Background
Recent reports have described favorable response rates for immune checkpoint inhibitors in prostate cancers with microsatellite instability (MSI). However, it is unclear whether MSI ...affects the entire tumor mass or is distributed heterogeneously, the latter potentially impairing treatment efficiency.
Methods
To identify prostate cancers with MSI, 316 advanced prostate cancers were analyzed by immunohistochemistry (IHC) for the mismatch repair (MMR) proteins MLH1, PMS2, MSH2, and MSH6 on a TMA format.
Results
Out of 200 interpretable cancers, IHC findings were consistent with MSI in 10 tumors. In 9 of these 10 cancers, tissue blocks were available for subsequent large section IHC, confirming MSI in 6 cases, each with combined protein loss of MSH2 and MSH6. One additional tumor with unequivocal loss of MLH1 and PMS2 on the TMA, for which further analyses could not be carried out due to lack of tissue, was also considered to exhibit MSI. In total, 7 of 200 interpretable advanced prostate cancers were found to exhibit MMR deficiency/MSI (3.5%). Subsequent analysis of all available cancer-containing archived tissue blocks (n=114) revealed consistent and homogeneous MMR protein loss in each case. Polymerase chain reaction (PCR)-based analysis using the “Bethesda panel” could be executed in 6 MMR deficient tumors of which 4 were MSI-high and 2 were MSI-low.
Conclusions
The absence of intratumoral heterogeneity for the MMR status suggests that MSI occurs early in prostate cancer. It is concluded that MMR analysis on limited biopsy material by IHC is sufficient to estimate the MMR status of the entire cancer mass.
Cytokeratin 18 (CK18) is an intermediate filament protein of the cytokeratin acidic type I group and is primarily expressed in single-layered or "simple" epithelial tissues and carcinomas of ...different origin.
To systematically determine CK18 expression in normal and cancerous tissues, 11,952 tumor samples from 115 different tumor types and subtypes (including carcinomas, mesenchymal and biphasic tumors) as well as 608 samples of 76 different normal tissue types were analyzed by immunohistochemistry in a tissue microarray format.
CK18 was expressed in normal epithelial cells of most organs but absent in normal squamous epithelium. At least an occasional weak CK18 positivity was seen in 90 of 115 (78.3%) tumor types. Wide-spread CK18 positivity was seen in 37 (31.9%) of tumor entities, including adenocarcinomas of the lung, prostate, colon and pancreas as well as ovarian cancer. Tumor categories with variable CK18 immunostaining included cancer types arising from CK18 positive precursor cells but show CK18 downregulation in a fraction of cases, tumor types arising from CK18 negative precursor cells occasionally exhibiting CK18 neo-expression, tumors derived from normal tissues with variable CK18 expression, and tumors with a mixed differentiation. CK18 downregulation was for example seen in renal cell cancers and breast cancers, whereas CK18 neo-expression was found in squamous cell carcinomas of various origins. Down-regulation of CK18 in invasive breast carcinomas of no special type and clear cell renal cell carcinomas (ccRCC) was related to adverse tumor features in both tumors (p ≤ 0.0001) and poor patient prognosis in ccRCC (p = 0.0088). Up-regulation of CK18 in squamous cell carcinomas was linked to high grade and lymph node metastasis (p < 0.05). In summary, CK18 is consistently expressed in various epithelial cancers, especially adenocarcinomas.
Down-regulation or loss of CK18 expression in cancers arising from CK18 positive tissues as well as CK18 neo-expression in cancers originating from CK18 negative tissues is linked to cancer progression and may reflect tumor dedifferentiation.
p16 (CDKN2A) is a member of the INK4 class of cell cycle inhibitors, which is often dysregulated in cancer. However, the prevalence of p16 expression in different cancer types is controversial. ...15,783 samples from 124 different tumor types and 76 different normal tissue types were analyzed by immunohistochemistry in a tissue microarray format. p16 was detectable in 5,292 (45.0%) of 11,759 interpretable tumors. Except from adenohypophysis in islets of Langerhans, p16 staining was largely absent in normal tissues. In cancer, highest positivity rates were observed in uterine cervix squamous cell carcinomas (94.4%), non-invasive papillary urothelial carcinoma, pTaG2 (100%), Merkel cell carcinoma (97.7%), and small cell carcinomas of various sites of origin (54.5%-100%). All 124 tumor categories showed at least occasional p16 immunostaining. Comparison with clinico-pathological data in 128 vulvar, 149 endometrial, 295 serous ovarian, 396 pancreatic, 1365 colorectal, 284 gastric, and 1245 urinary bladder cancers, 910 breast carcinomas, 620 clear cell renal cell carcinomas, and 414 testicular germ cell tumors revealed only few statistically significant associations. Comparison of human papilloma virus (HPV) status and p16 in 497 squamous cell carcinomas of different organs revealed HPV in 80.4% of p16 positive and in 20.6% of p16 negative cancers (p<0.0001). It is concluded, that a positive and especially strong p16 immunostaining is a feature for malignancy which may be diagnostically useful in lipomatous, urothelial and possibly other tumors. The imperfect association between p16 immunostaining and HPV infection with high variability between different sites of origin challenges the use of p16 immunohistochemistry as a surrogate for HPV positivity, except in tumors of cervix uteri and the penis.
Background
Microsatellite instability (MSI) has emerged as a predictive biomarker for immune checkpoint inhibitor therapy. Cancer heterogeneity represents a potential obstacle for the analysis of ...predicitive biomarkers. MSI has been reported in pancreatic cancer, but data on the possible extent of intratumoral heterogeneity are lacking.
Methods
To study MSI heterogeneity in pancreatic cancer, a tissue microarray (TMA) comprising 597 tumors was screened by immunohistochemistry with antibodies for the mismatch repair (MMR) proteins MLH1, PMS2, MSH2, and MSH6.
Results
In six suspicious cases, large section immunohistochemistry and microsatellite analysis (Bethesda panel) resulted in the identification of 4 (0.8%) validated MSI cases out of 480 interpretable pancreatic ductal adenocarcinomas. MSI was absent in 55 adenocarcinomas of the ampulla of Vater and 7 acinar cell carcinomas. MMR deficiency always involved MSH6 loss, in three cases with additional loss of MSH2 expression. Three cancers were MSI-high and one case with isolated MSH6 loss was MSS in PCR analysis. The analysis of 44 cancer-containing tumor blocks revealed that the loss of MMR protein expression was always homogeneous in affected tumors. Automated digital image analysis of CD8 immunostaining demonstrated markedly higher CD8 + tumor infiltrating lymphocytes in tumors with (mean = 685, median = 626) than without (mean = 227; median = 124) MMR deficiency (
p
< 0.0001), suggesting a role of MSI for immune response.
Conclusions
Our data suggest that MSI occurs early in a small subset of ductal adenocarcinomas of the pancreas and that immunohistochemical MMR analysis on limited biopsy or cytology material may be sufficient to estimate MMR status of the entire cancer mass.
High homogeneity of MMR deficiency in ovarian cancer Fraune, Christoph; Rosebrock, Janina; Simon, Ronald ...
Gynecologic oncology,
March 2020, 2020-Mar, 2020-03-00, 20200301, Volume:
156, Issue:
3
Journal Article
Peer reviewed
Mismatch repair (MMR) deficiency and Bethesda panel microsatellite instability (MSI) are increasingly analyzed to identify tumors that might benefit from immune checkpoint inhibitors, but tumor ...heterogeneity is a potential obstacle for such analyses. In ovarian cancer, data on intratumoral heterogeneity of MMR deficiency/MSI are lacking.
N = 582 ovarian cancers were screened for MMR deficiency by immunohistochemistry (IHC) on a tissue microarray. 10 cases suspect for MMR deficiency were identified among 478 interpretable cancers and repeated IHC on large sections combined with polymerase chain reaction (PCR)-based MSI analysis validated MMR deficiency/MSI in 9 of these tumors.
MMR deficiency/MSI was predominantly seen in endmetrioid cancers (8 of 35, 23%) and also in 1 of 358 serous carcinomas (0.3%), but was absent in 34 mucinous carcinomas, 23 clear cell carcinomas, 17 malignant mixed Mullerian tumors (carcinosarcomas), and 11 mixed carcinomas. MMR deficiency involed protein loss of PMS2/MLH1 in 6 cases and of MSH2 and/or MSH6 in 3 cases. 7 MMR deficient cancers were MSI-high (all endometrioid), one was MSI-low (endometrioid) and one cancer with unequivocal MMR protein loss exhibited microsatellite stability (serous). MLH1 promotor methylation was observed in 4 of 5 endometrioid cancers with MLH1 protein loss. Immunostaining of all available cancer-containing tissue blocks (n = 114) of tumors with confirmed MMR deficiency/MSI revealed uniform MMR status throughout the entire tumor mass.
Our data show that MSI is present in a substantial proportion of endometrioid ovarian cancers but can also occur in other tumor subtypes. MMR deficiency/MSI typically involves the entire tumor mass, suggesting that MMR inactivation occurs early in tumorigenesis in a subset of ovarian cancers.
•Microsatellite instability (MSI) occurs in about 20% of endometrial ovarian cancers.•Because MSI is an early event in this tumor type, analysis of biopsies can yield representative results.•MSI should be routinely determined at least in every newly diagnosed endometrioid ovarian cancer.
Chymotrypsin-like elastase family member 3B (CELA3B, elastase-3B) is a pancreatic enzyme with digestive function in the intestine. Since RNA analyses of normal tissues suggest that CELA3B expression ...is limited to the pancreas, the potential diagnostic utility of CELA3B immunohistochemistry for the distinction of pancreatic from extrapancreatic cancers and in the distinction of acinar cell carcinoma from ductal adenocarcinoma was assessed. CELA3B expression was successfully analyzed in 13,223 tumor samples from 132 different tumor types and subtypes as well as 8 samples each of 76 different normal tissue types by immunohistochemistry in a tissue microarray format (TMA). In normal tissues, CELA3B immunostaining was only seen in acinar cells and in a fraction of ductal cells of the pancreas as well as on some apical membranes of surface epithelial cells of the intestine. Among tumors, CELA3B immunostaining was seen in 12 of 16 (75%) acinar cell carcinoma of the pancreas including 6 cases with strong staining (37.5%) as well as in 5 of 13,207 other tumors (0.04%). These included 1.2% of 91 adenoid cystic carcinomas, 1.2% of 246 mucoepidermoid carcinomas and 0.8% of 127 acinic cell carcinomas of salivary glands. Our data show a good sensitivity (75%) and a high specificity (99.9%) of CELA3B immunohistochemistry for diagnosing acinar cell carcinoma of the pancreas.
Biglycan (BGN), a proteoglycan of the extracellular matrix, is included in mRNA signatures for prostate cancer aggressiveness. To understand the impact of BGN on prognosis and its relationship to ...molecularly defined subsets, we analyzed BGN expression by immunohistochemistry on a tissue microarray containing 12,427 prostate cancers. Seventy-eight percent of 11,050 interpretable cancers showed BGN expression, which was considered as low intensity in 47.7% and as high intensity in 31.1% of cancers. BGN protein expression rose with increasing pathological tumor stage, Gleason grade, lymph node metastasis and early PSA recurrence (P<.0001 each). Comparison with our molecular database attached to the TMA revealed that BGN expression was linked to presence of TMPRRS2:ERG fusion and PTEN deletion (P<.0001 each). In addition, BGN was strongly linked to androgen-receptor (AR) levels (P<.0001), suggesting a hormone-depending regulation of BGN. BGN up-regulation is a frequent feature of prostate cancer that parallels tumor progression and may be useful to estimate tumor aggressiveness particularly if combined with other molecular markers.
The transcriptional coactivator YAP1 controls the balance between cell proliferation and apoptosis. YAP1 overexpression is linked to poor prognosis in many cancer types, yet its role in prostate ...cancer is unknown. Here, we applied YAP1 immunohistochemistry to a tissue microarray containing 17,747 clinical prostate cancer specimens. Cytoplasmic and nuclear YAP1 staining was seen in 81% and 63% of tumours. For both cytoplasmic and nuclear YAP1 staining, high levels were associated with advanced tumour stage, classical and quantitative Gleason grade, positive nodal stage, positive surgical margin, high KI67 labelling index, and early biochemical recurrence (p < 0.0001 each). The prognostic role of YAP1 staining was independent of established prognostic features in multivariate models (p < 0.001). Comparison with previously studied molecular markers identified associations between high YAP1 staining, TMPRSS2:ERG fusion (p < 0.0001), high androgen receptor (AR) expression (p < 0.0001), high Ki67 labelling index (p < 0.0001), and PTEN and 8p deletions (p < 0.0001 each). In conclusion, high YAP1 protein expression is an independent predictor of unfavourable disease course in prostate cancer. That cytoplasmic and nuclear YAP1 staining is equally linked to phenotype and prognosis fits well to a model where YAP1 activation during tumour progression includes up regulation, cytoplasmic accumulation and subsequent translocation to the nucleus.
Deletions of chromosome arm 13q belong to the most frequent molecular alterations in prostate cancer. To better understand the role of 13q deletion in prostate cancer we took advantage of our large ...prostate cancer tissue microarray comprising more than 12 000 cancer samples with full pathological and clinical follow‐up data. Fluorescence in situ hybridization with probes for ENOX1 (13q14.11) and the retinoblastoma gene (RB1, 13q14.2) was employed. A 13q deletion was found in 21% of 7375 analyzable cancers. Deletions were always heterozygous and associated with high Gleason grade (P < .0001), advanced tumor stage (P < .0001), high preoperative prostate‐specific antigen (PSA) levels (P = .0125), lymph node metastasis (P = .0377), positive resection margin (P = .0064), and early biochemical recurrence (P < .0001). 13q deletions were marginally more frequent in prostate cancers with negative ERG status (22.9%) than in ERG‐positive tumors (18.7%; P < .0001). Loss of 13q predicted patient prognosis independently from established prognostic parameters that are available at the time of biopsy (P = .0004), including preoperative PSA level, clinical tumor stage, and biopsy Gleason grade. In summary, the results of our study identify 13q deletion as a frequent event in prostate cancer, which is linked to an adverse phenotype and poor prognosis in this disease.
TIGIT is an inhibitory immune checkpoint receptor and a putative target for novel immune therapies. Here, we analysed two different types of tissue microarrays of healthy lymphatic and various ...inflamed tissues, colorectal and lung cancers, as well as >1700 tumour samples from 86 different tumour entities for TIGIT and/or PD-1 by bright field and/or multiplex fluorescence immunohistochemistry. TIGIT was detected in CD8+ cytotoxic T cells, CD4+ T helper cells, FOXP3+ regulatory T cells, and NK cells, but not in CD11c+ dendritic cells, CD68+ macrophages, and CD20+ B lymphocytes. TIGIT expression paralleled that of PD-1. More than 70% of TIGIT+ cells were PD-1+, and more than 90% of the PD-1+ cells were TIGIT+. Expression varied between different tissue compartments. TIGIT expression in tonsil gradually increased from the interfollicular area over the marginal/mantle zone to the germinal centre in all T cell subtypes. In inflammatory diseases, the strongest expression of TIGIT/PD-1 was found in Hashimoto thyroiditis. TIGIT+ lymphocytes were seen in all 86 different tumour entities with considerable high variability of TIGIT positivity within and between different cancer entities. Particularly, high densities of TIGIT+ lymphocytes were, for example, seen in squamous cell cancers of various origins. In summary, the variable expression levels of TIGIT and PD-1 in cell types and tissue compartments illustrate the high complexity of immune microenvironments. The high frequency of TIGIT (and PD-1) expressing lymphocytes in cancers highlights considerable opportunities for cotargeting with checkpoint inhibitors.
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