Molecular mechanisms of late cancer relapse

The development of immunotherapy has proven to be a tremendous step toward successful cancer therapy. Despite this achievement, some patients relapse and develop resistance to the treatment. Understanding the molecular mechanisms underlying these failures is critical to increase the rate of positive treatment outcomes. Dr. Aude Chapuis (Clinical Research Division) has been treating cancer patients using adoptive T cell therapy, a strategy that isolates and expands T lymphocytes to recognize and eradicate tumor cells. In a recent study published in Nature Communications, Dr. Aude Chapuis and fellow Dr. Kelly Paulson, in collaboration with Dr. Paul Nghiem, investigated late relapse in two patients with Merkel cell carcinoma.

Merkel cell carcinoma is an aggressive skin cancer associated with infection by Merkel cell polyomavirus (MCPγV). The two patients were treated with a combination of adoptive T cell therapy and immune checkpoint inhibitor antibodies. In the first patient, CD8⁺ lymphocytes were isolated from the blood. CD8⁺ T cells recognizing a recently identified HLA-B restricted epitope from MCPγV were specifically expanded ex vivo and then infused back into the patient. The cells were injected twice with a 33-day interval, followed by regular anti-CTLA4 and anti-PD-1 checkpoint inhibitor antibodies to hamper tumor growth. The tumor size shrank by more than 90%. However, 22 months after cell infusion the patient relapsed.

At the time of relapse, the autologous transplanted T cells could still be detected and represented more than 25% of the total CD8⁺ T cells in the blood. This result excluded the hypothesis that loss of the transferred T cells might be responsible for the relapse. The targeted viral proteins were still expressed in the tumor tissues, suggesting that the cancer did not escape by loss of antigen.

With the help of Dr. Raphael Gottardo, the authors performed single cell RNA sequencing (scRNAseq), a technique that characterizes single-cell gene expression from thousands of cells in a sample. Peripheral blood mononuclear cells collected from the patient at different time points (pre-treatment, early post-treatment at day 27, responding post-treatment at day 376, relapse post-treatment at day 614) were analyzed. scRNAseq revealed that a population of cytotoxic CD8⁺ T cells detected at the time of response disappeared by the time of relapse. Activation markers such as IL32 were also lower in the relapse samples, suggesting that the transferred CD8⁺ T cells might not encounter the targeted antigen anymore. This was further corroborated by immunofluorescence analyses of biopsies showing a disappearance of CD8⁺ T cells from the tumor between the time of response and relapse.

Additional scRNAseq analyses of tumor samples demonstrated that the transcriptional landscape in cells from the surrounding microenvironment was similar before and after the relapse but significant changes were detected in the tumor cells. Importantly, a significant decrease in HLA-B antigen expression, recognized by the transferred T cells, was observed and might explain loss of T cells stimulation and recruitment to the tumor tissues. Similar observations were made in a second patient whose T cells were directed at HLA-A2 instead of HLA-B major histocompatibility complex.

In the absence of HLA gene mutations, DNA methylation provides a possible explanation for the gene repression. Indeed, ex vivo treatment of the tumor cells with hypomethylating agents was successful at restoring HLA-B gene expression. “We hope that these agents would be potentially applicable to patients, as they are already used in other oncology settings. We are eagerly testing the combination of these agents and T cells preclinically, with hopes to move this forward into the clinic when we have additional data about efficacy and likely safety”, explained Drs. Paulson and Chapuis.

More patients could be tested using scRNAseq: ”This is a super cool technology that allows us to look at the microenvironment all at once. We know that looking at tumor cells alone is not enough, and this escape mechanism identified is surely not the only new one out there for the finding”, explained the two researchers. When asked about future directions, Drs. Paulson and Chapuis replied that they were looking forward to “Testing ways to restore antigen presentation in tumors, to improve immunotherapies of all types” and “making more cell-based therapies that recognize antigens and epitopes presented by different HLA. If we put in effective T cells of multiple restrictions, it is much harder for the tumor to get around the T cells”.

Funding for this study was provided by the National Institutes of Health, Immunotherapy Integrated Research Center at the Fred Hutch, Damon Runyon, MCC gift fund at University of Washington, EMD Serono, and 10x Genomics.

Fred Hutch/UW Cancer Consortium faculty members Drs. Aude Chapuis, Paul Nghiem, Raphael Gottardo, Philip Greenberg and Jason Bielas contributed to this research.

KG Paulson, V Voillet, MS McAfee, DS Hunter, FD Wagener, M Perdicchio, WJ Valente, SJ Koelle, CD Church, N Vandeven, H Thomas, AG Colunga, JG Iyer, C Yee, R Kulikauskas, DM Koelle, RH Pierce, JH Bielas, PD Greenberg, S Bhatia, R Gottardo, P Nghiem, AG Chapuis. 2018. Acquired cancer resistance to combination immunotherapy from transcriptional loss of class I HLA. Nature communications. 9(1):3868.