Mutually Exclusive Expression of COL11A1 by CAFs and Tumour Cells in a Large panCancer and a Salivary Gland Carcinoma Cohort

For the SGC cohort, all formalin-fixed paraffin-embedded (FFPE) SGC specimen, which were resected at the Department of Oto-Rhino-Laryngology, Head and Neck Surgery of the University of Cologne between 1998 and 2018 were retrieved from the archives of the Institute of Pathology, University Hospital of Cologne (n = 110). All cases were revised in detail by two pathologists (CA, AQ). Immunohistochemical staining for CK7, p63, NOR-1, S100, androgen receptor and HER2 as well as FISH break-apart analysis for MYB, MYBL1, MAML2 and ETV6 genes were employed in case the original diagnosis would not be unequivocal. For the panCancer cohort, 25 cases of each tumour type as well as five tissue blocks with the respective normal tissue were retrieved from the archives. These cases comprised the ten most prevalent carcinoma types according to the US National Cancer Institute [29] as well as the different lymphomas (Table S1). Cases were selected in order to mirror the actual relative prevalence of each grade and tumour subtype (e.g., growth pattern, ER positivity in case of breast carcinomas) while also including rare forms. If available, basic clinicopathological data as sex, age and TNM classification were recorded. Before TMA preparation, it was ensured that sufficient residual tumour material for possible complementary diagnostic tests would be available afterwards. TMAs were prepared as described before [25]. Briefly, four tissue cylinders in case of the SGC cohort and two tissue cylinders in case of the panCancer cohort were punched out from FFPE block and transferred to an empty paraffin block. All procedures were in accordance with the ethical standards of the University of Cologne and the Helsinki Declaration from 1975 and its revision in 1983. Patients gave their written informed consent to participate in this study.

The RNAscope assays were performed according to the manufacturer’s instruction, as previously reported [30]. Briefly, 5 μm sections were cut from FFPE blocks, pre-treated (30 min for pre-treatment 2 and 3), digested and hybridised at 40 °C with mRNA probes specific for human COL11A1 (each ready to use, Advanced Cell Diagnostics, Bio-Techne, Minneapolis, MN, USA) using a Ventana Discovery system (Roche, Switzerland). Subsequently, the slides were counterstained for 10 s in haematoxylin. Immunohistochemical staining of CD8 + T cells and p53 mutation status was carried out before for this cohort. Tumours were termed “inflamed” if the amount of CD8 + T cells exceeded > 100 per core [25].

CAFs_COL11A1 in the tumour tissue were morphologically identified. The percentage of COL11A1-positive stromal surface as indicator for CAFs_COL11A1 was recorded by two pathologists (C.A. and A.Q.) as follows: 0: < 1%; 1: 1–20%; 2: 21–50%; 3: > 50%; 4: > 50% and high staining intensity due to frequently merged RNA-ISH signals. As two (panCancer cohort) and four (SGC cohort) TMA-cores of 1 mm diameter were scored per case, the upper median was chosen as resulting score for further statistical analysis. The percentage of TC_COL11A1 was noted for each spot and the mean percentage was calculated. For binary analyses, cases with CAFs_COL11A1 score ≥ 1 or ≥ 1% TC_COL11A1 were designated stroma-positive or tumour cell-positive, respectively.

For the detection of the protein expression, TMAs with samples from 41 patients of the SGC cohort and from 100 patients of the panCancer cohort (breast, prostate, lung, colorectum n = 25 each) were used. Sample preparation, MSI data acquisition as well as data and imaging analysis were performed as previously published [28]. We detected one COL11A1 peptide after tryptic cleavage [(R)GEKGEAGPPGAAGPPGAK(G); m/z value: 1545.82]. From this peptide, four adducts, [M + H] + (1546.828 m/z), [M + NH4] + (1563.854 m/z), [M + Na] + (1568.81 m/z) and [M + K] + (1584.784 m/z) were recognised. As all adducts were distributed equally among the different tumour types, only the measurements for the [M + H] + adduct were used for further comparisons.

Statistical analysis was carried out with SPSS statistical software (IBM SPSS 25.0, Armonk, NY). Bar plots and dot plots were drawn with RStudio (R Foundation for Statistical Computing, Vienna, Austria.) and the R package ggplot2 [31]. Interdependencies between categorical variables were tested using Fisher’s exact test or Pearson’s Chi-square test depending of the group size. Correlations between ordinal variables were calculated with spearman’s rank correlation coefficient. A Kruskal–Wallis-Test with p value correction was employed for the comparisons of COL11A1 protein expression between different tumour types. P values below 0.05 were considered significant.

The panCancer cohort (n = 275) comprised the ten most prevalent carcinoma types as well as lymphomas (25 cases each). Additionally, five samples of normal tissue of each respective organ were included (n = 55). We measured the infiltration by CAFs_COL11A1 using a semiquantitative score (Score 0—4) that reflected the percentage of stromal surface stained by the COL11A1 RNA-ISH. Additionally, we assessed the percentage of tumour cells with COL11A1 staining (TC_COL11A1). CAFs_COL11A1 were almost exclusively observed in tumour tissues with an overall positivity rate of 34.5% compared to 1.6% in normal tissue (one positive endometrium sample). We observed dramatic differences of CAFs_COL11A1 infiltration across different primary sites (Fig. 1 and Table S1). The highest frequencies of positive tumours were noted for CRC (79%), MC (75%), gastric (48%) and esophageal carcinomas (44%), while only 8% and 4% of lymphomas and prostate carcinomas were infiltrated by CAFs_COL11A1. Interestingly, CRC, MC and esophageal carcinomas exhibited very similar frequencies of highly positive cases (Score 3), reaching 29.2%, 33.3% and 28%, respectively. Conversely, only 8% of all stomach carcinomas were assigned to this category. None of the cases from the panCancer cohort exhibited confluent stromal COL11A1 staining required for the maximum CAFs_COL11A1 Score of 4, which was observed in several SGC subgroups.

When all cases of the panCancer cohort were pooled (Table 1), the CAFs_COL11A1 score strongly correlated with tumour grade (p = 0.001) and tumour stage (p < 0.001). To a lesser extent, but still significantly, CAFs_COL11A1 score also correlated with nodal spread (p = 0.048). Moreover, a trend for a higher rate of lymphatic invasion and higher age and was observed for tumours with infiltration by CAFs_COL11A1 (p values: 0.067 and 0.081, respectively). The trend for a higher percentage of CAFs_COL11A1 infiltrated carcinomas among females (p = 0.078) resulted from the high percentage of MCs.

MC and CRC were infiltrated by CAFs_COL11A1 in over 50% of the cases and were consecutively studied in more detail. For MC (Table S2), a significant positive correlation with stromal CAFs_COL11A1 score was observed for tumour grade and Ki67 index (0.478, p = 0.018 and 0.656, p = 0.002, respectively). Also, the presence of CAFs_COL11A1 was associated with negativity for oestrogen receptor (ER), as 100% of ER- and only 54.5% of ER + MC were infiltrated by CAFs_COL11A1 (p = 0.011). In this subgroup analysis, no significant dependency between CAFs_COL11A1 score and progesterone or androgen receptor status or clinicopathological criteria was observed. Also, no significant association between CAFs_COL11A1 score and clinicopathological parameters, including microsatellite instability was observed for CRC. Of note, tumour cells with staining for COL11A1 (TC_COL11A1) were detected in one OC as well as one endometrial carcinoma with a percentage of 5% and 70% of tumour cells, respectively.

110 SGC were available for COL11A1 mRNA-ISH, containing 29 MuEp, 25 AdCy, 19 salivary duct carcinomas (SaDu), 10 acinic cell carcinomas (Acin), 7 secretory carcinomas (Sec), 6 epithelial-myoepithelial carcinomas (EpMy), 6 adenocarcinomas not otherwise specified (ANOS) as well as 4 basal cell (Bas) and 4 myoepithelial carcinomas (MyEp). We observed CAFs_COL11A1 in the peritumoural stroma with marked differences across the different subtypes (Figs. 1 and 2). While frequencies of more or equal to 50% of cases with CAFs_COL11A1 were detected among MuEp (89.7%), SaDu (78.9%) and Acin (50.0%), MyEp and Bas showed no CAFs_COL11A1 at all. A heterogeneous distribution was observed in EpMy (33.3%), AdCy (28.0%) and Sec (14.3%). A CAFs_COL11A1 score of 4 was only detected among MuEp and SaDu—none of the tumours from other organs exhibited such intense staining.

When all SGC cases were pooled, CAFs_COL11A1 score positively correlated with higher N-status and CD8 + T cell infiltration (p = 0.015 and 0.017, respectively, Table 2). Interestingly, all cases with vascular invasion by the tumour cells also exhibited CAFs_COL11A1 (p = 0.004). Moreover, the presence of CAFs_COL11A1 was associated with perineural spread (p = 0.004). No association between CAFs_COL11A1 and either tumour grade or pT-status was observed in the SGC cohort. On single-entity level, AdCy with CAFs_COL11A1 were more likely to exhibit perineural spread (p = 0.007) or a TP53 mutation (p = 0.003; Table S3). No other significant associations of CAFs_COL11A1 with clinicopathological parameters were observed in the other tumour types.

For 14 cases, both the primary and a nodal metastasis were analysed (Table S4). In 50% of the cases, both the primary and the metastasis were either positive or negative for CAFs_COL11A1. 35.7% were positive in the primary but not in the nodal metastasis while in the opposite was the case in 14.3%.

In addition to a staining of CAFs, some SGC types also exhibited TC_COL11A1. The highest rate of tumours with TC_COL11A1 was observed among MyEp (75.0%, n = 4), AdCy (52.0%, n = 25) and EpMy (50.0%, n = 6). Similarly, the mean percentage of TC_COL11A1 in individual tumours was highest in MyEp followed by EpMy and AdCy (Table S5). Conversely, these entities showed low frequencies of cases with CAFs_COL11A1 of 0.0%, 28.0% and 33.3%, respectively (Fig. 3A). As expected, TC_COL11A1were inversely correlated with the presence of CAFs_COL11A1 (p < 0.001, Fig. 3B). This nearly mutually exclusive pattern was sustained by the fact, that only 4% of all cases were positive for both tumour cell and stromal COL11A1, while 69% stained exclusively for either of the two patterns (Fig. 3C).

Neither stromal nor intratumoural COL11A1 staining was associated with an alteration of 5-year event-free survival.

Using MSI, COL11A1 protein was measured in TMA tissue samples from both cohorts. After tryptic cleavage, we detected one COL11A1 peptide [(R)GEKGEAGPPGAAGPPGAK(G); m/z value: 1545.82]. Among the different primary sites, the highest intensities were noted for SGC, breast and colon carcinomas, which largely confirmed our results from the RNA-ISH analysis (Fig. 4A). When comparing the different SGC types, the highest intensities for COL11A1 protein were measured in SaDu and AdCy, resulting in a significant difference to the other histologic groups (p value < 0.001) (Fig. 4B). Even though this finding supports the notion that COL11A1 is markedly upregulated in SaDu, it is remarkable that the moderate frequency of CAFs_COL11A1 and TC_COL11A1 in AdCy results in such pronounced protein expression. This seeming discrepancy between ISH and MSI results might be traced back to a slow turnover of COL11A1 in AdCy, possibly due to a relative absence of CAFs in these tumours.