Reversible downregulation of HLA class I in adenoid cystic carcinoma

Abstract

Background: Adenoid cystic carcinoma (ACC) is a rare, but lethal cancer with low response rates to systemic therapies, such as cytotoxic chemotherapy and immune-checkpoint inhibitors (ICIs). Despite extensive clinical trials, no effective treatments for patients with recurrent or metastatic ACC are available, and ACC mortality rates remain poor.

Methods: We employed automated multiplex immunofluorescence (mIF), single-cell RNA sequencing (scRNA-seq) Gene Expression analysis, RNA in-situ hybridization, and spatial transcriptomics analysis to characterize the immune landscape of ACC tumors, ACC metastasis, and normal tissues from regions where ACCs arise. Based on results from these studies, we treated freshly resected ACCs with interferon-γ or a stimulator of the interferon genes (STING) agonist in vitro. Additionally, we included one patient with ACC in a phase 1 clinical study of a novel STING agonist (dazostinag) plus pembrolizumab.

Results: The mIF analysis revealed that ACC tumors are immunologically "cold", with few tumor-infiltrating T-lymphocytes and low programmed death-ligand 1 (PD-L1) expression. The most striking finding was a very low beta-2-microglobulin (B2M) expression in nearly all ACCs, with only focal expression found in some ACC metastases. mIF and RNA sequencing analyses of normal salivary gland and breast tissues revealed a p63+, NFIB+, basal duct cell population, with similarly low B2M/human leukocyte antigen (HLA) class I expression. Spatial transcriptomics analysis of the focally B2M-positive ACC metastases uncovered the genetic pathway driving upregulation of B2M, an interferon-γ program mediating the reintroduction of HLA-I/B2M; the significantly upregulated genes included IRF1, GBP1, and TAP1. On short-term treatment of primary ACC tissues in vitro with interferon-γ or a STING agonist, we observed strongly upregulated HLA class I/B2M expression. Moreover, treatment of a patient with recurrent, metastatic breast ACC with a STING agonist and pembrolizumab led to a partial response with a 70% tumor reduction.

Conclusions: Low B2M/HLA class I expression may explain why ACCs are immunologically cold and the lack of response to ICIs. Our findings suggest that the normal cell of ACC origin exists in a B2M/HLA-class I low state, and that pharmacologic manipulation with immune activators, such as STING agonists, can restore HLA/B2M in ACCs, as supported by the promising response observed in a patient with metastatic ACC. These findings indicate a potential path to urgently needed immunotherapies.

Keywords: Head and Neck Cancer; Human leukocyte antigen - HLA; Immune modulatory; Major histocompatibility complex - MHC.

Figure 1. Multiplex immunofluorescence panel and immune landscape of ACC tumors and comparison cohort. (A) 10-plex immunofluorescence antibody panel of a representative ACC and (B) an HNSCC case. (C, D) Maps showing the spatial distribution of classified epithelial cancer cells and immune cells, plotted by the x-centroid y-centroid coordinates. Panels in (E) and (F) display expressions of individual immune cell markers on the same region of interest. (G) Complete list of protein targets detected by the multiplex immunofluorescence panel. Markers are used for cell classification, cell subtype classification, basal cell detection, and checkpoint marker expression stratification. (H) Percentage of immune cells (macrophages, cytotoxic T cells, T-helper cells, and B cells) to total cells in the tumor area. (I) Relative composition of immune cells. (J) Percentage of immune cells to total cells within the malignant epithelium. (K) Percentage of T cells to epithelial cells within the malignant epithelium. (L) Combined positive score (CPS) and tumor proportion scores (TPS) for PD-L1 expression. Cases with scores of TPS>1% and CPS>1 are labeled with*. ACC, adenoid cystic carcinoma; HNSCC, head and neck squamous cell carcinoma; PD-L1, programmed death-ligand 1.

Figure 2. B2M, HLA Class I, and NFIB Expression in ACCs and HNSCCs. (A) Comparative images for B2M expression obtained by the multiplex immune platform, manual immunohistochemistry, and B2M RNA-ISH on an ACC case showing tumor (T) and a surrounding lymph follicle (LF). (B–E) Immunohistochemical stains of ACC and HNSCC cases of B2M, HLA-class I, B2M RNA, and NFIB expression. Representative images are shown at 20× magnification. Scale bar, 100 µm. ACC, adenoid cystic carcinoma; B2M, beta-2-microglobulin; HNSCC, head and neck squamous cell carcinoma; HLA-class I, human leukocyte antigen class I; IHC, immunohistochemistry; NFIB, nuclear factor I B; RNA-ISH, RNA in-situ hybridization.

Figure 3. Single-cell RNA sequencing analysis of normal salivary gland tissue and ACC reveals a B2M-low population of basal cells. (A) UMAP representation of reanalyzed scRNA-seq data from tumor-only cells of a single ACC case. Cells with matches to known cell types are labeled by color and tumor epithelial cells are circled in red. (B) UMAP of the cells represented in 3A, labeled with cluster IDs produced with Seurat’s Louvain algorithm. (C) UMAPs identifying cells within subclusters (labeled in 3B) with basal-like expression of TP63+, ACTA2+, and NFIB+, luminal-like expression of KIT+, as well as cells with particularly low expression of B2M (arrow, C3). (D) Box-plot quantification of transcripts across subclusters, highlighting low B2M expression in clusters C3 and C4. (E) UMAP representation of reanalyzed Human Protein Atlas scRNA-seq data of normal salivary gland tissue. Cells with matches to known cell types are labeled via color, with salivary duct cells boxed in yellow. (F) UMAP of the replotted salivary duct cells highlighted in 3E, labeled with cluster IDs produced with Seurat’s Louvain algorithm. (G) UMAPs identifying cells within subclusters (labeled in 3F), with basal-like expression of TP63+, luminal-like expression of KIT+, as well as cells with particularly low expression of ACTA2 and B2M. (H) Box-plot quantification of transcripts within salivary duct cells showing TP63 positive cells have lower expression of B2M. ns, not significant; ***p<0.001. ACC, adenoid cystic carcinoma; scRNA-seq, single-cell RNA sequencing; UMAP, Uniform Manifold Approximation and Projection.

Figure 4. NFIB+/p63+/CK5+basal duct cells express low HLA/B2M. (A) Representative images of a normal breast gland duct with p63, SMA, NFIB, and HLA staining, (B) with NFIB and HLA staining, and (C) with p63 and SMA staining. (D) Representative image for p63, CK5, NFIB, and HLA-class I immunofluorescence analysis in a normal salivary gland duct. (E) p63 and HLA-class I stains highlight low HLA-class I expression in p63+ basal cells. (F) Centroid map of normal salivary gland duct depicting the p63− duct cells (blue) and p63+ duct cells (orange). (G) Box plots showing quantification for all normal ducts on tissue slide. p63+ duct cells are significantly lower in HLA and B2M expression. (H) Representative image for p63, SMA, NFIB, and HLA-class I immunofluorescence analysis in a normal breast gland duct. SMA and p63 staining highlight myoepithelial/basal cells in pink and yellow. (I) p63 and HLA-class I stains highlight low HLA-class I expression in p63+ myoepithelial/basal cells. (J) Centroid map of normal breast gland duct depicting the p63− duct cells (blue) and p63+ duct cells (orange). (K) Box plots showing quantification for all normal ducts on tissue slide. p63+ duct cells are significantly lower in B2M and HLA expression. *p<0.05; ***p<0.001. B2M, beta-2-microglobulin; HLA, human leukocyte antigen; NFIB, nuclear factor I B; SMA, smooth muscle actin.

Figure 5. Analysis of Visium HD data for ACC24. (A) UMAP representation of Visium HD 8 µm bin-level expression data colored by cluster. (B) Heatmap showing scaled expression of select cluster marker genes (genes significantly upregulated with logFC>1 for a given cluster vs all others). (C) UMAP colored by log-normalized B2M expression. (D) Bins colored by log-normalized B2M expression in their original spatial location overlaid on H&E. (E) Volcano plot depicting results of differential expression analysis between tumor cluster C5 and other tumor clusters (C0 and C3). The genes selected for IHC validation are labeled. (F) All tumor bins colored by cluster in their original spatial location overlaid on H&E. (G) Dot plot showing one representative GO:BP term from each of 15 biological process clusters derived from semantic similarity clustering of all GO:BP terms positively enriched by GSEA. The lowest p value term in each cluster was selected as representative for visualization purposes. (H) 3 of 143 significant GO:BP terms shown as individual GSEA plots, where the x-axis represents the genes in the logFC-ranked list and the y-axis shows the running enrichment score for the given gene set. (I–L) Immunohistochemical staining was performed to validate a subset of the identified upregulated genes in the HLA class I-high ACC tumor cells. 20× images of TAP1, IRF1, and GBP1 stains are shown for HLA class I-high and HLA class I-low regions in the metastasis ACC24. Scale bar, 100 µm. ACC, adenoid cystic carcinoma; B2M, beta-2-microglobulin; GO:BP, Gene Ontology biological process; GSEA, Gene Set Enrichment Analysis; HLA, human leukocyte antigen; IHC, immunohistochemistry; UMAP, Uniform Manifold Approximation and Projection.

Figure 6. Restoration of B2M expression with immune activator treatment. (A–E) Histochemical images of HLA-class I, B2M, B2M RNA in-situ hybridization, PD-L1, and NFIB of three ACC slice cultures treated with DMSO (control), interferon-γ, or STING agonist. Representative images are shown at 20× magnification. Scale bar, 100 µm. ACC, adenoid cystic carcinoma; B2M, beta-2-microglobulin; DMSO, dimethyl sulfoxide; HLA, human leukocyte antigen; IFN, interferon; NFIB, nuclear factor I B; PD-L1, programmed death-ligand 1; STING, stimulator of the interferon genes.

Figure 7. Patient with ACC with recurrent and metastatic disease treated with intravenous STING agonist, dazostinag, and pembrolizumab in the dose escalation cohort of a phase 1 clinical study. (A) Multiplex-immunofluorescence of the primary tumor of an index patient with ACC, showing the CK, MYB, NFIB, p63, SOX2, HLA-class I, B2M, and PD-L1 expression. Representative images are shown at 20× magnification. Scale bar, 100 µm. (B) CT scans of the patient with ACC with pulmonary metastasis at baseline and after a 9-month treatment course of intravenous STING agonist, dazostinag, in combination with pembrolizumab intravenous. The patient achieved a partial response, with a 70% reduction of the target lesion. ACC, adenoid cystic carcinoma; B2M, beta-2-microglobulin; HLA, human leukocyte antigen; NFIB, nuclear factor I B; PD-L1, programmed death-ligand 1; STING, stimulator of the interferon genes.