples tested also had B2M protein loss. There was no differential methylation of B2M promoter regions observed compared to lymph node controls. Results indicate that mechanisms other than mutation and methylation status contribute to loss of B2M surface expression and that a more comprehensive assessment of B2M expression within tumor tissues is achieved by B2M digital gene quantification. Consistent with this, there were no significant differences in expression of intra-tumoral immune markers between B2MMut and B2MWT tissues, whereas gene expression of B2M was significantly associated and positively correlated with the gene expression of CD4, CD8, CD56, CD137, CD68, CD163, PD1, PDL1, LAG3 and TIM3 (all p &lt;0.01) and with the protein expression of CD8, CD56, CD137, PDL1 and LAG3 (all p &lt;0.01), irrespective of their classification as an immune-effector, immune-checkpoint or macrophage markers. These observations are consistent with a co-ordinately regulated immune response, indicating an adaptive immune-checkpoint response to regulate immune-effector activation. In keeping with this, the housekeeper genes did not correlate with immune gene expression indicating that high or low co-ordinate expression of immune genes was not reflecting tissue RNA quality or quantity (Fig 1). Next, we tested the discovery cohort for relationships with the TCR repertoire. High B2M gene expression was significantly associated with reduced TCR diversity (p = 0.0101) compared to low B2M gene expression, suggesting that clonal T cell expansions are more likely with intact antigen presentation. The validation cohort also demonstrated that B2M gene expression correlated with immune cell infiltration and additionally showed that B2M positively correlated with the gene expression of HLA Class I//II molecules and a range of regulatory, transport and assembly molecules involved in the antigen presentation machinery pathway. However, no differential survival benefit was observed in patients with high versus low B2M. Conclusions In summary, digital gene expression is a robust measure of B2M quantification in the TME. Our data show that high B2M gene expression reflects an immunologically active or 'hot' tumor microenvironment in DLBCL characterised by higher levels of immune cell infiltration. These findings indicate that B2M gene expression level could be used as a biomarker of an active intra-tumoral immune response in DLBCL. Further studies are required to determine if B2M gene expression may have a role in stratifying the selection of patients in whom immune-based therapies are more likely to be effective. Disclosures Gould: NovoNordisk: Other: Travel funding - domestic flights to attend education, May 2018. Keane:MSD: Consultancy; BMS: Research Funding; Celgene: Consultancy; Gilead: Consultancy; Roche: Consultancy, Other: Travel Grant. Hertzberg:Takeda: Consultancy, Honoraria; MSD: Consultancy; Roche: Consultancy, Honoraria. Gandhi:Gilead: Honoraria, Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Honoraria, Other: Travel Support; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria.

Measurable Residual Disease (MRD) Monitoring in Acute Myeloid Leukemia (AML) with t(8;21)(q22;q22.1) RUNX1-RUNX1T1 Identifies Patients at High Risk of Relapse: Results of the AML Study Group (AMLSG)

Measurable Residual Disease (MRD) Monitoring in Acute Myeloid Leukemia (AML) with t(8;21)(q22;q22.1) RUNX1-RUNX1T1 Identifies Patients at High Risk of Relapse: Results of the AML Study Group (AMLSG)

Serial ultra-deep sequencing of circulating tumor DNA reveals the clonal evolution in non-small cell lung cancer patients treated with anti-PD1 immunotherapy.

BackgroundImmune‐therapy with anti‐PD1 inhibitors, such as pembrolizumab, is revolutionizing the treatment of non‐small cell lung cancers (NSCLC). However, identifying patients for the potential therapeutic response and predicting therapy resistance and early relapse remains a challenge.MethodsBetween 2016 and 2018, 60 patients were treated with pembrolizumab, among who 12 NSCLC patients had both baseline (before treatment) and serial (on treatment) periodical circulating tumor DNA (ctDNA) samples. Those samples were sequenced on a 329 pan cancer‐related gene panel. Analyses of tumor burden, blood tumor mutational burden (bTMB), maximum somatic allele frequency (MSAF), and tumor clonal structure were performed in association with clinical response. Candidate resistance mutations involved in relapse and metastases were further investigated.ResultsctDNA was detected and mutational profiling was performed for each patient. Those with a high baseline bTMB level showed significantly improved progression‐free survival (PFS) after pembrolizumab treatment. Tumor burden and therapeutic response significantly correlated with the MSAF instead of the bTMB. Clone analysis detected tumor progression about 2‐4 months ahead of computed tomography (CT) scan. One mutation in gene PTCH1 (Protein patched homolog 1) and two acquired anti‐PD1 candidate resistance mutations of gene B2M (β2 microglobulin) were identified in association with distant metastasis. The evolutionary tree of a representative patient was also described.ConclusionThis pilot study showed that MSAF could be another good indicator of therapeutic response, and clonal analysis could be clinically useful in monitoring clonal dynamics and detecting remote metastasis and early relapse.

Optimized Biobanking Procedures for Preservation of RNA in Tissue: Comparison of Snap-Freezing and RNAlater-Fixation Methods.

Introduction: Personalized treatment, supported by biomarkers, would improve survival of ovarian cancer patients. RNA molecules are potentially important biomarkers. The Danish CancerBiobank provides an infrastructure for handling and storage of biological material, including RNA, from Danish cancer patients. The aim of this study was to investigate the effects of handling-time and fresh-freezing versus RNAlater® fixation on RNA degradation in solid tissue from pelvic mass samples. Materials and Methods: We evaluated RNA quality in surgical tissue from patients with a pelvic mass. Corresponding samples were either fresh-frozen or fixed in RNAlater, at eight different time points after the surgery. Integrity was measured using a bioanalyzer, and the amount and quality were further investigated by quantitative reverse transcription-polymerase chain reaction measuring the expression of housekeeping genes B2M and HPRT1. Results: Our results show that tissue RNA is stable up to at least 180 minutes after the surgery, as the quality was comparable to the quality of RNA handled immediately. Likewise, patient RNA was of acceptable quality after both fresh-frezing and RNAlater fixation, but RNAlater fixation was slightly more effective for RNA preservation. Discussion and Conclusion: Our data suggest that RNA in pelvic mass samples is relatively stable. Knowledge about RNA stability is an important prerequisite for research in RNA biomarkers, where the challenge is to balance the need for careful RNA handling and storage with the need for effective large-scale biobanking in a busy clinical setting where patient treatment is the main priority.

646P - Mutation analysis of B2M gene in colorectal cancer patients with microsatellite instability

BackgroundMutations in genes involved into antigen presentation such as B2M are enriched among colorectal cancer (CRC) patients with high microsatellite instability (MSI-H) and can become negative predictors for checkpoint inhibitor therapy. The aim of this study was the determination of prevalence of B2M somatic mutations in MSI-H CRC patients and relationship between B2M mutations and lymphocytes infiltration and other clinicopathological features. We focused also for methylation status of B2M promoter as another possible mechanism of gene expression downregulation. MethodsFrom our study group enrolling 420 CRC patients, 37 MSI-H and 5 MSI-L patients were selected for screening of B2M mutational status. The characterization of patients was based on standard histopathological diagnosis, TNM classification, tumor-infiltrating lymphocytes and peritumoral lymphoid reactions were determined. MSI analysis was performed using fragment analysis. BRAF, KRAS and B2M mutational status were identified by Sanger sequencing. For methylation analysis of B2M promoter region was used nested methylation specific PCR. ResultsB2M mutations were detected in 5 MSI-H patients (13.5%). In four cases, heterozygous mutation c.45_48delTTCT located in exon 1 was present and 2 patients exhibited heterozygous mutation c.276delC in exon 2. From group of 5 MSI-H patients, one case was compound heterozygote for both these mutations. Methylation of B2M promoter was observed only in 1 MSI-H case from our study group. ConclusionsThe presence of B2M mutations should be correlated with the response to the checkpoint inhibitor therapy in order to improve patient’s stratification and also development of new immunotherapeutic approaches. Our pilot study recommends detection of CpG methylation in B2M gene for the future studies in the area of colorectal cancer. Editorial acknowledgementBiomedical Center Martin (ITMS 26220220187), APVV-16-0066 and VEGA 1/0380/18. Legal entity responsible for the studyThe authors. DisclosureAll authors have declared no conflicts of interest.

P2.04-53 Serial Ultra-Deep Sequencing of ctDNA Reveals the Clonal Evolution in Non-Small Cell Lung Cancer Patients Treated with Pembrolizumab

Immune-therapy with anti-PD1 inhibitors, such as pembrolizumab, is revolutionizing the treatment of non-small cell lung cancers (NSCLC). However, identifying patients for the potential therapeutic response and predicting therapy resistance and early relapse remains a challenge. Between 2016 and 2018, 60 patients were treated with pembrolizumab, among who 12 NSCLC patients had both baseline (before treatment) and serial (on treatment) periodical circulating tumor DNA (ctDNA) samples. Those samples were sequenced on a 329 pan cancer-related gene panel. Analyses of tumor burden, blood tumor mutational burden (bTMB), maximum somatic allele frequency (MSAF), and tumor clonal structure were performed in association with clinical response. Resistance mutations involved in relapse and metastases were further investigated. ctDNA was detected and mutational profiling was performed for each patient. Those with a high baseline bTMBlevel showed significantly improved progression-freesurvival (PFS) after pembrolizumab treatment. Tumor burden and therapeutic response significantly correlated with the MSAF instead of the bTMB. Clone analysis detected tumor progression about 2-4 months ahead of computedtomography (CT) scan. One mutation in gene PTCH1 (Protein patched homolog 1) and two acquired anti-PD1 resistance mutations of gene B2M (β2 microglobulin) were identified in association with distant metastasis. The evolutionary tree of a representative patient was also described. This pilot study showed thatMSAF could be another good indicator of therapeutic response, and clonal analysis could be clinically useful in monitoring clonal dynamics, and detecting resistance and early relapse.

Characterization of the continuous skin fibroblastoid cell line, WE‐skin11f, from walleye (Sander vitreus)

A walleye dermal fibroblastoid cell line, WE-skin11f, was established and characterized. WE-skin11f was immunocytochemically positive for two known dermal fibroblast protein markers: vimentin and collagen I. At passage 26, WE-skin11f cultures contained both diploid and aneuploid populations. Ascorbic acid was required to produce extracellular collagen I fibres. Both of the skin fibroblastoid cell lines, WE-skin11f and rainbow trout-derived RTHDF, were not as good as the walleye caudal fin fibroblastoid cell line, WE-cfin11f, at forming abundant dense extracellular collagen matrices. The thermobiology of WE-skin11f was similar to that of other walleye cell lines with 26°C showing best temperature for growth and 4°C showing no growth but 100% viability. The transcript levels of b2m and mhIa genes of the major histocompatibility class I receptor in WE-skin11f were largely similar at all temperatures examined (4, 14, 20 and 26°C). Cortisol had a variety of effects on WE-skin11f cells: growth inhibition, morphological change from fibroblastoid to epithelioid, and enhancement of barrier function. Treatment of WE-skin11f cells with the physiologically relevant concentration of 100ng/ml cortisol inhibited collagen I synthesis and matrix formation. Thus, WE-skin11f cell line could be useful in fish dermatology, endocrinology, and immunology research.

Safety and Biodistribution of Human Bone Marrow-Derived Mesenchymal Stromal Cells Injected Intrathecally in Non-Obese Diabetic Severe Combined Immunodeficiency Mice: Preclinical Study.

Mesenchymal stromal cells (MSCs) have potent immunomodulatory and neuroprotective properties, and have been tested in neurodegenerative diseases resulting in meaningful clinical improvements. Regulatory guidelines specify the need to perform preclinical studies prior any clinical trial, including biodistribution assays and tumourigenesis exclusion. We conducted a preclinical study of human bone marrow MSCs (hBM-MSCs) injected by intrathecal route in Non-Obese Diabetic Severe Combined Immunodeficiency mice, to explore cellular biodistribution and toxicity as a privileged administration method for cell therapy in Friedreich's Ataxia. For this purpose, 3 × 105 cells were injected by intrathecal route in 12 animals (experimental group) and the same volume of culture media in 6 animals (control group). Blood samples were collected at 24h (n = 9) or 4months (n = 9) to assess toxicity, and nineorgans were harvested for histology and safety studies. Genomic DNA was isolated from all tissues, and mouse GAPDH and human β2M and β-actin genes were amplified by qPCR to analyze hBM-MSCs biodistribution. There were no deaths nor acute or chronic toxicity. Hematology, biochemistry and body weight were in the range of normal values in all groups. At 24h hBM-MSCs were detected in 4/6 spinal cords and 1/6 hearts, and at 4months in 3/6 hearts and 1/6 brains of transplanted mice. No tumours were found. This study demonstrated that intrathecal injection of hBM-MSCs is safe, non toxic and do not produce tumors. These results provide further evidence that hBM-MSCs might be used in a clinical trial in patients with FRDA.

Abstract 1052: The human immune system reconstituted B-NDG mouse models are ideal tools for CAR-T and therapeutic antibody preclinical efficacy evaluation

Abstract The goal of our study is to establish mouse models for fast in vivo efficacy studies of biologics, such as CAR-T and therapeutic antibody candidates. B-NDG mice (NOD-Prkdcscid Il2rgtm1/Bcgen) were independently developed by Biocytogen to knockout the IL2rg gene in the NOD-SCID mouse background. Here we demonstrated that B-NDG, as one of the most severely immunodeficient mice, completely lack mature T, B, and NK cells, and were deficient in cytokine signaling. We have established more than 24 CDX models using B-NDG mice, including human solid tumor and blood tumor cell lines, such as bladder carcinoma, breast cancer, colon carcinoma, colorectal adenocarcinoma, glioblastoma, liver cancer, lung carcinoma, and various leukemia and lymphoma cell lines. We have previously demonstrated that B-NDG mice inoculated with human tumor cells can be used for CAR-T therapy evaluation. In addition, B-NDG mice are one of the best models for human immune system reconstitution using either PBMC or CD34+ hematopoietic stem cells. Human PBMCs reconstitute very well in B-NDG mice, with the percentage of hCD45+ cells reaching well over 50% within five weeks; moreover, the majority of these cells were CD3+ T lymphocytes. Human PBMC reconstituted B-NDG mice have also been successfully used for multiple therapeutic antibody efficacy studies. In order to reduce the GvHD, we have generated B-NDG B2M mice, on which the B2M gene was knocked-out. B-NDG B2M mice have a longer therapeutic window for PBMC reconstitution when compared with B-NDG mice, and have been successfully used for antibody efficacy studies. Other than PBMCs, human CD34+ hematopoietic stem cells have been successfully grafted in B-NDG mice, in which all major human myeloid and lymphoid lineage cell types were fully developed, reconstituting a functional human immune system. At 10 weeks post CD34+ cell implantation, the percentage of hCD45+ cells reached over 25%, containing human T, B, and NK cells. These CD34+ reconstituted B-NDG mice are powerful tools for the fields of hematopoiesis, immunology, and autoimmunity, as well to evaluate clinically-relevant therapeutic antibody drugs in vivo. Without any significant rejection of human-derived cells, the B-NDG mouse becomes an ideal model for transplantation of PDX, which retain the high heterogeneic properties typical of human tumor tissues. Using B-NDG mice, we have successfully established more than 300 PDX models, covering breast, colon, gastric, leukemia, lung, pancreatic cancers, and particularly blood tumor samples. Here we provide examples that use human immune reconstituted B-NDG PDX models to successfully evaluate the in vivo efficacy of human immune checkpoint antibodies in mono-or in combination therapy. In conclusion, we have developed more powerful and predictive mouse models for preclinical pharmacological evaluation of CAR-T and therapeutic antibodies. Citation Format: Qingcong Lin, Madeline Lee, Yanan Guo, Tian Gan. The human immune system reconstituted B-NDG mouse models are ideal tools for CAR-T and therapeutic antibody preclinical efficacy evaluation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1052.

Abstract 746: Genomic alteration of Chinese dMMR/MSI-H gastric cancer

Abstract Background: Gastric cancer is a high incidence malignancy often diagnosed at an advanced stage with limited therapeutic options and poor prognosis. A small fraction of gastric cancer with dMMR/MSI-H has extraordinary response to the immune checkpoint inhibitor treatment attributing to the immunogenicity generated by the elevated tumor mutation burden. At the meantime, the extensive genomic variants also cause profound impact on many other cancer related genes. Thus the understanding of dMMR/MSI-H gastric cancer genomic profiling is urgent for exploring clinical strategy to this cohort. Methods: Formalin Fixed Paraffin Embedded (FFPE) samples of 30 Chinese gastric cancer patients were collected for 450 gene panel based next-generation sequencing (NGS) assay. Genomic alterations including single base substitution, short and long insertions/deletions, copy number variations, gene fusions and rearrangement were assessed. Microsatellite instability (MSI) status and tumor mutational burden (TMB) were also calculated by NGS algorithm. Results: There were 15 males (50%) and 15 females (50%) diagnosed as gastric cancer with a median age of 67 years old. The TMB value of this cohort ranged from 29 to 118 muts/Mb, with the median value of 58 muts/Mb. The most frequent genomic alterations in Chinese MSI-H gastric cancer patients were revealed as KMT2D (50%), RNF43 (50%), CIC (50%), PIK3CA (50%), KMT2C (43%), ACVR2A (40%), TP53 (30%), ATM (33%), and ERBB3 (33%). Truncation has been found the most frequent gene alteration among these top mutations due to the deficiency of mismatch repair system caused reading frame shift. Epigenetic modifier genes with functions of chromatin remodeling and histone methylation have been found extensively mutated in MSI-H gastric cancer. Among eighteen (60%=18/30) patients carrying truncations in ARID1A whose coding protein participates in chromatin remodeling, seven of them possess loss-of-function biallelic truncations. Moderate frequency of mutation also occur at other genes in chromatin remodeling such as ARID1B (23%), PBRM1 (17%) and SMARCA4 (26%). About 77% patients in this cohort carry at least one truncation in genes belonging to the histone lysine methyltransferase 2 (KMT2) family such as KMT2A, KMT2C and KMT2D. Mutations of B2M gene which are commonly associated with resistance to immune checkpoint inhibitor have been found in 23% MSI-H gastric cancer. About 66% of the patients in this cohort carrying mutations in FBXW7, PIK3CA, PTEN, STK11 and TSC may benefit from the mTOR inhibitors. Conclusions: In general, dMMR/MSI-H gastric cancers possess elevated and dispersive TMB value. Epigenetic modifier genes involving in chromatin remodeling and histone methylation mutate frequently in MSI-H gastric cancer. About 66% of the patients in this cohort may benefit from the mTOR inhibitors. Citation Format: Jia Wei, Jian Wang, Xiangshan Fan, Yue Wang, Yao Fu, Kai Wang, Nandie Wu, Qin Liu, Yang Yang, Weifeng Wang, Baorui Liu. Genomic alteration of Chinese dMMR/MSI-H gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 746.

Abstract 4581: High endothelial venules are associated with immune evasion and hereditary background in mismatch repair-deficient colorectal cancers

Abstract DNA mismatch repair (MMR)-deficient cancers can develop in the context of Lynch syndrome or sporadically. MMR-deficient cancers accumulate a high load of immunogenic frameshift peptide neoantigens as a consequence of insertion/deletion mutations at coding microsatellites (microsatellite instability, MSI). Whether enhanced lymphocyte recruitment contributes to the high immune infiltration and the pronounced local immune response typically observed in MSI colorectal cancers (CRCs) is not known. Here we analyzed the density of high endothelial venules (HEVs), postcapillary blood vessels specialized for lymphocyte trafficking, in MSI CRC of sporadic and hereditary origin. Immunohistochemical staining with rat monoclonal MECA-79 antibody was used to detect HEVs in tumor specimens from MSI (hereditary, n=20 and sporadic, n=28) and MSS CRCs (n=35). Lymph follicle-associated HEVs were normalized per mm2 of tumor and peritumoral area. Mutations of the B2M gene were determined by Sanger sequencing. Significantly elevated HEV densities were detected in MSI (n=48) compared to MSS CRCs (n=35) (0.09 vs 0.02 counts per mm2, p=0.0002). Lynch syndrome-associated MSI CRCs presented with significantly higher HEV densities compared to sporadic counterparts (0.15 vs 0.05 counts per mm2, p=0.025). This difference remained significant when age-matched groups of hereditary and sporadic MSI CRCs were compared. MSI CRCs with B2M mutations (n=12) presented with significantly higher HEV densities than their B2M-wild type counterparts (n=14, p=0.016). Our results demonstrate that a high density of HEVs correlates with the MSI phenotype in CRC, underlining the elevated local immune activation in these tumors. Within MSI CRC, Lynch syndrome was a predictor of high HEV counts, suggesting a likely role of lymphocyte recruitment in immune surveillance of Lynch syndrome CRCs. The observation of high HEV counts particularly in MSI CRCs harboring B2M mutations supports the model that immune evasion occurs particularly in an activated local immune environment. The prognostic value of HEVs in MSI CRCs should be addressed in the future studies. Citation Format: Aysel Ahadova, Pauline Pfuderer, Alexej Ballhausen, Ian Frayling, Ann Ager, Magnus von Knebel Doeberitz, Matthias Kloor. High endothelial venules are associated with immune evasion and hereditary background in mismatch repair-deficient colorectal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4581.

Abstract LB-011: Fit-for-purpose quantitative programmed cell death ligand -1 (PD-L1) droplet digital PCR assay development using PAXgene RNA blood samples

Abstract Purpose Programmed death ligand receptor 1, (PD-L1) is a key immune checkpoint inhibitory receptor expressed on activated tumor specific immune cells. While immunohistochemistry assays have been developed for the detection of PD-L1 expression in several cancer types for patient treatment decision, a blood-based test for PD-L1 expression evaluation would be beneficial in providing complimentary evidence for potential clinical application. This study developed a fit-for-purpose clinical biomarker assay for the detection of PD-L1 mRNA expression in blood using droplet digital polymerase chain reaction (ddPCR) methods. Study Procedure Taqman primer/probe assays were selected based on coverage of PD-L1 gene and were tested for linearity and efficiency using a cDNA construct. Four housekeeping genes were analyzed in positive control cell line (A549 treated with interferon gamma), negative control cell line (A549 untreated) and PAXgene blood from healthy volunteers. Real-time quantitative PCR method was tested first, following initial experiments there were concerns with the performance of our qPCR assay prompting the switch to ddPCR which offers absolute quantification and a lower limit of detection. Assay performance characteristics of the ddPCR assay were evaluated including linearity, limit of blank (LOB), limit of detection (LOD), and precision. Thirty-five healthy volunteer samples were evaluated to establish a baseline level of PD-L1 expression. Data Summary Conducting linearity comparison between Taqman primer/probe kits using cDNA titrations showed no significant difference in linearity (r2=0.99) or efficiency (~95%) among the three primer/probe kits. Housekeeping genes B2M, GUSB, RPLP0, and TBP were compared and GUSB was most consistently expressed between IFN gamma treated and untreated cells; it was selected for use thereafter. In ddPCR, the LOB was determined to be 0 copies and the LOD was determined to be 19 copies of PD-L1. The average intra-run coefficient of variation (CV) was 7.44% and average inter-run CV was 7.70%. Analysis of healthy control samples yielded an average of 2415 PD-L1 copies with a range of 768-7510 copies. Treatment of A549 cells with IFN gamma resulted in a 6.7-fold increase in PD-L1 expression (21,580 copies in untreated cDNA versus 145,000 copies in treated cDNA). Conclusion Our fit-for purpose clinical biomarker assay development efforts have demonstrated that the ddPCR assay is capable of reliably and accurately detecting PD-L1 in PAXgene blood with low limit of detection and minimal inter-and-intra run variability. Compared to real-time PCR, ddPCR is a purely quantitative and offers a much broader dynamic range and provides more sensitive detection of PD-L1 in blood. Further evaluation and comparison of cancer to healthy volunteer samples is underway for more information on assay performance and disease biology. Citation Format: Dennis O'Rourke, Danyi Wang, Maria Cusano, Jorge F. Sanchez-Garcia, Ti Cai, Juergen Scheuenpflug, Zheng Feng. Fit-for-purpose quantitative programmed cell death ligand -1 (PD-L1) droplet digital PCR assay development using PAXgene RNA blood samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-011.

Effect of exonic microsatellite instability of B2M on the predictability of MSI/dMMR for immunotherapy.

2576 Background: Microsatellite instability/mismatch repair deficiency (MSI/dMMR) has been approved as a biomarker for immune checkpoint inhibitor therapy (ICI) . However, the deficiency of mismatch repair system also caused wide-spreading insertion/deletion (indel) mutations in the exonic microsatellite sites of critical immunotherapy related genes such as β-2-macroglobulin (B2M) whose product is critical to antigen presentation. Cases of aquired resistance to ICI treatment caused by the inactivation of B2M have been reported. Therefore, we investigated the mutational profile of B2M across the MSI/dMMR high prevalent cancers such as colorectal cancer (CRC), gastric cancer (GC) and endometrial cancer (EC). Methods: FFPE tumor samples from 37 CRC, 46 GC and 25 EC patients with matched normal tissues were collected for next-generation sequencing (NGS)-based 450 genes panel assay. Genomic alterations including single base substitution, short and long insertions/deletions, copy number variations, and gene rearrangement and tumor mutational burden were assessed. Immunohistochemistry (IHC) with antibody against B2M were performed on available samples to estimate the expression and localization of these proteins. Results: Exonic microsatellite sites of B2M gene have been found instable in 51% (19/37) of CRC, 22% (10/46) of GC and 8% (2/25) of EC. MSI caused small indels at B2M coding region leads to reading frame shift and the production of nonfunctional truncated proteins. Biallelic frameshift mutations, causing non-functional proteins, were found in 47% (9/19) of CRC and 30% (3/10) of GC patients carrying Indels in B2M gene. IHC assays showed the impaired expression of B2M proteins due to frameshift mutations. In addition, NGS test confirmed concurrent mutation in B2M for a MSI-H CRC patient with primary resistance to forth-line anti-PD1 treatment. Conclusions: The extensive mutations in the coding region of genes caused by the dMMR render the cancer cell sensitive to ICI. However, the concomitant variants in exonic microsatellite sites of B2M gene may compromise the predictability of MSI/dMMR as an ICI biomarker. Biallelic mutation of B2M may cause primary resistance to ICI while the mono-allelic mutation of B2M may gain acquired resistance. Therefore, B2M status could be considered when using MSI/dMMR as a biomarker for ICI.

774 Analysis of the tumor reactivity of tumor-infiltrating lymphocytes in a metastatic melanoma lesion that lost MHC class I expression after anti-PD-1 therapy

Several mechanisms for delayed relapses in melanoma patients long after the initial positive responses to anti-PD-1 therapy have been suggested. These include the escape of tumor cells from the immune system. However, the tumor-specific reactivity of TILs in the escaped tumor lesion, especially after MHC-class-I- loss has not been well evaluated. A 55-year-old Japanese woman with metastatic melanoma in her left supraclavicular lymph node and left accessory nerve lymph node was started on treatment with anti-PD-1 antibody therapy. Tumor progression was prevented by the therapy for 12 months. However, then one metastatic lesion started to grow again despite ongoing response in another lesion and the absence of new metastatic sites. We found no remarkable differences in the phenotypes of freshly isolated TILs from the two sites. Immunohistochemistry showed that MHC class I expression was diminished in the relapsed lesion. The tumor cell line established from the lesion also lost MHC class I expression completely. The copy-number assay revealed a deletion of the B2M gene. When we transduced the normal B2M gene into these cells, we confirmed the recovery of MHC class I molecule expression on their surface. Next, we expanded TILs from the left accessory nerve lymph node lesion and, assessed their reactivity to the autologous tumor cell line, with or without B2M gene transduction. We verified that TILs derived from the relapsed lesion could strongly recognize the autologous tumor cells in a MHC-class I-dependent manner. Our report emphasizes the limitations of T-cell-based immunotherapy and highlights the importance of developing alternative strategies for such cases.

Targeted Disruption of HLA Genes via CRISPR-Cas9 Generates iPSCs with Enhanced Immune Compatibility.

Induced pluripotent stem cells (iPSCs) have strong potential in regenerative medicine applications; however, immune rejection caused by HLA mismatching is a concern. B2M gene knockout and HLA-homozygous iPSC stocks can address this issue, but the former approach may induce NK cell activity and fail to present antigens, and it is challenging to recruit rare donors for the latter method. Here, we show two genome-editing strategies for making immunocompatible donor iPSCs. First, we generated HLA pseudo-homozygous iPSCs with allele-specific editing of HLA heterozygous iPSCs. Second, we generated HLA-C-retained iPSCs by disrupting both HLA-A and -B alleles to suppress the NK cell response while maintaining antigen presentation. HLA-C-retained iPSCs could evade Tcells and NK cells invitro and invivo. We estimated that 12 lines of HLA-C-retained iPSCs combined with HLA-class II knockout are immunologically compatible with >90% of the world's population, greatly facilitating iPSC-based regenerative medicine applications.