Germline homozygosity and allelic imbalance of HLA-I are common in esophagogastric adenocarcinoma and impair the repertoire of immunogenic peptides.

BACKGROUND: The individual HLA-I genotype is associated with cancer, autoimmune diseases and infections. This study elucidates the role of germline homozygosity or allelic imbalance of HLA-I loci in esophago-gastric adenocarcinoma (EGA) and determines the resulting repertoires of potentially immunogenic peptides. METHODS: HLA genotypes and sequences of either (1) 10 relevant tumor-associated antigens (TAAs) or (2) patient-specific mutation-associated neoantigens (MANAs) were used to predict good-affinity binders using an in silico approach for MHC-binding (www.iedb.org). Imbalanced or lost expression of HLA-I-A/B/C alleles was analyzed by transcriptome sequencing. FluoroSpot assays and TCR sequencing were used to determine peptide-specific T-cell responses. RESULTS: We show that germline homozygosity of HLA-I genes is significantly enriched in EGA patients (n=80) compared with an HLA-matched reference cohort (n=7605). Whereas the overall mutational burden is similar, the repertoire of potentially immunogenic peptides derived from TAAs and MANAs was lower in homozygous patients. Promiscuity of peptides binding to different HLA-I molecules was low for most TAAs and MANAs and in silico modeling of the homozygous to a heterozygous HLA genotype revealed normalized peptide repertoires. Transcriptome sequencing showed imbalanced expression of HLA-I alleles in 75% of heterozygous patients. Out of these, 33% showed complete loss of heterozygosity, whereas 66% had altered expression of only one or two HLA-I molecules. In a FluoroSpot assay, we determined that peptide-specific T-cell responses against NY-ESO-1 are derived from multiple peptides, which often exclusively bind only one HLA-I allele. CONCLUSION: The high frequency of germline homozygosity in EGA patients suggests reduced cancer immunosurveillance leading to an increased cancer risk. Therapeutic targeting of allelic imbalance of HLA-I molecules should be considered in EGA.

Immune responses against shared antigens are common in esophago-gastric cancer and can be enhanced using CD40-activated B cells.

BACKGROUND: Specific immune response is a hallmark of cancer immunotherapy and shared tumor-associated antigens (TAAs) are important targets. Recent advances using combined cellular therapy against multiple TAAs renewed the interest in this class of antigens. Our study aims to determine the role of TAAs in esophago-gastric adenocarcinoma (EGA). METHODS: RNA expression was assessed by NanoString in tumor samples of 41 treatment-naïve EGA patients. Endogenous T cell and antibody responses against the 10 most relevant TAAs were determined by FluoroSpot and protein-bound bead assays. Digital image analysis was used to evaluate the correlation of TAAs and T-cell abundance. T-cell receptor sequencing, in vitro expansion with autologous CD40-activated B cells (CD40Bs) and in vitro cytotoxicity assays were applied to determine specific expansion, clonality and cytotoxic activity of expanded T cells. RESULTS: 68.3% of patients expressed ≥5 TAAs simultaneously with coregulated clusters, which were similar to data from The Cancer Genome Atlas (n=505). Endogenous cellular or humoral responses against ≥1 TAA were detectable in 75.0% and 53.7% of patients, respectively. We found a correlation of T-cell abundance and the expression of TAAs and genes related to antigen presentation. TAA-specific T-cell responses were polyclonal, could be induced or enhanced using autologous CD40Bs and were cytotoxic in vitro. Despite the frequent expression of TAAs co-occurrence with immune responses was rare. CONCLUSIONS: We identified the most relevant TAAs in EGA for monitoring of clinical trials and as therapeutic targets. Antigen-escape rather than missing immune response should be considered as mechanism underlying immunotherapy resistance of EGA.

Regulatory B10 cells display an altered homoeostasis in acute graft-versus-host disease.

OBJECTIVE: The role of B cells and the subgroup of IL-10 producing B cells, known to have a regulatory function, in patients following a haematopoietic stem cell transplant (alloSCT) has not been clearly understood to date. METHODS: We prospectively recruited 95 patients following an alloSCT and studied the B-cell reconstitution on days 30, 90 and 150. Regulatory B10 cells could be analysed in 22 consecutively recruited patients on day 30 post-transplant. RESULTS: The total B-cell percentages in transplant recipients (median 0.33; range 0.01-5.9) were significantly reduced than the controls (P = 0.0001) and constituted predominantly of transitional CD24high CD38high B cells. Regulatory B10 cells could be analysed in 22 consecutively recruited patients on day 30 post-transplant. The percentages of B10 cells (median 1.35; 0.0-4.5) were significantly reduced in the transplant recipients in comparison with the control cohort (P < 0.0001). Interestingly, the percentages of B10 cells in patients with acute GvHD (median 1.7; 0.33-4.5) were significantly higher than those without GvHD (median 0.7; 0-1.9) (P = 0.0003). CONCLUSION: This is the first report demonstrating B10 cells in stem cell transplant recipients in the early post-alloSCT (30 d) period. Our data suggest a possible role for B10 cells in the pathophysiology of acute GvHD. Further longitudinal studies are warranted to understand the implications of our findings.

Immunological effects in patients with steroid-refractory graft-versus-host disease following treatment with basiliximab, a CD25 monoclonal antibody.

Steroid-refractory graft-versus-host disease (GvHD) is a complication following an allogeneic stem cell transplantation with limited therapeutic options. Studies have shown a response in up to 80% of patients with this condition after treatment with the CD25 monoclonal antibody, basiliximab. Despite the good responses to treatment, around 50% of the patients experience recurrence of their GvHD symptoms 4-6 wk following cessation of therapy. The in vivo changes in the following treatment with this antibody have not been elucidated so far. We treated 14 patients with severe steroid-refractory GvHD with basiliximab weekly for 4 wk and monitored the changes in the T-, B-, NK- and dendritic cell subsets over this time period. The overall response to treatment was 92% (13/14) with 50% (7/14) achieving a complete response. Fifty four percentage (7/13) of the patients who responded showed recurrence of their GvHD symptoms. Contrary to expectations, our observations showed a significant depletion of the regulatory T-cell subset following treatment. Our findings suggest that the undesirable depletion of the regulatory T cells along with the CD25(+) acute inflammatory cells might be responsible for the high incidence of GvHD recurrence in this cohort of patients.