PMC7142242_01
Lack of acinar cells and presence of AQP5+ CK7+ hybrid epithelial structures in the parotid salivary gland following anti-PD-L1 therapy. (a,b,c,e) Ki67 staining in striated ducts of sicca control, pSS and post-anti-PD-L1 therapy tissue. Panels (c,d) (immunostained for high molecular weight cytokeratins (hmwCK) to mark SD cells, outlined in dashed line) show high resolution of area in inset in (b). (f-h) AQP5 and K7 double immunostaining of sicca control, pSS and post-anti-PD-L1 therapy tissue. Insets in (f) shows normal K7 and apical AQP5 staining patterns of intercalated ducts and acinar cell, respectively. Inset in (g) show acinar cell clusters in pSS with occasional CK7+ cell presence. Block arrows in (h) denote AQP5-K7 double-positive cells. (i) Quantification of AQP5 and CK7 cell content of epithelial cell structures following anti-PD-L1 blockade. (j) Quantification of composition of intercalated duct in sicca control tissue. (k) Quantification of composition of intercalated ducts in pSS tissue. (l,m) CD4 immunostaining. (o-q) CD8 immunostaining. All scale bars represent 50 mum, unless otherwise stated.
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The SDs of the sicca control patient demonstrated minimal presence of Ki67+ proliferative cells (Figure 1a). Proliferative epithelial cells, defined by immunostaining for Ki67 and high molecular weight cytokeratins (hmwCK) in serial sections, were detected in the pSS tissue (Figures 1b-d). No effect of anti-PD-L1 therapy was apparent on SDs, which displayed a phenotype similar to the sicca control and minimal proliferative cells, marked by Ki67+ (Figures 1a,e). AQP5+CK7- acinar cells with large cytoplasm:nucleus ratios and are easily identifiable (280/mm2 sicca control; 170/mm2 pSS; Figures 1f,g). AQP5 is localized apically in healthy acinar cell clusters (Figure 1f, inset). In pSS tissue, these conventional acini demonstrated some dysregulation of AQP5 localization, in line with other studies of the minor SGs, but maintained their large cytoplasm:nucleus ratio. Acinar clusters in pSS tissue also contained occasional CK7+ cells. (Figure 1g inset). In tissue following PD-L1 blockade, AQP5+CK7- acinar clusters with large cytoplasm:nucleus ratio and apically located AQP5 were not detectable (Figure 1h). The parenchyma post-PD-L1 blockade was instead dominated by mixture of AQP5+CK7-, AQP5-CK7+, and AQP5+CK7+ cell clusters, whereby AQP5 localization was homogenous throughout the cells and cells maintained a low cytoplasm:nucleus ratio (Figures 1h,i). Interestingly, "hybrid" epithelial structures comprising a mixture of AQP5+ and CK7+ cells comprised 70% of total epithelial cell clusters (33 structures /mm2; Figures 1h,j). IDs, 2 IDs/mm2; pSS 12%, 10/mm2; Figures 1j,k). In sicca control and pSS tissue, CD4+ and CD8+ T cells were detected sporadically in SG parenchyma (acinar cells plus IDs; Figures 1l,m,o,p). Both T cell subsets were present dispersed at visibly higher frequencies, both in-between and inside epithelial structures following anti-PD-L1 therapy (Figures 1n,q). In pSS tissue, p16+ cells were mostly located in striated ducts, in line with our previous studies (13/mm2 in SDs, 8/mm2 IDs, 9/mm2 acinar cells; Figures 2f,h). p16+ cells were present in SG tissue following anti-PD-L1 therapy at a frequency of 51/mm2, higher than both sicca control and pSS, of which 95% (equal to 48 cells/mm2) were located in the ID-like epithelial structures described in Figures 1, 2g,h. No LELs, germinal centers or IgG secreting plasma cells were observed in this biopsy (Figures 1p, 3, Table 1).
Hybrid epithelial structures following PD-L1 therapy are proliferative, potentially senescent and express PD-L1. (a-c) Ki67 immunostaining of sicca control, pSS and post PD-L1 therapy SG tissue. Inset in (c) shows high resolution image of boxed area in main panel. (d) Quantification of Ki67+ epithelial cell types (sicca control and pSS tissue) or hybrid epithelial structures (str; anti-PD-L1 blockade). (e-g) Immunostaining of sicca control, pSS and post-PD-L1 blockade tissue for p16. (h) Quantification of frequency of p16+ cells in epithelial cell types (sicca control and pSS tissue), and hybrid epithelial structures (str; anti-PD-L1 tissue). SDs, striated ducts; ID, intercalated ducts; Ac, acini clusters. (i) PD-L1 immunostaining in sicca control. (j) PD-L1 immunostaining in pSS tissue. (k) PD-L1 immunostaining in germinal center of pSS SG tissue, showing PD-L1+ lymphocytes. (l) Low resolution microscopy of PD-L1 immunostaining in patient taking durvalumab. (m) High resolution image of patient taking durvalumab, showing intensely PD-L1+ epithelial structures (white arrow), lightly positive epithelial structures (black arrow) and positive striated duct cells (block arrow). All scale bars represent 50 muM.
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In the sicca control and pSS parotid SGs, Ki67+ proliferating cells were detected at a total frequency of 6/mm2 and 24/mm2, respectively (Figures 2a,b). IDs, and acini) in sicca control tissue, and 66% (equal to 16 cells/mm2) in pSS tissue (Figure 2d). In tissue following anti PD-L1 therapy, 275 Ki67+ cells/mm2 were detected, of which 85% (equal to 230 cells /mm2) were located within the ID-like epithelial structures (Figures 2c,d). The remaining Ki67+ cells were located in SDs (Figures 2c,d). p16 can be used to denote senescent cells. p16+ cells were present at frequencies of 31/mm2 in sicca control tissue (Figures 2e,h), and were mostly stromal/non epithelial in nature. Indeed, p16+ cells were detected in pSS tissue at a total frequency of 37/mm2 (Figures 2f,h). In pSS tissue, p16+ cells were mostly located in striated ducts, in line with our previous studies (13/mm2 in SDs, 8/mm2 IDs, 9/mm2 acinar cells; Figures 2f,h). p16+ cells were present in SG tissue following anti-PD-L1 therapy at a frequency of 51/mm2, higher than both sicca control and pSS, of which 95% (equal to 48 cells/mm2) were located in the ID-like epithelial structures described in Figures 1, 2g,h. PD-L1+ cells were present in the SG parenchyma (Figures 2i,j). PD-L1+ presumptive dendritic cells were detected in the germinal center of pSS tissue (Figure 2k). In tissue following PD-L1 blockade, unconventional epithelial structures were either lightly or intensely PD-L1+ (Figures 2l,m). Striated ducts also demonstrated some PD-L1 positivity (Figure 2m).
The histology of a parotid salivary gland following prolonged anti PD-L1 therapy contains focal lymphocytic sialadentitis, but does not resemble the parotid gland in pSS. (a) CD45 immunohistochemical of sicca control. (b-d) CD45, CD20 and CD3 immunohistochemical staining of a parotid salivary gland from a pSS patient. (e) High resolution images of a lymphoepithelial lesion, whereby B cells are seen to invade striated ducts. Through as yet unclear mechanisms, both B cells and epithelial cells proliferate, and may represent a precursor stadium to MALT lymphomas. White dashed line demarcates boundaries of original striated duct. Inset box shows CD20+ B cells in between CD20- epithelial cells. (f) High resolution image of a germinal center, indicated by Bcl-6 positivity. Bcl-6 positive B cells are characteristic of germinal centers. Dashed white line outlines boundaries of germinal center. Inset shows Bcl-6-positive nuclei of B cells. (g) Double immunohistochemical staining for IgA and IgG classes of antibodies. IgA represents the isotype normally found in mucosal tissues such as the SG (red label). Where >80% of labeled cells are IgG positive (brown), class switching is assumed to have occurred, such as the present example. (h-n) CD45, CD20, CD8, and CD4 immunohistochemical staining of a parotid salivary gland from patient following prolonged anti-PD-L1 therapy. Insets in (h) shows high resolution of boxed area. (k) High resolution image of diffuse CD8 immunostaining. (l) High resolution image of focally organized CD8 immunostaining. (n) High resolution image of focally organized CD4 immunostaining. (o) High resolution image of focal CD8 immunostaining. (p) IgA IgG double immunostaining following prolonged anti-PD-L1 therapy, showing majority of pink, IgA class antibodies.
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SG histopathological findings (i.e., nature of sialadentitis, presence of LELs, IgG plasma cells, and germinal centers) in a patient with sicca following PD-L1 blockade to that observed in pSS, we performed immunohistochemical staining for infiltrating lymphoid cells. Sicca control parotid SGs show dispersed, scarce CD45+ cell presence, no focal CD45+ cell presence, no focal lymphocytic sialadentitis, no LELs, germinal centers or IgG plasma cell presence (Figure 3a). These features were all present in the parotid biopsy of the pSS patient examined (Figures 3b-g; clinical details in Table 1). CD45+ leukocytes were present throughout the biopsy post PD-L1 blockade, both periductally and dispersed between the epithelial cells of the parenchyma (Figure 3h). CD4+ T cells, in addition to considerable presence of CD8+ T cells, and a minor contribution to total infiltrate from CD20+ B cells (Figures 3i-o). CD4+ and CD8+ lymphocytes present were both diffusely dispersed through the tissue and focally clustered around the striated ducts, in a similar manner to pSS ("focal sialadentitis"; Figures 3k,l,n,o). No LELs, germinal centers or IgG secreting plasma cells were observed in this biopsy (Figures 1p, 3, Table 1).