# Diagnostic and Prognostic Impact of Progesterone Receptor Immunohistochemistry: A Study Evaluating More Than 16,000 Tumors

Authors: Florian Viehweger; Lisa-Marie Tinger; David Dum; Natalia Gorbokon; Anne Menz; Ria Uhlig; Franziska Büscheck; Andreas M. Luebke; Claudia Hube-Magg; Andrea Hinsch; Doris Höflmayer; Christoph Fraune; Patrick Lebok; Sören Weidemann; Maximilian Lennartz; Frank Jacobsen; Till S. Clauditz; Rainer Krech; Till Krech; Andreas H. Marx; Ronald Simon; Eike Burandt; Stefan Steurer; Guido Sauter; Sarah Minner; Christian Bernreuther
Journal: Analytical Cellular Pathology
 (2022)

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## Abstract

Progesterone receptor (PR) is a member of the nuclear/steroid hormone receptor family of ligand-dependent transcription factors. It plays an important role in reproduction and mammary gland development and has various tissue-specific effects in nonreproductive organs. In diagnostic pathology, positive PR immunostaining is used to support a diagnosis of breast or gynecologic origin in a tumor. In this study, the expression of PR was analyzed by immunohistochemistry in 18,176 (interpretable: 16,445) samples from 147 different tumor types and subtypes in a tissue microarray format. PR immunostaining was detected in 57.4% of breast tumors, 28.6% of other gynecological tumors, and 1.8% of nongynecological and nonmammary tumors. Among the group of nongynecological and nonmammary tumors, particularly high rates of PR positivity were seen in neuroendocrine tumors (54.3%) and neuroendocrine carcinomas (35.7%) of the pancreas. A comparison with clinico-pathological parameters showed that reduced PR immunostaining was significantly associated with adverse histopathological and clinical features in breast carcinoma, endometrioid endometrial carcinoma, and pancreatic neuroendocrine tumors. In summary, our analysis of 147 different tumor types for PR immunostaining provides a ranking list of tumor entities according to their prevalence of PR positivity, helps to better understand the diagnostic utility of PR, and highlights the distinct PR positivity among neuroendocrine neoplasms of pancreatic origin.


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## Body

## 1. Introduction

Progesterone receptor (PR) is a member of the nuclear/steroid hormone receptor family of ligand-dependent transcription factors. PR mediates the physiological effects of progesterone which plays an important role in the establishment and maintenance of pregnancy, hence the hormone’s name, which comes from the Latinpro gestationem. In addition to its effects in reproduction and mammary gland development [1], PR is involved in the regulation of various genes, affects cellular proliferation and differentiation in various nonreproductive tissues, exerts a neurosteroid activity in the central nervous system (reviewed in [2]), inhibits smooth muscle contractile activity in the gastrointestinal tract [3], and plays a role in development and maturation of the lung [4].In diagnostic pathology, immunohistochemical detection of PR supports the diagnosis of a carcinoma of breast or gynecologic origin if cancers of unknown primary (CUP) are being evaluated [5]. However, many studies have shown that nonbreast and nongynecological tumors can also express estrogen and/or progesterone receptor. Data on PR immunostaining in the literature typically lacks associations with patient age, gender, or stage in cancer [6–14] and is overall highly variable. For example, the reported range of PR positivity ranges from 0 to 76% in colorectal cancer [6–8], from 0 to 52% in adenocarcinoma of the gall bladder [9, 10, 15, 16], from 0 to 85% in prostate cancer [11, 17, 18], from 0 to 63% in non-small-cell lung cancer [12, 19, 20], from 38.7 to 75.8% in papillary thyroid carcinoma [13, 14, 21–24], and from 15.2 to 100% in angiomyolipoma of the kidney [25–27]. These conflicting data are likely to be caused by the use of different antibodies, staining protocols, and interpretation criteria in these studies.To better understand the diagnostic impact of PR immunohistochemistry, a comprehensive and highly standardized study analyzing a large number of tumors, especially from nongynecological and nonbreast tissues, is needed. Therefore, PR expression was successfully analyzed in more than 16,000 tumor tissue samples from 147 different tumor types and subtypes as well as 76 different nonneoplastic tissue types by immunohistochemistry in a tissue microarray (TMA) format in this study.


## 2. Material and Methods

### 2.1. Tissue Microarrays (TMAs)

The normal tissue TMA was composed of 8 samples from 8 different donors for each of 76 different normal tissue types (608 samples on one slide). The tumor TMAs contained a total of 18,176 primary tumors from 147 tumor types and subtypes. Detailed histopathological data on grade, pT, and pN status (HER2 status for breast cancer) were available from 2,139 breast cancers, 259 endometrial cancers, 192 neuroendocrine neoplasms, and 524 ovarian tumors. Clinical follow-up data were available from 877 patients with breast cancer. In these patients, the median follow-up time was 43 (range 1-88) months. The composition of both normal and tumor TMAs is described in detail in the results section. All samples were from the archives of the Institutes of Pathology, University Hospital of Hamburg, Germany; the Institute of Pathology, Clinical Center Osnabrueck, Germany; and Department of Pathology, Academic Hospital Fuerth, Germany. Tissues were fixed in 4% buffered formalin and then embedded in paraffin. One tissue spot (diameter: 0.6 mm) was transmitted from a tumor containing donor block in an empty recipient paraffin block. The use of archived remnants of diagnostic tissues for manufacturing of TMAs and their analysis for research purposes as well as patient data analysis has been approved by local laws (HmbKHG, §12) and by the local ethics committee (Ethics commission Hamburg, WF-049/09). All work has been carried out in compliance with the Helsinki Declaration.


### 2.2. Immunohistochemistry (IHC)

Freshly prepared TMA sections were immunostained in one day in one experiment. Slides were deparaffinized with xylol, rehydrated through a graded alcohol series, and exposed to heat-induced antigen retrieval for 5 minutes in an autoclave at 121°C in pH 7.8 buffer. Endogenous peroxidase activity was blocked with Dako Peroxidase Blocking Solution™ (Agilent, CA, USA; #52023) for 10 minutes. Primary antibody specific against PR (rabbit recombinant, MSVA-570R, #3332-570R; MS Validated Antibodies GmbH, Hamburg, Germany) was applied at 37°C for 60 minutes at a dilution of 1 : 50 (final concentration: 4μg/ml). Bound antibody was then visualized using the EnVision Kit™ (Agilent, CA, USA; #K5007) according to the manufacturer’s directions. The sections were counterstained with haemalaun. For the purpose of antibody validation, immunohistochemical staining of the normal tissue TMA was performed with a different antiprogesterone antibody (mouse monoclonal, PgR636, Agilent, CA, USA; # IR068) on the DAKO autostainer system. Only nuclear staining was scored. For normal tissues, the staining intensity of positive cells was semiquantitively recorded (+, ++, +++). For tumor tissues, the percentage of PR positive tumor cells was estimated, and the staining intensity was semiquantitatively recorded (0, 1+, 2+, 3+). For statistical analyses, the staining results were categorized into four groups as follows: negative: no staining at all, weak staining: staining intensity of 1+ in ≤70% or staining intensity of 2+ in ≤30% of tumor cells, moderate staining: staining intensity of 1+ in >70%, staining intensity of 2+ in >30% but in ≤70% or staining intensity of 3+ in ≤30% of tumor cells, and strong staining: staining intensity of 2+ in >70% or staining intensity of 3+ in >30% of tumor cells.


### 2.3. Statistics

Statistical calculations were performed with JMP 14 software (SAS Institute Inc., NC, USA). Contingency tables and the chi2-test were performed to search for associations between PR and tumor phenotype. Survival curves were calculated according to Kaplan-Meier. The log-rank test was applied to detect significant differences between groups. A p value of ≤0.05 was defined as significant.


## 2.1. Tissue Microarrays (TMAs)

The normal tissue TMA was composed of 8 samples from 8 different donors for each of 76 different normal tissue types (608 samples on one slide). The tumor TMAs contained a total of 18,176 primary tumors from 147 tumor types and subtypes. Detailed histopathological data on grade, pT, and pN status (HER2 status for breast cancer) were available from 2,139 breast cancers, 259 endometrial cancers, 192 neuroendocrine neoplasms, and 524 ovarian tumors. Clinical follow-up data were available from 877 patients with breast cancer. In these patients, the median follow-up time was 43 (range 1-88) months. The composition of both normal and tumor TMAs is described in detail in the results section. All samples were from the archives of the Institutes of Pathology, University Hospital of Hamburg, Germany; the Institute of Pathology, Clinical Center Osnabrueck, Germany; and Department of Pathology, Academic Hospital Fuerth, Germany. Tissues were fixed in 4% buffered formalin and then embedded in paraffin. One tissue spot (diameter: 0.6 mm) was transmitted from a tumor containing donor block in an empty recipient paraffin block. The use of archived remnants of diagnostic tissues for manufacturing of TMAs and their analysis for research purposes as well as patient data analysis has been approved by local laws (HmbKHG, §12) and by the local ethics committee (Ethics commission Hamburg, WF-049/09). All work has been carried out in compliance with the Helsinki Declaration.


## 2.2. Immunohistochemistry (IHC)

Freshly prepared TMA sections were immunostained in one day in one experiment. Slides were deparaffinized with xylol, rehydrated through a graded alcohol series, and exposed to heat-induced antigen retrieval for 5 minutes in an autoclave at 121°C in pH 7.8 buffer. Endogenous peroxidase activity was blocked with Dako Peroxidase Blocking Solution™ (Agilent, CA, USA; #52023) for 10 minutes. Primary antibody specific against PR (rabbit recombinant, MSVA-570R, #3332-570R; MS Validated Antibodies GmbH, Hamburg, Germany) was applied at 37°C for 60 minutes at a dilution of 1 : 50 (final concentration: 4μg/ml). Bound antibody was then visualized using the EnVision Kit™ (Agilent, CA, USA; #K5007) according to the manufacturer’s directions. The sections were counterstained with haemalaun. For the purpose of antibody validation, immunohistochemical staining of the normal tissue TMA was performed with a different antiprogesterone antibody (mouse monoclonal, PgR636, Agilent, CA, USA; # IR068) on the DAKO autostainer system. Only nuclear staining was scored. For normal tissues, the staining intensity of positive cells was semiquantitively recorded (+, ++, +++). For tumor tissues, the percentage of PR positive tumor cells was estimated, and the staining intensity was semiquantitatively recorded (0, 1+, 2+, 3+). For statistical analyses, the staining results were categorized into four groups as follows: negative: no staining at all, weak staining: staining intensity of 1+ in ≤70% or staining intensity of 2+ in ≤30% of tumor cells, moderate staining: staining intensity of 1+ in >70%, staining intensity of 2+ in >30% but in ≤70% or staining intensity of 3+ in ≤30% of tumor cells, and strong staining: staining intensity of 2+ in >70% or staining intensity of 3+ in >30% of tumor cells.


## 2.3. Statistics

Statistical calculations were performed with JMP 14 software (SAS Institute Inc., NC, USA). Contingency tables and the chi2-test were performed to search for associations between PR and tumor phenotype. Survival curves were calculated according to Kaplan-Meier. The log-rank test was applied to detect significant differences between groups. A p value of ≤0.05 was defined as significant.


## 3. Results

### 3.1. Technical Issues

An interpretable result was found in 16,445 (90.5%) tumors. Noninterpretable samples were due to lack of unequivocal tumor cells or loss of the tissue spot during technical procedures for one or both of the markers. A sufficient number of samples of each normal tissue type was evaluable.


### 3.2. Progesterone Receptor Immunostaining in Normal Tissues

In normal tissues, PR was expressed in various organs of the female reproductive organs, such as ovarian stroma, corpus luteum of the ovary, epithelial and stromal cells of the fallopian tube, stromal cells and basal cell layer of the squamous epithelium of the ectocervix, stromal and epithelial cells of the endocervix, stromal and epithelial cells of the endometrium, and decidual cells. In the female breast, some epithelial cells showed a moderate to strong staining. A positive immunostaining was also observed in islets of Langerhans of the pancreas, in a subset of epithelial cells of the adenohypophysis, a subset of adrenocortical cells, in a small number of epithelial cells of the submandibular gland, in subsets of glomerular, tubular and stromal cells of the kidney, in epithelial cells of the cauda epididymis, and a fraction of smooth muscle cells of the ileum, esophagus, and aorta. In some organs, only stromal cells showed a positive immunostaining. This included the prostate gland, the seminal vesicle, and the urinary bladder. PR staining was completely absent in skeletal muscle, heart muscle, fat, skin (including hair follicle and sebaceous glands), oral mucosa of the lip, oral cavity, surface epithelium of the tonsil, and transitional mucosa of the anal canal, squamous epithelium of the esophagus, urothelium of the renal pelvis and urinary bladder, corpus spongiosum of the penis, placental trophoblastic cells, mucosa of the stomach, duodenum, ileum, appendix, colon, rectum and gall bladder, liver, parotid gland, sublingual gland, Brunner gland of the duodenum, testis, respiratory epithelium and glands of bronchi and sinus paranasales, lung, thyroid and parathyroid gland, spleen, lymph node, thymus, cerebellum, and cerebrum. Images of PR staining in normal tissues are shown in Figure1. By using the antibody PgR636, all positive stainings described above were confirmed. An additional staining of occasional mast cells, intracellular mucin within goblet cells in the tubular gut, and colloid of the thyroid gland was only seen by this antibody and was considered a tolerable antibody-specific cross-reactivity (Supplementary Figure 1).Figure 1
PR immunostaining in normal tissues. Positive PR immunostaining in (a) stromal cells and epithelial cells in proliferative endometrium, (b) stromal cells and epithelial cell in endocervix, (c) luminal cells of breast epithelium, (d) islets of Langerhans in pancreas, (e) stromal cells of the prostate, and (f) stromal cells of seminal vesicle.
(a)(b)(c)(d)(e)(f)


### 3.3. Progesterone Receptor Immunostaining in Neoplastic Tissues

A PR immunostaining was found in 1,856 (11.3%) of 16,445 cases (573 weak, 333 moderate, 950 strong; Table1). 55 of 147 (37.4%) different tumor entities included at least one PR-positive case and 31 (21%) entities contained at least one tumor with strong PR staining. PR immunostaining was detected in 57.4% of breast tumors, 28.6% of other gynecological tumors, and 1.8% of nongynecological and nonmammary tumors. A ranking of tumor categories according to the rate of PR positivity is given in Table 2. Particularly, high rates of PR positivity were seen in neuroendocrine tumors (54.3%) and neuroendocrine carcinomas (35.7%) of the pancreas. The group of nonbreast and nongynecological tumors expressing PR in at least 10% of cases included also Leydig cell tumor of the testis (36.7%), medullary thyroid carcinoma (20.4%), small cell neuroendocrine carcinoma of the prostate (16.7%), small cell carcinoma of the lung (12.5%), angiomyolipoma (12.5%), adrenal cortical carcinoma (11.5%), follicular thyroid carcinoma (11.3%), and papillary thyroid carcinoma (10.6%). Images of progesterone receptor staining in “nonmammary” and “nongynecological” tumors are shown in Figure 2.Table 1
PR immunostaining in tumors.
Tumor entityOn TMA (n)PR immunostainingAnalyzable (n)Negative (%)Weak (%)Moderate (%)Strong (%)Tumors of the skin (n=410)Pilomatrixoma3534100.00.00.00.0Basal cell carcinoma8882100.00.00.00.0Benign nevus2929100.00.00.00.0Squamous cell carcinoma of the skin9090100.00.00.00.0Malignant melanoma4646100.00.00.00.0Malignant melanoma lymph node metastasis8684100.00.00.00.0Merkel cell carcinoma4645100.00.00.00.0Tumors of the head and neck (n=1,188)Squamous cell carcinoma of the larynx6054100.00.00.00.0Squamous cell carcinoma of the pharynx6059100.00.00.00.0Oral squamous cell carcinoma (floor of the mouth)8080100.00.00.00.0Warthin tumor of the parotid gland5553100.00.00.00.0Adenocarcinoma, NOS (papillary cystadenocarcinoma)1411100.00.00.00.0Salivary duct carcinoma1513100.00.00.00.0Acinic cell carcinoma of the salivary gland181135100.00.00.00.0Adenocarcinoma NOS of the salivary gland1098197.50.01.21.2Adenoid cystic carcinoma of the salivary gland180126100.00.00.00.0Basal cell adenocarcinoma of the salivary gland2521100.00.00.00.0Basal cell adenoma of the salivary gland8666100.00.00.00.0Epithelial-myoepithelial carcinoma of the salivary gland5352100.00.00.00.0Mucoepidermoid carcinoma of the salivary gland34332799.70.00.30.0Myoepithelial carcinoma of the salivary gland211794.10.05.90.0Myoepithelioma of the salivary gland1110100.00.00.00.0Oncocytic carcinoma of the salivary gland128100.00.00.00.0Polymorphous adenocarcinoma, low grade, of the salivary gland4134100.00.00.00.0Pleomorphic adenoma of the salivary gland5341100.00.00.00.0Tumors of the lung, pleura, and thymus (n=382)Adenocarcinoma of the lung196191100.00.00.00.0Squamous cell carcinoma of the lung8075100.00.00.00.0Small cell carcinoma of the lung161687.56.36.30.0Mesothelioma, epitheloid3930100.00.00.00.0Mesothelioma, other types7670100.00.00.00.0Tumors of the female genital tract (n=1,534)Squamous cell carcinoma of the vagina7874100.00.00.00.0Squamous cell carcinoma of the vulva130123100.00.00.00.0Squamous cell carcinoma of the cervix12912699.20.80.00.0Adenocarcinoma of the cervix212190.54.84.80.0Endometrioid endometrial carcinoma23619733.521.813.731.0Endometrial serous carcinoma826879.414.72.92.9Carcinosarcoma of the uterus484187.84.90.07.3Endometrial carcinoma, high grade, G3131283.38.30.08.3Endometrial clear cell carcinoma86100.00.00.00.0Endometrioid carcinoma of the ovary1109238.018.514.129.3Serous carcinoma of the ovary55952067.922.14.25.8Mucinous carcinoma of the ovary967794.80.00.05.2Clear cell carcinoma of the ovary504588.96.72.22.2Carcinosarcoma of the ovary474465.922.76.84.5Granulosa cell tumor of the ovary373718.929.729.721.6Leydig cell tumor of the ovary4450.050.00.00.0Sertoli cell tumor of the ovary11100.00.00.00.0Sertoli Leydig cell tumor of the ovary3333.366.70.00.0Steroid cell tumor of the ovary33100.00.00.00.0Brenner tumor414095.00.00.05.0Tumors of the breast (n=2,051)Invasive breast carcinoma of no special type1764160542.910.710.735.8Lobular carcinoma of the breast36330243.09.99.637.4Medullary carcinoma of the breast343387.93.03.06.1Tubular carcinoma of the breast292317.48.78.765.2Mucinous carcinoma of the breast655123.57.87.860.8Phyllodes tumor of the breast503727.00.018.954.1Tumors of the digestive system (n=3,911)Adenomatous polyp, low-grade dysplasia5050100.00.00.00.0Adenomatous polyp, high-grade dysplasia5050100.00.00.00.0Adenocarcinoma of the colon2482214699.90.00.00.0Gastric adenocarcinoma, diffuse type176150100.00.00.00.0Gastric adenocarcinoma, intestinal type174157100.00.00.00.0Gastric adenocarcinoma, mixed type6249100.00.00.00.0Adenocarcinoma of the esophagus8383100.00.00.00.0Squamous cell carcinoma of the esophagus7575100.00.00.00.0Squamous cell carcinoma of the anal canal8988100.00.00.00.0Cholangiocarcinoma5050100.00.00.00.0Gallbladder adenocarcinoma3131100.00.00.00.0Gallbladder klatskin tumor4139100.00.00.00.0Hepatocellular carcinoma300299100.00.00.00.0Ductal adenocarcinoma of the pancreas61250597.81.00.60.6Pancreatic/ampullary adenocarcinoma8975100.00.00.00.0Acinar cell carcinoma of the pancreas1615100.00.00.00.0Gastrointestinal stromal tumor (GIST)5049100.00.00.00.0Tumors of the urinary system (n=3,181)Noninvasive papillary urothelial carcinoma, pTa G2 low grade177170100.00.00.00.0Noninvasive papillary urothelial carcinoma, pTa G2 high grade141135100.00.00.00.0Noninvasive papillary urothelial carcinoma, pTa G3219195100.00.00.00.0Urothelial carcinoma, pT2-4 G373563699.70.00.00.3Squamous cell carcinoma of the bladder2222100.00.00.00.0Small cell neuroendocrine carcinoma of the bladder232395.74.30.00.0Sarcomatoid urothelial carcinoma2523100.00.00.00.0Urothelial carcinoma of the kidney pelvis626198.41.60.00.0Clear cell renal cell carcinoma1287117999.90.10.00.0Papillary renal cell carcinoma36832999.70.30.00.0Clear cell (tubulo) papillary renal cell carcinoma262495.80.04.20.0Chromophobe renal cell carcinoma17015395.43.30.01.3Oncocytoma25723193.16.50.40.0Tumors of the male genital organs (n=1,350)Adenocarcinoma of the prostate, Gleason3+38383100.00.00.00.0Adenocarcinoma of the prostate, Gleason4+48080100.00.00.00.0Adenocarcinoma of the prostate, Gleason5+58585100.00.00.00.0Adenocarcinoma of the prostate (recurrence)25825799.60.40.00.0Small cell neuroendocrine carcinoma of the prostate191883.316.70.00.0Seminoma621586100.00.00.00.0Embryonal carcinoma of the testis5045100.00.00.00.0Leydig cell tumor of the testis303063.333.33.30.0Sertoli cell tumor of the testis210.0100.00.00.0Sex cord stromal tumor of the testis110.00.0100.00.0Spermatocytic tumor of the testis11100.00.00.00.0Yolk sac tumor5044100.00.00.00.0Teratoma504195.10.00.04.9Squamous cell carcinoma of the penis8078100.00.00.00.0Tumors of endocrine organs (n=1,171)Adenoma of the thyroid gland11411292.94.52.70.0Papillary thyroid carcinoma39237989.49.01.10.5Follicular thyroid carcinoma15415188.75.33.32.6Medullary thyroid carcinoma11110879.616.71.91.9Parathyroid gland adenoma4342100.00.00.00.0Anaplastic thyroid carcinoma454397.72.30.00.0Adrenal cortical adenoma504495.52.30.02.3Adrenal cortical carcinoma262688.511.50.00.0Phaeochromocytoma5049100.00.00.00.0Appendix, neuroendocrine tumor (NET)221794.10.05.90.0Colorectal, neuroendocrine tumor (NET)1211100.00.00.00.0Ileum, neuroendocrine tumor (NET)4949100.00.00.00.0Lung, neuroendocrine tumor (NET)1918100.00.00.00.0Pancreas, neuroendocrine tumor (NET)979445.717.09.627.7Colorectal, neuroendocrine carcinoma (NEC)1210100.00.00.00.0Gallbladder, neuroendocrine carcinoma (NEC)44100.00.00.00.0Pancreas, neuroendocrine carcinoma (NEC)141464.37.114.314.3Tumors of haematopoetic and lymphoid tissues (n=353)Hodgkin lymphoma5853100.00.00.00.0Small lymphocytic lymphoma, B-cell type (B-SLL/B-CLL)5044100.00.00.00.0Diffuse large B cell lymphoma (DLBCL)113103100.00.00.00.0Follicular lymphoma8880100.00.00.00.0T-cell non-Hodgkin lymphoma2524100.00.00.00.0Mantle cell lymphoma1817100.00.00.00.0Marginal zone lymphoma1614100.00.00.00.0Diffuse large B-cell lymphoma (DLBCL) in the testis1616100.00.00.00.0Burkitt lymphoma52100.00.00.00.0Tumors of soft tissue and bone (n=914)Tenosynovial giant cell tumor4545100.00.00.00.0Granular cell tumor5345100.00.00.00.0Leiomyosarcoma383789.22.70.08.1Liposarcoma132130100.00.00.00.0Malignant peripheral nerve sheath tumor (MPNST)1313100.00.00.00.0Myofibrosarcoma2626100.00.00.00.0Angiosarcoma7366100.00.00.00.0Angiomyolipoma918887.59.10.03.4Dermatofibrosarcoma protuberans2117100.00.00.00.0Ganglioneuroma1414100.00.00.00.0Kaposi sarcoma85100.00.00.00.0Neurofibroma11710495.24.80.00.0Sarcoma, not otherwise specified (NOS)747098.60.00.01.4Paraganglioma4141100.00.00.00.0Ewing sarcoma2318100.00.00.00.0Rhabdomyosarcoma66100.00.00.00.0Schwannoma121112100.00.00.00.0Synovial sarcoma1211100.00.00.00.0Osteosarcoma4339100.00.00.00.0Chondrosarcoma3822100.00.00.00.0Rhabdoid tumor55100.00.00.00.0Table 2
Ranking of PR immunostaining in tumors (only tumor entities with≥3 evaluable tumors were included in the ranking. Mammary tumors are italicized. Gynecological tumors are in bold).
Ranking PR≥ weak (%)≥ mod (%)Strong (%)Tubular carcinoma of the breast82.673.965.2Granulosa cell tumor of the ovary81.151.421.6Mucinous carcinoma of the breast76.568.660.8Phyllodes tumor of the breast73.073.054.1Sertoli Leydig cell tumor of the ovary66.70.00.0Endometrioid endometrial carcinoma66.544.731.0Endometrioid carcinoma of the ovary62.043.529.3Invasive breast carcinoma of no special type57.146.535.8Lobular carcinoma of the breast57.047.037.4Pancreas, neuroendocrine tumor (NET)54.337.227.7Leydig cell tumor of the ovary50.00.00.0Leydig cell tumor of the testis36.73.30.0Pancreas, neuroendocrine carcinoma (NEC)35.728.614.3Carcinosarcoma of the ovary34.111.44.5Serous carcinoma of the ovary32.110.05.8Endometrial serous carcinoma20.65.92.9Medullary thyroid carcinoma20.43.71.9Endometrial carcinoma, high grade, G316.78.38.3Small cell neuroendocrine carcinoma of the prostate16.70.00.0Small cell carcinoma of the lung12.56.30.0Angiomyolipoma12.53.43.4Carcinosarcoma of the uterus12.27.37.3Medullary carcinoma of the breast12.19.16.1Adrenal cortical carcinoma11.50.00.0Follicular thyroid carcinoma11.36.02.6Clear cell carcinoma of the ovary11.14.42.2Leiomyosarcoma10.88.18.1Papillary thyroid carcinoma10.61.60.5Adenocarcinoma of the cervix9.54.80.0Adenoma of the thyroid gland7.12.70.0Oncocytoma6.90.40.0Myoepithelial carcinoma of the salivary gland5.95.90.0Appendix, neuroendocrine tumor (NET)5.95.90.0Mucinous carcinoma of the ovary5.25.25.2Brenner tumor5.05.05.0Teratoma4.94.94.9Neurofibroma4.80.00.0Chromophobe renal cell carcinoma4.61.31.3Adrenal cortical adenoma4.52.32.3Small cell neuroendocrine carcinoma of the bladder4.30.00.0Clear cell (tubulo) papillary renal cell carcinoma4.24.20.0Adenocarcinoma NOS of the salivary gland2.52.51.2Anaplastic thyroid carcinoma2.30.00.0Ductal adenocarcinoma of the pancreas2.21.20.6Urothelial carcinoma of the kidney pelvis1.60.00.0Sarcoma, not otherwise specified (NOS)1.41.41.4Squamous cell carcinoma of the cervix0.80.00.0Adenocarcinoma of the prostate (recurrence)0.40.00.0Urothelial carcinoma, pT2-4 G30.30.30.3Mucoepidermoid carcinoma of the salivary gland0.30.30.0Papillary renal cell carcinoma0.30.00.0Adenocarcinoma of the colon0.10.00.0Clear cell renal cell carcinoma0.10.00.0Figure 2
Moderate to strong PR immunostaining in “nonmammary” and “nongynecological” tumors. (a) Neuroendocrine tumor of the pancreas. (b) Neuroendocrine carcinoma of the pancreas. (c) Small cell carcinoma of the lung. (d) Leydig cell tumor of the testis. (e) Medullary thyroid carcinoma. (f) Follicular thyroid carcinoma. (g) Papillary thyroid carcinoma. (h) Angiomyolipoma.
(a)(b)(c)(d)(e)(f)(g)(h)


### 3.4. Progesterone Receptor Immunostaining, Tumor Phenotype, and Prognosis

Reduced PR immunostaining was significantly associated with adverse histopathological and clinical features in breast carcinoma, endometroid endometrial carcinoma, and pancreatic neuroendocrine tumors (Table3). In breast carcinomas of no special type, reduced PR immunostaining was linked to advanced tumor stage (p<0.0001), lymph node metastasis (p<0.0001), high tumor grade (p<0.0001), distant metastasis (p<0.0001), positive HER2 status (p<0.0001), and shorter overall survival (negative vs. any positivity, HR 1.8, 95% CI 1.3-2.5, p=0.0127; Supplementary Figure 2). In endometroid endometrial carcinoma, low PR immunostaining was linked to lymph node metastasis (p=0.0327). In 49 pancreatic neuroendocrine tumors, low PR immunostaining was linked to lymph node metastasis (p=0.0345). PR staining was unrelated to histopathological features in 343 serous ovarian carcinomas. Within nonmammary, nongynecological, and nonprostate tumors, PR positivity was more common in tumors from female (3.2% of 3,085) than from male patients (1.6% of 4,752; p<0.0001).Table 3
PR immunostaining and tumor phenotype in breast carcinoma of no special type, endometrioid endometrial carcinoma, high-grade serous ovarian carcinoma, and pancreatic neuroendocrine tumors.
nProgesterone receptor IHC resultpNegative (%)Weak (%)Moderate (%)Strong (%)Breast carcinoma of no special typeTumor stagepT174936.79.911.941.5<0.0001pT261344.511.411.432.6pT3-412254.911.54.129.5GradeG118323.010.912.653.6<0.0001G279934.310.413.441.9G354359.710.77.222.5Nodal stagepN068240.28.89.841.2<0.0001pN132538.214.514.233.2pN211447.413.210.528.9pN36863.213.25.917.6Distant metastasispM019938.77.510.643.2<0.0001pM110464.411.55.818.3HER2 statusNegative85037.810.511.140.7<0.0001Positive12063.312.56.717.5Endometrioid endometrial carcinomaTumor stagepT19429.822.313.834.00.8885pT22334.817.48.739.1pT3-42934.524.117.224.1Nodal stagepN04320.925.618.634.90.0327pN+2556.016.08.020.0Serous ovarian carcinomaTumor stagepT13351.518.29.121.20.0750pT24369.820.92.37.0pT326772.318.73.75.2Nodal stagepN08365.126.56.02.40.0534pN117176.015.82.35.8Pancreatic neuroendocrine tumorsTumor stagepT11040.010.020.030.00.0954pT21526.733.36.733.3pT32268.24.513.613.6pT420.050.00.050.0Nodal stagepN02429.220.816.733.30.0345pN+2171.49.59.59.5


## 3.1. Technical Issues

An interpretable result was found in 16,445 (90.5%) tumors. Noninterpretable samples were due to lack of unequivocal tumor cells or loss of the tissue spot during technical procedures for one or both of the markers. A sufficient number of samples of each normal tissue type was evaluable.


## 3.2. Progesterone Receptor Immunostaining in Normal Tissues

In normal tissues, PR was expressed in various organs of the female reproductive organs, such as ovarian stroma, corpus luteum of the ovary, epithelial and stromal cells of the fallopian tube, stromal cells and basal cell layer of the squamous epithelium of the ectocervix, stromal and epithelial cells of the endocervix, stromal and epithelial cells of the endometrium, and decidual cells. In the female breast, some epithelial cells showed a moderate to strong staining. A positive immunostaining was also observed in islets of Langerhans of the pancreas, in a subset of epithelial cells of the adenohypophysis, a subset of adrenocortical cells, in a small number of epithelial cells of the submandibular gland, in subsets of glomerular, tubular and stromal cells of the kidney, in epithelial cells of the cauda epididymis, and a fraction of smooth muscle cells of the ileum, esophagus, and aorta. In some organs, only stromal cells showed a positive immunostaining. This included the prostate gland, the seminal vesicle, and the urinary bladder. PR staining was completely absent in skeletal muscle, heart muscle, fat, skin (including hair follicle and sebaceous glands), oral mucosa of the lip, oral cavity, surface epithelium of the tonsil, and transitional mucosa of the anal canal, squamous epithelium of the esophagus, urothelium of the renal pelvis and urinary bladder, corpus spongiosum of the penis, placental trophoblastic cells, mucosa of the stomach, duodenum, ileum, appendix, colon, rectum and gall bladder, liver, parotid gland, sublingual gland, Brunner gland of the duodenum, testis, respiratory epithelium and glands of bronchi and sinus paranasales, lung, thyroid and parathyroid gland, spleen, lymph node, thymus, cerebellum, and cerebrum. Images of PR staining in normal tissues are shown in Figure1. By using the antibody PgR636, all positive stainings described above were confirmed. An additional staining of occasional mast cells, intracellular mucin within goblet cells in the tubular gut, and colloid of the thyroid gland was only seen by this antibody and was considered a tolerable antibody-specific cross-reactivity (Supplementary Figure 1).Figure 1
PR immunostaining in normal tissues. Positive PR immunostaining in (a) stromal cells and epithelial cells in proliferative endometrium, (b) stromal cells and epithelial cell in endocervix, (c) luminal cells of breast epithelium, (d) islets of Langerhans in pancreas, (e) stromal cells of the prostate, and (f) stromal cells of seminal vesicle.
(a)(b)(c)(d)(e)(f)


## 3.3. Progesterone Receptor Immunostaining in Neoplastic Tissues

A PR immunostaining was found in 1,856 (11.3%) of 16,445 cases (573 weak, 333 moderate, 950 strong; Table1). 55 of 147 (37.4%) different tumor entities included at least one PR-positive case and 31 (21%) entities contained at least one tumor with strong PR staining. PR immunostaining was detected in 57.4% of breast tumors, 28.6% of other gynecological tumors, and 1.8% of nongynecological and nonmammary tumors. A ranking of tumor categories according to the rate of PR positivity is given in Table 2. Particularly, high rates of PR positivity were seen in neuroendocrine tumors (54.3%) and neuroendocrine carcinomas (35.7%) of the pancreas. The group of nonbreast and nongynecological tumors expressing PR in at least 10% of cases included also Leydig cell tumor of the testis (36.7%), medullary thyroid carcinoma (20.4%), small cell neuroendocrine carcinoma of the prostate (16.7%), small cell carcinoma of the lung (12.5%), angiomyolipoma (12.5%), adrenal cortical carcinoma (11.5%), follicular thyroid carcinoma (11.3%), and papillary thyroid carcinoma (10.6%). Images of progesterone receptor staining in “nonmammary” and “nongynecological” tumors are shown in Figure 2.Table 1
PR immunostaining in tumors.
Tumor entityOn TMA (n)PR immunostainingAnalyzable (n)Negative (%)Weak (%)Moderate (%)Strong (%)Tumors of the skin (n=410)Pilomatrixoma3534100.00.00.00.0Basal cell carcinoma8882100.00.00.00.0Benign nevus2929100.00.00.00.0Squamous cell carcinoma of the skin9090100.00.00.00.0Malignant melanoma4646100.00.00.00.0Malignant melanoma lymph node metastasis8684100.00.00.00.0Merkel cell carcinoma4645100.00.00.00.0Tumors of the head and neck (n=1,188)Squamous cell carcinoma of the larynx6054100.00.00.00.0Squamous cell carcinoma of the pharynx6059100.00.00.00.0Oral squamous cell carcinoma (floor of the mouth)8080100.00.00.00.0Warthin tumor of the parotid gland5553100.00.00.00.0Adenocarcinoma, NOS (papillary cystadenocarcinoma)1411100.00.00.00.0Salivary duct carcinoma1513100.00.00.00.0Acinic cell carcinoma of the salivary gland181135100.00.00.00.0Adenocarcinoma NOS of the salivary gland1098197.50.01.21.2Adenoid cystic carcinoma of the salivary gland180126100.00.00.00.0Basal cell adenocarcinoma of the salivary gland2521100.00.00.00.0Basal cell adenoma of the salivary gland8666100.00.00.00.0Epithelial-myoepithelial carcinoma of the salivary gland5352100.00.00.00.0Mucoepidermoid carcinoma of the salivary gland34332799.70.00.30.0Myoepithelial carcinoma of the salivary gland211794.10.05.90.0Myoepithelioma of the salivary gland1110100.00.00.00.0Oncocytic carcinoma of the salivary gland128100.00.00.00.0Polymorphous adenocarcinoma, low grade, of the salivary gland4134100.00.00.00.0Pleomorphic adenoma of the salivary gland5341100.00.00.00.0Tumors of the lung, pleura, and thymus (n=382)Adenocarcinoma of the lung196191100.00.00.00.0Squamous cell carcinoma of the lung8075100.00.00.00.0Small cell carcinoma of the lung161687.56.36.30.0Mesothelioma, epitheloid3930100.00.00.00.0Mesothelioma, other types7670100.00.00.00.0Tumors of the female genital tract (n=1,534)Squamous cell carcinoma of the vagina7874100.00.00.00.0Squamous cell carcinoma of the vulva130123100.00.00.00.0Squamous cell carcinoma of the cervix12912699.20.80.00.0Adenocarcinoma of the cervix212190.54.84.80.0Endometrioid endometrial carcinoma23619733.521.813.731.0Endometrial serous carcinoma826879.414.72.92.9Carcinosarcoma of the uterus484187.84.90.07.3Endometrial carcinoma, high grade, G3131283.38.30.08.3Endometrial clear cell carcinoma86100.00.00.00.0Endometrioid carcinoma of the ovary1109238.018.514.129.3Serous carcinoma of the ovary55952067.922.14.25.8Mucinous carcinoma of the ovary967794.80.00.05.2Clear cell carcinoma of the ovary504588.96.72.22.2Carcinosarcoma of the ovary474465.922.76.84.5Granulosa cell tumor of the ovary373718.929.729.721.6Leydig cell tumor of the ovary4450.050.00.00.0Sertoli cell tumor of the ovary11100.00.00.00.0Sertoli Leydig cell tumor of the ovary3333.366.70.00.0Steroid cell tumor of the ovary33100.00.00.00.0Brenner tumor414095.00.00.05.0Tumors of the breast (n=2,051)Invasive breast carcinoma of no special type1764160542.910.710.735.8Lobular carcinoma of the breast36330243.09.99.637.4Medullary carcinoma of the breast343387.93.03.06.1Tubular carcinoma of the breast292317.48.78.765.2Mucinous carcinoma of the breast655123.57.87.860.8Phyllodes tumor of the breast503727.00.018.954.1Tumors of the digestive system (n=3,911)Adenomatous polyp, low-grade dysplasia5050100.00.00.00.0Adenomatous polyp, high-grade dysplasia5050100.00.00.00.0Adenocarcinoma of the colon2482214699.90.00.00.0Gastric adenocarcinoma, diffuse type176150100.00.00.00.0Gastric adenocarcinoma, intestinal type174157100.00.00.00.0Gastric adenocarcinoma, mixed type6249100.00.00.00.0Adenocarcinoma of the esophagus8383100.00.00.00.0Squamous cell carcinoma of the esophagus7575100.00.00.00.0Squamous cell carcinoma of the anal canal8988100.00.00.00.0Cholangiocarcinoma5050100.00.00.00.0Gallbladder adenocarcinoma3131100.00.00.00.0Gallbladder klatskin tumor4139100.00.00.00.0Hepatocellular carcinoma300299100.00.00.00.0Ductal adenocarcinoma of the pancreas61250597.81.00.60.6Pancreatic/ampullary adenocarcinoma8975100.00.00.00.0Acinar cell carcinoma of the pancreas1615100.00.00.00.0Gastrointestinal stromal tumor (GIST)5049100.00.00.00.0Tumors of the urinary system (n=3,181)Noninvasive papillary urothelial carcinoma, pTa G2 low grade177170100.00.00.00.0Noninvasive papillary urothelial carcinoma, pTa G2 high grade141135100.00.00.00.0Noninvasive papillary urothelial carcinoma, pTa G3219195100.00.00.00.0Urothelial carcinoma, pT2-4 G373563699.70.00.00.3Squamous cell carcinoma of the bladder2222100.00.00.00.0Small cell neuroendocrine carcinoma of the bladder232395.74.30.00.0Sarcomatoid urothelial carcinoma2523100.00.00.00.0Urothelial carcinoma of the kidney pelvis626198.41.60.00.0Clear cell renal cell carcinoma1287117999.90.10.00.0Papillary renal cell carcinoma36832999.70.30.00.0Clear cell (tubulo) papillary renal cell carcinoma262495.80.04.20.0Chromophobe renal cell carcinoma17015395.43.30.01.3Oncocytoma25723193.16.50.40.0Tumors of the male genital organs (n=1,350)Adenocarcinoma of the prostate, Gleason3+38383100.00.00.00.0Adenocarcinoma of the prostate, Gleason4+48080100.00.00.00.0Adenocarcinoma of the prostate, Gleason5+58585100.00.00.00.0Adenocarcinoma of the prostate (recurrence)25825799.60.40.00.0Small cell neuroendocrine carcinoma of the prostate191883.316.70.00.0Seminoma621586100.00.00.00.0Embryonal carcinoma of the testis5045100.00.00.00.0Leydig cell tumor of the testis303063.333.33.30.0Sertoli cell tumor of the testis210.0100.00.00.0Sex cord stromal tumor of the testis110.00.0100.00.0Spermatocytic tumor of the testis11100.00.00.00.0Yolk sac tumor5044100.00.00.00.0Teratoma504195.10.00.04.9Squamous cell carcinoma of the penis8078100.00.00.00.0Tumors of endocrine organs (n=1,171)Adenoma of the thyroid gland11411292.94.52.70.0Papillary thyroid carcinoma39237989.49.01.10.5Follicular thyroid carcinoma15415188.75.33.32.6Medullary thyroid carcinoma11110879.616.71.91.9Parathyroid gland adenoma4342100.00.00.00.0Anaplastic thyroid carcinoma454397.72.30.00.0Adrenal cortical adenoma504495.52.30.02.3Adrenal cortical carcinoma262688.511.50.00.0Phaeochromocytoma5049100.00.00.00.0Appendix, neuroendocrine tumor (NET)221794.10.05.90.0Colorectal, neuroendocrine tumor (NET)1211100.00.00.00.0Ileum, neuroendocrine tumor (NET)4949100.00.00.00.0Lung, neuroendocrine tumor (NET)1918100.00.00.00.0Pancreas, neuroendocrine tumor (NET)979445.717.09.627.7Colorectal, neuroendocrine carcinoma (NEC)1210100.00.00.00.0Gallbladder, neuroendocrine carcinoma (NEC)44100.00.00.00.0Pancreas, neuroendocrine carcinoma (NEC)141464.37.114.314.3Tumors of haematopoetic and lymphoid tissues (n=353)Hodgkin lymphoma5853100.00.00.00.0Small lymphocytic lymphoma, B-cell type (B-SLL/B-CLL)5044100.00.00.00.0Diffuse large B cell lymphoma (DLBCL)113103100.00.00.00.0Follicular lymphoma8880100.00.00.00.0T-cell non-Hodgkin lymphoma2524100.00.00.00.0Mantle cell lymphoma1817100.00.00.00.0Marginal zone lymphoma1614100.00.00.00.0Diffuse large B-cell lymphoma (DLBCL) in the testis1616100.00.00.00.0Burkitt lymphoma52100.00.00.00.0Tumors of soft tissue and bone (n=914)Tenosynovial giant cell tumor4545100.00.00.00.0Granular cell tumor5345100.00.00.00.0Leiomyosarcoma383789.22.70.08.1Liposarcoma132130100.00.00.00.0Malignant peripheral nerve sheath tumor (MPNST)1313100.00.00.00.0Myofibrosarcoma2626100.00.00.00.0Angiosarcoma7366100.00.00.00.0Angiomyolipoma918887.59.10.03.4Dermatofibrosarcoma protuberans2117100.00.00.00.0Ganglioneuroma1414100.00.00.00.0Kaposi sarcoma85100.00.00.00.0Neurofibroma11710495.24.80.00.0Sarcoma, not otherwise specified (NOS)747098.60.00.01.4Paraganglioma4141100.00.00.00.0Ewing sarcoma2318100.00.00.00.0Rhabdomyosarcoma66100.00.00.00.0Schwannoma121112100.00.00.00.0Synovial sarcoma1211100.00.00.00.0Osteosarcoma4339100.00.00.00.0Chondrosarcoma3822100.00.00.00.0Rhabdoid tumor55100.00.00.00.0Table 2
Ranking of PR immunostaining in tumors (only tumor entities with≥3 evaluable tumors were included in the ranking. Mammary tumors are italicized. Gynecological tumors are in bold).
Ranking PR≥ weak (%)≥ mod (%)Strong (%)Tubular carcinoma of the breast82.673.965.2Granulosa cell tumor of the ovary81.151.421.6Mucinous carcinoma of the breast76.568.660.8Phyllodes tumor of the breast73.073.054.1Sertoli Leydig cell tumor of the ovary66.70.00.0Endometrioid endometrial carcinoma66.544.731.0Endometrioid carcinoma of the ovary62.043.529.3Invasive breast carcinoma of no special type57.146.535.8Lobular carcinoma of the breast57.047.037.4Pancreas, neuroendocrine tumor (NET)54.337.227.7Leydig cell tumor of the ovary50.00.00.0Leydig cell tumor of the testis36.73.30.0Pancreas, neuroendocrine carcinoma (NEC)35.728.614.3Carcinosarcoma of the ovary34.111.44.5Serous carcinoma of the ovary32.110.05.8Endometrial serous carcinoma20.65.92.9Medullary thyroid carcinoma20.43.71.9Endometrial carcinoma, high grade, G316.78.38.3Small cell neuroendocrine carcinoma of the prostate16.70.00.0Small cell carcinoma of the lung12.56.30.0Angiomyolipoma12.53.43.4Carcinosarcoma of the uterus12.27.37.3Medullary carcinoma of the breast12.19.16.1Adrenal cortical carcinoma11.50.00.0Follicular thyroid carcinoma11.36.02.6Clear cell carcinoma of the ovary11.14.42.2Leiomyosarcoma10.88.18.1Papillary thyroid carcinoma10.61.60.5Adenocarcinoma of the cervix9.54.80.0Adenoma of the thyroid gland7.12.70.0Oncocytoma6.90.40.0Myoepithelial carcinoma of the salivary gland5.95.90.0Appendix, neuroendocrine tumor (NET)5.95.90.0Mucinous carcinoma of the ovary5.25.25.2Brenner tumor5.05.05.0Teratoma4.94.94.9Neurofibroma4.80.00.0Chromophobe renal cell carcinoma4.61.31.3Adrenal cortical adenoma4.52.32.3Small cell neuroendocrine carcinoma of the bladder4.30.00.0Clear cell (tubulo) papillary renal cell carcinoma4.24.20.0Adenocarcinoma NOS of the salivary gland2.52.51.2Anaplastic thyroid carcinoma2.30.00.0Ductal adenocarcinoma of the pancreas2.21.20.6Urothelial carcinoma of the kidney pelvis1.60.00.0Sarcoma, not otherwise specified (NOS)1.41.41.4Squamous cell carcinoma of the cervix0.80.00.0Adenocarcinoma of the prostate (recurrence)0.40.00.0Urothelial carcinoma, pT2-4 G30.30.30.3Mucoepidermoid carcinoma of the salivary gland0.30.30.0Papillary renal cell carcinoma0.30.00.0Adenocarcinoma of the colon0.10.00.0Clear cell renal cell carcinoma0.10.00.0Figure 2
Moderate to strong PR immunostaining in “nonmammary” and “nongynecological” tumors. (a) Neuroendocrine tumor of the pancreas. (b) Neuroendocrine carcinoma of the pancreas. (c) Small cell carcinoma of the lung. (d) Leydig cell tumor of the testis. (e) Medullary thyroid carcinoma. (f) Follicular thyroid carcinoma. (g) Papillary thyroid carcinoma. (h) Angiomyolipoma.
(a)(b)(c)(d)(e)(f)(g)(h)


## 3.4. Progesterone Receptor Immunostaining, Tumor Phenotype, and Prognosis

Reduced PR immunostaining was significantly associated with adverse histopathological and clinical features in breast carcinoma, endometroid endometrial carcinoma, and pancreatic neuroendocrine tumors (Table3). In breast carcinomas of no special type, reduced PR immunostaining was linked to advanced tumor stage (p<0.0001), lymph node metastasis (p<0.0001), high tumor grade (p<0.0001), distant metastasis (p<0.0001), positive HER2 status (p<0.0001), and shorter overall survival (negative vs. any positivity, HR 1.8, 95% CI 1.3-2.5, p=0.0127; Supplementary Figure 2). In endometroid endometrial carcinoma, low PR immunostaining was linked to lymph node metastasis (p=0.0327). In 49 pancreatic neuroendocrine tumors, low PR immunostaining was linked to lymph node metastasis (p=0.0345). PR staining was unrelated to histopathological features in 343 serous ovarian carcinomas. Within nonmammary, nongynecological, and nonprostate tumors, PR positivity was more common in tumors from female (3.2% of 3,085) than from male patients (1.6% of 4,752; p<0.0001).Table 3
PR immunostaining and tumor phenotype in breast carcinoma of no special type, endometrioid endometrial carcinoma, high-grade serous ovarian carcinoma, and pancreatic neuroendocrine tumors.
nProgesterone receptor IHC resultpNegative (%)Weak (%)Moderate (%)Strong (%)Breast carcinoma of no special typeTumor stagepT174936.79.911.941.5<0.0001pT261344.511.411.432.6pT3-412254.911.54.129.5GradeG118323.010.912.653.6<0.0001G279934.310.413.441.9G354359.710.77.222.5Nodal stagepN068240.28.89.841.2<0.0001pN132538.214.514.233.2pN211447.413.210.528.9pN36863.213.25.917.6Distant metastasispM019938.77.510.643.2<0.0001pM110464.411.55.818.3HER2 statusNegative85037.810.511.140.7<0.0001Positive12063.312.56.717.5Endometrioid endometrial carcinomaTumor stagepT19429.822.313.834.00.8885pT22334.817.48.739.1pT3-42934.524.117.224.1Nodal stagepN04320.925.618.634.90.0327pN+2556.016.08.020.0Serous ovarian carcinomaTumor stagepT13351.518.29.121.20.0750pT24369.820.92.37.0pT326772.318.73.75.2Nodal stagepN08365.126.56.02.40.0534pN117176.015.82.35.8Pancreatic neuroendocrine tumorsTumor stagepT11040.010.020.030.00.0954pT21526.733.36.733.3pT32268.24.513.613.6pT420.050.00.050.0Nodal stagepN02429.220.816.733.30.0345pN+2171.49.59.59.5


## 4. Discussion

Our successful analysis of more than 16,000 tumors revealed PR expression in 57.4% of breast tumors, 28.6% of other gynecological tumors, and 1.8% of nongynecological and nonmammary tumors.Given the large size of our study, particular emphasis was placed on the validation of our reagents and protocols. The International Working Group for Antibody Validation (IWGAV) has proposed that antibody validation for immunohistochemistry on formalin fixed tissues should include either a comparison of the findings obtained by two independent antibodies or a comparison with expression data obtained by another independent method [28–30]. Both methods were applied in this project. A comparison of our IHC data with RNA data provided from three independent publicly available databases (Human Protein Atlas (HPA) RNA-seq tissue dataset [31], FANTOM5 project [32, 33], and Genotype-Tissue Expression (GTEx) project [34]) revealed IHC positivity in all tissues with unequivocal RNA expression such as the organs of the female genital tract, prostate, seminal vesicle, epididymis, and the pituitary gland. RNA expression had previously not been recorded for several tissues with a positive PR immunostaining such as the aortic wall, pancreatic islet cells, kidney, duodenum, adrenal gland, stroma cells of urinary bladder and pyelon mucosa, smooth muscle cells of gastrointestinal tract, or salivary glands. These tissues had previously either not been analyzed on the RNA level (aortic wall, Brunner glands of the duodenum) or the PR positive cells constitute such small fractions of their respective organs total number of cells that their PR RNAs may not have occurred at detectable quantities. True PR expression in all these cell types is supported by identical stainings obtained by the antibody PgR636 (Supplementary Figure 1). Additional positivity obtained by PgR636 in goblet cells of the gut and of thyroidal colloid was considered an antibody cross-reactivity specific to PgR636 because these tissues remained unstained by MSVA-570R.The PR immunostaining results in breast and other gynecological tumors were in the range of most previous studies which is another confirmation of our experimental approach. The slightly lower PR positivity rate of breast tumors in our study (57.4%) compared to the 60-70% positivity rate described in previous studies (reviewed in [35]) may reflect a TMA effect. TMAs generally result in slightly lower positivity rates than seen in large section analysis. In a highly standardized study comparing PR immunostaining between TMAs and traditional sections in more than 500 breast cancers, Torhorst et al. [36] had found a PR positivity of 41-53% in multiple TMAs and 60% PR positivity in large sections. Although progesterone receptors are widely expressed in ovarian cancers, their distribution varies significantly by histology. Particularly, sex cord stromal tumors showed high PR positivity (50-81%), which fits well with previous studies [37–39]. PR positivity was found in 62% of endometroid but only in 32% of high grade serous ovarian carcinomas. This is in line with earlier studies describing 41-67% PR positivity in endometroid [40–43] but only 25-50% PR positivity in high-grade serous carcinomas of the ovary [43, 44]. In the uterus, endometrioid carcinomas (67%) also showed a much higher rate of PR positivity than serous carcinomas (21%). Consistent with these data, earlier reports have described PR positivity in 62.3-81.3% of endometroid [45–47] but only in 20-46% of serous carcinomas of the endometrium [48, 49]. As expected from previous studies (reviewed in [35, 50]) an absent or low expression of PR in breast and endometrium cancer was linked to unfavorable patient outcome. This observation seemingly reflects a loss of PR expression during cellular dedifferentiation as part of tumor progression.Positive PR immunostaining was found in 239 tumors from 30 different categories in nonbreast and nongynecological tumors. In this group, a particularly high rate of PR positivity was observed in sex cord stromal tumors of the testis and in several neuroendocrine neoplasms. Among neuroendocrine tumors, there was a noticeable accumulation of positive cases among tumors originating from the pancreas. PR positivity was found in 54% of neuroendocrine tumors and in 36% of neuroendocrine carcinomas of the pancreas which is consistent with earlier studies describing PR positivity in 58-82% of pancreatic neuroendocrine tumors [51–53]. Given that only 0-6% of intestinal and none of the neuroendocrine tumors of the lung showed PR immunostaining, immunohistochemical PR analysis appears to represent a relevant diagnostic tool to determine the origin of metastases from neuroendocrine tumors. In concordance with our results, PR immunostaining has thus been proposed in the differential diagnosis between metastasis of small bowel neuroendocrine tumor and pancreatic neuroendocrine tumor [54]. The high rate of PR positive neuroendocrine tumors of the pancreas corresponds to the strong nuclear PR immunostaining in islets of Langerhans in normal pancreatic tissue [55, 56]. It is therefore not surprising that reduced PR staining, potentially a sign of dedifferentiation, was associated with the presence of lymph node metastasis in our pancreatic neuroendocrine tumors. Viale et al. also found reduced PR positivity associated with presence of metastases [53]. Another rare tumor entity of the pancreas, solid pseudopapillary neoplasm (which was not analyzed in this study), also was shown to express PR and therefore could come into differential diagnosis when evaluating a PR positive pancreatic tumor [51].Other neuroendocrine neoplasms that showed PR expression in a significant fraction of cases predominantly included poorly differentiated small-cell neuroendocrine carcinomas from various sites of origin and medullary carcinoma of the thyroid. It is of note that various other tumors of the thyroid gland also showed PR immunostaining in 7-12% of cases. Other investigators have reported even higher rates of PR positivity in 39-76% of papillary thyroid carcinoma [13, 14, 21–23] and 17% of follicular thyroid carcinoma [57]. A dependency of PR in the pathogenesis of at least some thyroid cancers could explain why thyroid cancer is more than twice as common in women compared to men [58] and is the second most common type of cancer in pregnancy [59]. Bertoni et al. have demonstrated a direct effect of progesterone on thyroid cells, upregulating genes involved in thyroid function and growth [60]. Furthermore, patients receiving mifepristone, a PR blocker, had a decrease in thyroid hormone levels [61]. Interestingly, among 7,657 nonmammary and nongynecological tumors, significantly, more PR-positive tumors were seen in women (3.2%) than in men (1.6%), although there are no great quantitative differences in the progesterone serum levels between women and men outside the luteal phase [62].In summary, our analysis of 147 different tumor types for PR immunostaining provides a ranking list of tumor entities according to their prevalence of PR positivity. Given the highly discordant literature data, such a ranking order would have been difficult to extract from the existing literature (summarized in Supplementary Figure3). These data help to better understand the diagnostic utility of PR IHC. The distinction of neuroendocrine neoplasms derived from the pancreas appears to represent a particularly strong and poorly known application of PR IHC.



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