Characterizing the role of tumor-specific B cells in Merkel cell carcinoma disease control

Abstract

Merkel cell carcinoma (MCC) is a rare neuroendocrine skin cancer with high recurrence and mortality rates. MCC is primarily driven by truncation and clonal integration of the Merkel cell polyomavirus (MCPyV) DNA into the host cell’s chromosomes. Viral integration leads to constitutive expression of the immunogenic T-antigen (T-Ag) oncoproteins, small and truncated large T-Ag, which promote MCPyV-driven MCC (VP-MCC). A second, less common form of non-viral MCC (VN-MCC) arises from accumulation of UV-mediated DNA mutations that affect tumor-suppressor genes. Independently of its origin, MCC is highly immunogenic and often recognized by T & B lymphocytes. Tumor-infiltrating and circulating cancer-specific T cells in MCC patients have been shown to be key promoters of tumor control. In contrast, the role B cells may play in anti-MCC tumor immunity remains unknown.In Chapter 1, we 1) describe MCC biology and therapy resistance, the most pressing issue in the field; 2) highlight the opportunity that MCC presents to investigate total and cancer-specific B cell responses across patients—the latter being extremely difficult to assess in most solid tumors; and 3) address how B cells may be harnessed to develop novel therapies aimed at improving MCC treatments for patients with refractory disease. In Chapter 2, we characterized total and cancer-specific B cell responses in 47 blood samples and 19 unmatched tumors from VP-MCC patients. MCC patient blood data revealed circulating B cell phenotypes that correlate with MCC progression only in female patients, and independently of specificity for MCC viral oncoproteins. In contrast, data from B cells in tumors revealed a strong association between high frequencies of viral oncoprotein-specific antibody-secreting cells and long-term MCC control. These MCC-specific antibody-secreting cells are primarily derived from germinal center B cells, whose detection in tumors also associated with improved disease control. In line with these findings, we identified higher frequencies of follicular helper CD4+ T cells in VP-MCC tumors from patients with better MCC outcomes. Finally, we demonstrated in vitro that B cells engineered to be specific for viral oncoproteins increase the sensitivity of oncoprotein-specific CD4+ T cells by over 50-fold. Together, these results suggest that synergy of viral oncoprotein-specific B and CD4+ T cell responses may promote MCC anti-tumor immunity. Given the association between MCC-specific antibody-secreting B cells and patient outcomes, Chapter 3 explores the T-antigen epitopes recognized by oncoprotein-specific antibodies in VP-MCC patient blood. Mapping of these antibodies revealed an association between preferential binding (immunodominance) against conformational epitopes on the “commonT” domain shared by all T-Ag isoforms and better MCC control. Importantly, this observation was lost when antibody binding against T-Ag linear epitopes was assessed. In line with these results, we detail a patient with progressive VP-MCC and no oncoprotein-specific serum antibody immunodominance, whose tumor was marked by a large expansion of antibody-secreting cells against unique domains of the large T-Ag and no detectable B cells against commonT. These results suggest that B cell binding to specific oncoprotein epitopes impact their ability to promote anti-tumor immunity. The importance of the adaptive immune response against viral oncoproteins in MCC led us to explore in Chapter 4 whether the length of truncated large T-Ag in VP-MCC tumors associates with patient outcomes. The truncation site of large T-Ag is clonal within a given patient’s tumor and results in an oncoprotein length between 228 to 787 amino acids. We found that most patients present with tumors in which truncated large T-Ag is under 350 amino acids. Importantly, analysis of T-antigen DNA sequences from 40 MCC patients with associated clinical data revealed that patients with longer large T-Ag (above 350 amino acids) were significantly less likely to recur and survived longer. Together, these data suggest that increased large T-Ag length may promote MCC immunogenicity as evidenced by lower frequency at presentation and improved disease control after diagnosis. Chapter 5 describes a patient with a lymph node invaded by VP-MCC and Chronic Lymphocytic Leukemia (CLL), a B cell malignancy. Interestingly, this patient has remained MCC- and CLL-free for over 10 years following tumor surgical excision and local radiation. Single-cell RNA-sequencing revealed that malignant B cells from CLL may enhance MCC tumor growth via cytokine-enhanced cell proliferation. This observation highlights a potential mechanism by which aberrant B cells may promote MCC tumorigenesis and progression. Finally, Chapters 6 and 7 describe additional immune mechanisms of MCC control and propose mechanistic studies to probe the role of B cells in MCC anti-tumor immunity. In Chapter 6, we show that certain subsets of myeloid cells in MCC tumors associate with progressive disease. Chapter 7 speculates how the newly developed “SLAP” MCC mouse can be used to test MCC-specific B cell responses in tumor control. Specifically, we propose using classic immunological approaches such as genetic knockout, depletion of immune cell types, and adoptive transfer of engineered B cells specific for the T-antigens to address the role of B cells in MCC. Collectively, our work provides the first in-depth analysis of cancer-specific B cell responses in MCC. By integrating clinical outcomes, immunophenotyping, antibody specificity, and viral oncoprotein structure, these studies reveal that the quality and epitope specificity of B cell responses may have a key role in controlling anti-tumor immunity. We conclude by summarizing how our findings lay the groundwork for mechanistic studies in MCC mouse models that may form the basis for future therapeutic strategies leveraging B cells against solid cancers.