Trophoblast Cell Surface Antigen 2 Expression in Human Tumors: A Tissue Microarray Study on 18,563 Tumors.

INTRODUCTION: Trophoblast cell surface antigen 2 (TROP2) is the target of sacituzumab govitecan, an antibody-drug conjugate approved for treatment of triple negative breast cancer and urothelial carcinoma.
METHODS: A tissue microarray containing 18,563 samples from 150 different tumor types and subtypes as well as 608 samples of 76 different normal tissue types was analyzed by TROP2 immunohistochemistry.
RESULTS: TROP2 positivity was found in 109 tumor categories, including squamous cell carcinomas of various origins, urothelial, breast, prostate, pancreatic, and ovarian cancers (>95% positive). High TROP2 expression was linked to advanced stage (p = 0.0069) and nodal metastasis (p < 0.0001) in colorectal cancer as well as to nodal metastasis in gastric adenocarcinoma (p = 0.0246) and papillary thyroid cancer (p = 0.0013). Low TROP2 expression was linked to advanced stage in urothelial carcinoma (p < 0.0001), high pT (p = 0.0024), and high grade (p < 0.0001) in breast cancer, as well as with high Fuhrmann grade (p < 0.0001) and pT stage (p = 0.0009) in papillary renal cell carcinomas.
CONCLUSION: TROP2 is expressed in many epithelial neoplasms. TROP2 deregulation can be associated with cancer progression in a tumor-type dependent manner. Since anti-TROP2 cancer drugs have demonstrated efficiency, they may be applicable to a broad range of tumor entities in the future. The Author(s). Published by S. Karger AG, Basel.

Introduction

Trophoblast cell surface antigen 2 (TROP2), also known as tumor-associated calcium signal transducer 2 or EpCAM 2, is a membrane glycoprotein coded by the tumor-associated calcium signal transducer 2 gene at chromosome 1p32 [1, 2]. TROP2 acts as a cell surface receptor with a role in cell self-renewal, proliferation, and transformation [3, 4, 5]. In embryonic development, TROP2 plays a critical role in placenta formation, embryo implantation, stem cell proliferation, and organ development [5]. TROP2 can be found overexpressed in many cancer types. Consistent with its role in embryogenesis, TROP2 expression is regulated by several oncogenic transcription factors such as CREB1, nuclear factor-κB, HOXA10, HNF4A, TP63, TP53, ERG, HNF1A/TCF-1, and FOXP3 [6, 7]. TROP2 knockout cells show a disturbed proliferation, while overexpression of TROP2 accelerates the cancer cell cycle and drives cancer growth [8].

TROP2 is the target of sacituzumab govitecan (SG; TrodelvyTM), an antibody-drug conjugate that specifically targets TROP2 via the humanized anti-TROP2 antibody, hRS7 IgG which is conjugated to SN-38, the active metabolite of irinotecan [9]. SG has demonstrated significant clinical benefit in metastatic triple negative breast cancer [10], hormone-positive breast cancer [11], small-cell lung cancer [12], nonsmall-cell lung cancer [13], and metastatic urothelial carcinomas [14]. SG has recently been granted accelerated approval by the FDA for metastatic triple negative breast cancer patients that have failed at least two prior therapies [15]. SG has also been granted Fast Track Designation from the FDA for the treatment of adult urothelial cancer patients in the neoadjuvant/adjuvant, locally advanced or metastatic setting who have previously received a programmed death receptor-1 or programmed death-ligand 1 inhibitor and a platinum-containing chemotherapy or who are platinum ineligible and have previously received a programmed death receptor-1 or programmed death-ligand 1 inhibitor [16]. A number of studies have analyzed TROP2 expression in cell lines and clinical samples of urothelial [17], salivary gland [18], ovarian [19, 20, 21], endometrial [22], gastric [23, 24, 25], oral squamous cell [26, 27], lung [28], and cervical [29] carcinomas. However, the predictive role of TROP2 expression for response to SG is not entirely clear. Although high response rates have been reported particularly from cancers with moderate to strong TROP2 expression, there are also studies suggesting response in tumors with low TROP2 levels [12, 30, 31].

Currently, most available data on TROP2 expression in cancer tissues are based on RNA profiling and are available from public databases such as The Cancer Genome Atlas (TCGA) Research Network (https://www.cancer.gov.tcga) or the Gene Expression Omnibus (GEO) [32]. Only few studies have so far analyzed large cohorts of cancers for protein expression by immunohistochemistry (IHC) [33, 34]. To better understand the prevalence of TROP2 protein expression in cancer, TROP2 expression was analyzed in more than 18,000 tumor tissue samples from 150 different tumor types and subtypes as well as 76 non-neoplastic tissue categories by IHC in a tissue microarray (TMA) format in this study.

Material and Methods

Tissue Microarrays

The normal TMA was composed of 8 samples from 8 different donors for each of 76 different normal tissue types (608 samples on one slide). The cancer TMAs contained a total of 18,563 primary tumors from 150 tumor types and subtypes. Detailed histopathological data on grade, pathological tumor (pT) stage, or pathological lymph node (pN) status were available from subsets of breast cancers (n = 2,139), urothelial carcinomas (n = 1,073), high-grade serous ovarian cancers (n = 344), endometroid endometrial cancers (n = 160), colorectal (n = 2,351), gastric (n = 327), and pancreatic carcinomas (n = 598) as well as clear cell (n = 1,224) and papillary (n = 310) renal cell carcinomas. Clinical follow-up data were available from 877 breast cancer, 850 kidney cancer, and 254 bladder cancer patients (treated by cystectomy) with a median follow-up time of 43/39/14 months (range 1–88; 1–250; 1–77). The composition of both normal and cancer TMAs is described in detail in the result section. All samples were retrieved 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. The TMA manufacturing process has previously been described in detail [35, 36]. In brief, one tissue spot (diameter: 0.6 mm) per patient was transmitted from a cancer containing donor block into 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.

Immunohistochemistry

Freshly prepared TMA sections were immunostained on 1 day in one experiment. Slides were deparaffinized with xylol, rehydrated through a graded alcohol series, and exposed to heat-induced antigen retrieval for 5 min in an autoclave at 121°C in pH 7.8 DakoTarget Retrieval SolutionTM (Agilent, Santa Clara, CA, USA; #S2367). Endogenous peroxidase activity was blocked with Dako Peroxidase-Blocking SolutionTM (Agilent, Santa Clara, CA, USA; #52023) for 10 min. Primary antibody-specific against TROP2 protein (recombinant rabbit monoclonal, MSVA-733R, MS Validated Antibodies, Hamburg, Germany) was applied at 37°C for 60 min at a dilution of 1:150. For the purpose of antibody validation, the normal tissue TMA was also analyzed with the polyclonal goat TROP2 antibody (R&D; # AF650) at a dilution of 1:450 and an otherwise identical protocol. Bound antibody was visualized using the EnVision KitTM (Agilent, Santa Clara, CA, USA; #K5007) according to the manufacturer's directions. The sections were counterstained with haemalaun. For tumor tissues, the percentage of TROP2-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.

Statistics

Statistical calculations were performed with JMP 14 software (SAS Institute Inc., Cary, NC, USA). Contingency tables and the χ2 were performed to search for associations between TROP2 immunostaining 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.

Results

Technical Aspects

A total of 16,024 (86.3%) of 18,563 tumor samples were interpretable in our TMA analysis. Noninterpretable samples demonstrated lack of unequivocal tumor cells or loss of the tissue spot during technical procedures. Enough samples of each normal tissue type were evaluable to enable a characterization of the normal tissue staining pattern.

TROP2 in Normal Tissues

TROP2 immunostaining was always membranous and found in many epithelial cell types. TROP2 staining was strong in all keratinizing and nonkeratinizing squamous epithelia. In the skin and the esophagus, the basal cell layers were either negative or markedly less stained, while all cell layers of the squamous epithelium of the uterine cervix and the tonsil surface showed an identical staining intensity. Skin appendices (sebaceous glands, hair follicles), tonsil crypt epithelium, and epithelial cells of the thymus including corpuscles of Hassall's also showed strong TROP2 staining. A strong TROP2 positivity was seen in all cells of the respiratory epithelium, while a moderate staining occurred in pneumocytes and in bronchial glands. In the placenta, staining was strong in amnion, chorion, and cytotrophoblast cells. TROP2 staining was largely absent in the gastrointestinal tract (GIT), with the exception of the superficial epithelial cell layers of the stomach and few scattered TROP2-positive epithelial cells (<0.1%) in the duodenum, ileum, appendix, and colon epithelium. In the pancreas, a strong positivity occurred in excretory and intercalated ducts, while acinar cells showed a variable staining, predominantly located at the apical membranes. Islets of Langerhans were TROP2 negative. A strong TROP2 staining was also seen in all epithelial cells of salivary glands, gallbladder epithelium, intrahepatic bile ducts, breast glands, fallopian tube (not all cells), endocervix, endometrium (not all glands of secretion phase), urothelium, prostate (less intensive staining in basal than in luminal cells), most epithelial cells of the seminal vesicle, and the epididymis. In the kidney, a strong TROP2 immunostaining occurred in distal tubuli and collecting ducts, while staining was weak to moderate in the parietal layer of the Bowman capsule. In the thyroid gland, a moderate to strong TROP2 staining of apical membranes was seen in a variable number of follicles. A small subset of epithelial cells of the adenohypophysis also showed a moderate TROP2 positivity. A small subset of positive cells was seen in the bone marrow, perhaps reflecting granulocyte precursor cells. Examples of TROP2 immunostaining in normal tissues are shown in Figure 1. TROP2 immunostaining was always absent in Brunner glands, hepatocytes, testis, decidua cells, ovary (stroma, follicular cysts, corpus luteum), adrenal gland, parathyroid gland, aorta, fat, muscles of all types, and the brain. All these findings were obtained by using the monoclonal rabbit recombinant antibody MSVA-733R and the goat polyclonal antibody AF650, although the polyclonal antibody resulted into somewhat more background staining (online suppl. Fig. 1; for all online suppl. material, see www.karger.com/doi/10.1159/000522206).

Fig. 1

TROP2 immunostaining of normal tissues. The panels show a strong TROP2 positivity of surface epithelial cells of the tonsil (a), urothelium of the urinary bladder (b), and the endometrium (c) as well as in acinar and basal cells of the prostate (d). TROP2 staining is somewhat weaker and largely limited to the most apical elements of the surface epithelium in the stomach antrum (e), distal tubuli and the visceral layer of the Bowman capsule of the kidney (f), and intrahepatic bile ducts of the liver (g). TROP2 immunostaining is lacking in colon epithelial cells (h).

TROP2 in Cancer

TROP2 immunostaining was detectable in 9,691 (60.5%) of the 16,024 analyzable tumors, including 2,259 (14.1%) with weak, 2,899 (18.1%) with moderate, and 4,533 (28.3%) with strong positivity. Overall, 109 (72.6%) of 150 tumor categories showed detectable TROP2 expression with 89 (59.3%) tumor categories including at least 1 case with strong positivity (Table 1). Among 86 epithelial tumor entities, 84 (97.7%) showed detectable TROP2 expression with 78 (90.7%) tumor categories including at least 1 case with strong positivity. The only exceptions were 4 cases of medullary thyroid carcinomas and one colorectal neuroendocrine tumor (NET), which did not show detectable TROP2 staining. Particularly high rates of positivity and high expression levels were seen in squamous cell carcinomas of various origins and various categories of urothelial, breast, prostate, pancreatic, and ovarian cancers. Representative images of TROP2 immunostaining in tumor tissues are shown in Figure 2. A graphical representation of a ranking order of TROP2-positive and strongly positive cancers is given in Figure 3. The relationship between TROP2 immunostaining and histopathological features is shown in Table 2. High TROP2 expression was linked to advanced stage (p = 0.0069), nodal metastasis (p < 0.0001), blood vessel (V+, p = 0.0012), and lymph vessel invasion (L1, p < 0.0001) in colorectal cancer and nodal metastasis in gastric adenocarcinoma (p = 0.0246) and papillary thyroid cancer (p = 0.0013). Low TROP2 expression was linked to advanced stage in urothelial carcinoma (p < 0.0001), high pT (p = 0.0024), high grade (p < 0.0001), and “triple negative receptor status” (p = 0.001) in breast cancer, as well as with high Fuhrmann grade (p < 0.0001) and pT stage (p = 0.0009) in papillary renal cell carcinomas. Associations between TROP2 expression and clinicopathological features were not found in clear cell renal cell carcinomas, high-grade serous ovarian carcinomas, pancreatic adenocarcinomas, and endometroid endometrial carcinomas.

Table 1

TROP2 immunostaining in human tumors

	Tumor entity	On TMA, n	TROP2 immunostaining result
			analyzable, n	negative, %	weak, %	moderate, %	strong, %
Tumors of the skin	Pilomatrixoma	35	26	46.2	38.5	0.0	15.4
	Basal cell carcinoma	88	79	21.5	54.4	16.5	7.6
	Benign nevus	29	26	100.0	0.0	0.0	0.0
	Squamous cell carcinoma of the skin	90	89	3.4	20.2	43.8	32.6
	Malignant melanoma	46	43	100.0	0.0	0.0	0.0
	Malignant melanoma lymph node metastasis	86	85	98.8	0.0	0.0	1.2
	Merkel cell carcinoma	46	33	100.0	0.0	0.0	0.0
Tumors of the head and neck	Squamous cell carcinoma of the larynx	110	88	39.8	4.5	15.9	39.8
	Squamous cell carcinoma of the pharynx	60	57	0.0	14.0	12.3	73.7
	Oral squamous cell carcinoma (floor of the mouth)	130	115	14.8	21.7	24.3	39.1
	Pleomorphic adenoma of the parotid gland	50	31	41.9	29.0	22.6	6.5
	Warthin tumor of the parotid gland	104	82	0.0	0.0	1.2	98.8
	Adenocarcinoma, NOS (papillary cystadenocarcinoma)	14	12	0.0	25.0	50.0	25.0
	Salivary duct carcinoma	15	11	0.0	27.3	36.4	36.4
	Acinic cell carcinoma of the salivary gland	181	131	17.6	38.2	28.2	16.0
	Adenocarcinoma NOS of the salivary gland	109	69	0.0	20.3	46.4	33.3
	Adenoid cystic carcinoma of the salivary gland	180	87	12.6	40.2	37.9	9.2
	Basal cell adenocarcinoma of the salivary gland	25	24	16.7	16.7	37.5	29.2
	Basal cell adenoma of the salivary gland	101	81	29.6	13.6	23.5	33.3
	Epithelial-myoepithelial carcinoma of the salivary gland	53	51	3.9	7.8	47.1	41.2
	Mucoepidermoid carcinoma of the salivary gland	343	242	5.8	4.5	37.2	52.5
	Myoepithelial carcinoma of the salivary gland	21	18	50.0	16.7	27.8	5.6
	Myoepithelioma of the salivary gland	11	9	66.7	11.1	0.0	22.2
	Oncocytic carcinoma of the salivary gland	12	11	27.3	27.3	18.2	27.3
	Polymorphous adenocarcinoma, low grade, of the salivary gland	41	37	10.8	10.8	67.6	10.8
	Pleomorphic adenoma of the salivary gland	53	43	51.2	11.6	23.3	14.0
Tumors of the lung, pleura, and thymus	Adenocarcinoma of the lung	196	181	6.1	4.4	33.7	55.8
	Squamous cell carcinoma of the lung	80	72	0.0	12.5	6.9	80.6
	Small-cell carcinoma of the lung	16	11	27.3	63.6	9.1	0.0
	Mesothelioma, epithelioid	39	31	83.9	12.9	3.2	0.0
	Mesothelioma, other types	76	52	84.6	13.5	1.9	0.0
	Thymoma	29	23	39.1	43.5	17.4	0.0
Tumors of the female genital tract	Squamous cell carcinoma of the vagina	78	46	0.0	10.9	23.9	65.2
	Squamous cell carcinoma of the vulva	130	109	0.9	12.8	27.5	58.7
	Squamous cell carcinoma of the cervix	129	114	0.0	2.6	18.4	78.9
	Adenocarcinoma of the cervix	21	21	0.0	4.8	19.0	76.2
	Endometrioid endometrial carcinoma	236	203	2.5	16.7	20.7	60.1
	Endometrial serous carcinoma	82	55	7.3	18.2	34.5	40.0
	Carcinosarcoma of the uterus	48	42	28.6	19.0	23.8	28.6
	Endometrial carcinoma, high grade, G3	13	12	33.3	33.3	16.7	16.7
	Endometrial clear cell carcinoma	8	6	16.7	16.7	16.7	50.0
	Endometrioid carcinoma of the ovary	110	93	4.3	22.6	29.0	44.1
	Serous carcinoma of the ovary	559	514	1.8	29.8	26.5	42.0
	Mucinous carcinoma of the ovary	96	75	20.0	16.0	32.0	32.0
	Clear cell carcinoma of the ovary	50	46	10.9	47.8	23.9	17.4
	Carcinosarcoma of the ovary	47	37	27.0	43.2	8.1	21.6
	Granulosa cell tumor of the ovary	37	35	100.0	0.0	0.0	0.0
	Leydig cell tumor of the ovary	4	4	100.0	0.0	0.0	0.0
	Sertoli cell tumor of the ovary	1	1	0.0	100.0	0.0	0.0
	Sertoli Leydig cell tumor of the ovary	3	3	100.0	0.0	0.0	0.0
	Steroid cell tumor of the ovary	3	3	100.0	0.0	0.0	0.0
	Brenner tumor	41	38	2.6	2.6	21.1	73.7
	Tumors of the breast	Invasive breast carcinoma of no special type	1,764	1,594	1.6	8.8	31.3	58.3
	Lobular carcinoma of the breast	363	309	1.3	7.8	51.5	39.5
	Medullary carcinoma of the breast	34	32	15.6	12.5	43.8	28.1
	Tubular carcinoma of the breast	29	17	11.8	0.0	47.1	41.2
	Mucinous carcinoma of the breast	65	46	21.7	10.9	17.4	50.0
	Phyllodes tumor of the breast	50	40	7.5	0.0	60.0	32.5
Tumors of the digestive system	Adenomatous polyp, low-grade dysplasia	50	27	44.4	51.9	3.7	0.0
	Adenomatous polyp, high-grade dysplasia	50	46	37.0	52.2	8.7	2.2
	Adenocarcinoma of the colon	2,482	2,171	52.3	32.5	10.9	4.2
	Gastric adenocarcinoma, diffuse type	176	144	29.2	20.8	41.7	8.3
	Gastric adenocarcinoma, intestinal type	174	160	16.9	36.3	30.6	16.3
	Gastric adenocarcinoma, mixed type	62	57	21.1	22.8	42.1	14.0
	Adenocarcinoma of the esophagus	83	75	18.7	16.0	41.3	24.0
	Squamous cell carcinoma of the esophagus	75	66	0.0	10.6	24.2	65.2
	Squamous cell carcinoma of the anal canal	89	69	5.8	10.1	20.3	63.8
	Cholangiocarcinoma	50	40	35.0	20.0	30.0	15.0
	Gallbladder adenocarcinoma	31	29	6.9	10.3	34.5	48.3
	Gallbladder Klatskin tumor	41	38	2.6	15.8	36.8	44.7
	Hepatocellular carcinoma	300	287	60.3	10.8	13.2	15.7
	Ductal adenocarcinoma of the pancreas	612	383	0.5	6.5	42.6	50.4
	Pancreatic/ampullary adenocarcinoma	89	66	7.6	16.7	40.9	34.8
	Acinar cell carcinoma of the pancreas	16	15	40.0	13.3	33.3	13.3
	GIST	50	47	100.0	0.0	0.0	0.0
Tumors of the urinary system	Noninvasive papillary urothelial carcinoma, pTa G2 low grade	177	125	0.0	1.6	29.6	68.8
	Noninvasive papillary urothelial carcinoma, pTa G2 high grade	141	106	0.0	1.9	14.2	84.0
	Noninvasive papillary urothelial carcinoma, pTa G3	219	162	0.0	4.9	22.2	72.8
	Urothelial carcinoma, pT2–4 G3	735	587	5.3	2.7	16.4	75.6
	Squamous cell carcinoma of the bladder	22	20	10.0	5.0	25.0	60.0
	Small-cell NEC of the bladder	23	21	85.7	4.8	4.8	4.8
	Sarcomatoid urothelial carcinoma	25	23	56.5	17.4	8.7	17.4
	Urothelial carcinoma of the kidney pelvis	62	60	5.0	6.7	15.0	73.3
	Clear cell renal cell carcinoma	1,287	1,180	87.1	6.0	6.0	0.8
	Papillary renal cell carcinoma	368	339	25.1	15.9	24.8	34.2
	Clear cell (tubulo) papillary renal cell carcinoma	26	23	30.4	21.7	21.7	26.1
	Chromophobe renal cell carcinoma	170	153	42.5	37.9	10.5	9.2
	Oncocytoma	257	228	67.5	22.8	4.8	4.8
Tumors of the male genital organs	Adenocarcinoma of the prostate, Gleason 3 + 3	83	83	0.0	2.4	24.1	73.5
	Adenocarcinoma of the prostate, Gleason 4 + 4	80	80	0.0	3.8	28.8	67.5
	Adenocarcinoma of the prostate, Gleason 5 + 5	85	84	4.8	10.7	36.9	47.6
	Adenocarcinoma of the prostate (recurrence)	258	250	2.4	8.4	31.6	57.6
	Small-cell NEC of the prostate	19	12	41.7	33.3	0.0	25.0
	Seminoma	621	592	99.7	0.3	0.0	0.0
	Embryonal carcinoma of the testis	50	47	87.2	10.6	2.1	0.0
	Leydig cell tumor of the testis	30	30	100.0	0.0	0.0	0.0
	Sertoli cell tumor of the testis	2	2	100.0	0.0	0.0	0.0
	Sex cord stromal tumor of the testis	1	1	100.0	0.0	0.0	0.0
	Spermatocytic tumor of the testis	1	1	100.0	0.0	0.0	0.0
	Yolk sac tumor	50	46	71.7	26.1	0.0	2.2
	Teratoma	50	36	66.7	2.8	8.3	22.2
	Squamous cell carcinoma of the penis	80	78	1.3	14.1	39.7	44.9
Tumors of endocrine organs	Adenoma of the thyroid gland	114	113	74.3	20.4	5.3	0.0
	Papillary thyroid carcinoma	392	374	16.0	13.9	11.8	58.3
	Follicular thyroid carcinoma	154	150	62.7	19.3	12.7	5.3
	Medullary thyroid carcinoma	111	108	84.3	13.9	0.9	0.9
	Parathyroid gland adenoma	43	40	90.0	7.5	2.5	0.0
	Anaplastic thyroid carcinoma	45	44	70.5	9.1	4.5	15.9
	Adrenal cortical adenoma	50	47	97.9	0.0	2.1	0.0
	Adrenal cortical carcinoma	26	22	90.9	4.5	0.0	4.5
	Phaeochromocytoma	50	47	100.0	0.0	0.0	0.0
	Appendix, NET	22	16	87.5	12.5	0.0	0.0
	Colorectal, NET	12	11	100.0	0.0	0.0	0.0
	Ileum, NET	49	49	100.0	0.0	0.0	0.0
	Lung, NET	19	18	88.9	5.6	5.6	0.0
	Pancreas, NET	97	83	81.9	12.0	3.6	2.4
	Colorectal, NEC	12	11	72.7	9.1	9.1	9.1
	Gallbladder, NEC	4	4	75.0	25.0	0.0	0.0
	Pancreas, NEC	14	14	78.6	21.4	0.0	0.0
Tumors of hematopoietic and lymphoid tissuesw	Hodgkin lymphoma	103	78	100.0	0.0	0.0	0.0
	B-SLL/B-CLL	50	47	100.0	0.0	0.0	0.0
	DLBCL	114	108	100.0	0.0	0.0	0.0
	Follicular lymphoma	88	87	100.0	0.0	0.0	0.0
	T-cell non-Hodgkin lymphoma	24	24	100.0	0.0	0.0	0.0
	Mantle cell lymphoma	18	18	100.0	0.0	0.0	0.0
	Marginal zone lymphoma	16	15	100.0	0.0	0.0	0.0
	DLBCL in the testis	16	16	100.0	0.0	0.0	0.0
	Burkitt lymphoma	5	3	100.0	0.0	0.0	0.0
Tumors of soft tissue and bone	Tenosynovial giant cell tumor	45	29	100.0	0.0	0.0	0.0
	Granular cell tumor	53	32	100.0	0.0	0.0	0.0
	Leiomyoma	50	49	100.0	0.0	0.0	0.0
	Leiomyosarcoma	87	76	100.0	0.0	0.0	0.0
	Liposarcoma	132	112	100.0	0.0	0.0	0.0
	MPNST	13	13	100.0	0.0	0.0	0.0
	Myofibrosarcoma	26	26	100.0	0.0	0.0	0.0
	Angiosarcoma	73	59	88.1	11.9	0.0	0.0
	Angiomyolipoma	91	88	100.0	0.0	0.0	0.0
	Dermatofibrosarcoma protuberans	21	17	100.0	0.0	0.0	0.0
	Ganglioneuroma	14	14	92.9	7.1	0.0	0.0
	Kaposi sarcoma	8	5	100.0	0.0	0.0	0.0
	Neurofibroma	117	104	99.0	1.0	0.0	0.0
	Sarcoma, NOS	74	72	100.0	0.0	0.0	0.0
	Paraganglioma	41	41	100.0	0.0	0.0	0.0
	Ewing sarcoma	23	18	100.0	0.0	0.0	0.0
	Rhabdomyosarcoma	6	6	83.3	16.7	0.0	0.0
	Schwannoma	121	113	93.8	6.2	0.0	0.0
	Synovial sarcoma	12	11	100.0	0.0	0.0	0.0
	Osteosarcoma	43	34	100.0	0.0	0.0	0.0
	Chondrosarcoma	38	19	100.0	0.0	0.0	0.0
	Rhabdoid tumor	5	5	100.0	0.0	0.0	0.0

NOS, not otherwise specified; MPNST, malignant peripheral nerve sheath tumor; DLBCL, diffuse large B-cell lymphoma; B-SLL/B-CLL, small lymphocytic lymphoma, B-cell type; NEC, neuroendocrine carcinoma; GIST, gastrointestinal stromal tumor.

Fig. 2

TROP2 immunostaining in cancer. The panels show a strong, membranous, and cytoplasmatic TROP2 immunostaining in a squamous cell carcinoma of the oral cavity (a), a recurrent adenocarcinoma (Gleason 5 + 5 = 10) of the prostate (b), a breast cancer of no special type (c), a gastric adenocarcinoma (d), a papillary carcinoma of the thyroid (e), and an adenocarcinoma of the lung (f). TROP2 staining is absent in an epithelioid pleural mesothelioma (g) and a colorectal adenocarcinoma (h).

Fig. 3

Ranking order of TROP2 immunostaining in cancers. Both the frequency of positive cases (blue dots) and the frequency of strongly positive cases (orange dots) are shown.

Table 2

TROP2 immunostaining and tumor phenotype in colon, papillary thyroid, breast, urinary bladder, stomach, and papillary kidney carcinomas

	n	TROP2 immunostaining result				p value
		negative, %	weak, %	moderate, %	strong, %
Colon adenocarcinoma
Primary tumor
pT1	79	54.4	35.4	8.9	1.3	0.0069
pT2	406	53.2	34.5	7.4	4.9
pT3	1,157	53.5	32.2	11.2	3.1
pT4	419	49.6	30.1	13.4	6.9
Regional lymph nodes
pN0	1,088	57.9	30.6	8.1	3.4	<0.0001
pN+	963	46.6	34.2	14.1	5.1
Venous invasion
V0	1,479	54.9	31.3	9.6	4.2	0.0012
V1	543	45.9	35.5	14.2	4.4
Lymphatic invasion
L0	661	58.9	31.2	7	3	<0.0001
L1	1,368	49.3	33	12.9	4.8
Tumor localization
Left colon	1,122	52	35	8.9	4.1	0.0273
Right colon	425	53.2	29.2	13.4	4.2
MMR status
Defective	86	51.2	34.9	10.5	3.5	0.9848
Proficient	1,071	51.1	35.5	9.4	4
RAS mutation status
Mutated	325	48.3	38.5	9.8	3.4	0.2722
Wild type	414	54.8	32.6	8.5	4.1
BRAF mutation status
Mutated	14	42.9	14.3	28.6	14.3	0.1262
Wild type	90	56.7	28.9	10	4.4
Papillary thyroid carcinomas
Primary tumor
pT1	151	11.9	17.9	9.3	60.9	0.0487
pT2	76	26.3	14.5	11.8	47.4
pT3–4	96	12.5	11.5	7.3	68.8
Regional lymph nodes
pN0	89	20.2	13.5	7.9	58.4	0.0013
pN+	122	4.1	10.7	7.4	77.9
Breast carcinoma of no special type
Primary tumor
pT1	899	0.9	6.8	30.1	62.2	0.0024
pT2	796	2	8.3	33.5	56.2
pT3–4	182	4.9	8.8	35.2	51.1
Grade
G1	215	0.5	2.8	22.3	74.4	<0.0001
G2	1,050	1.8	5.6	33.2	59.3
G3	659	2.3	12.3	33.2	52.2
Regional lymph nodes
pN0	872	1.9	7.1	32.3	58.6	0.286
pN1	406	1.5	8.6	31.3	58.6
pN2	148	1.4	6.1	35.8	56.8
pN3	100	4	6	44	46
HER2 status
Negative	995	1.9	9.9	30.3	57.9	0.6044
Positive	125	0.8	12	32.8	54.4
ER status
Negative	233	1.7	19.7	24.9	53.6	<0.0001
Positive	822	1.9	8	31.4	58.6
PR status
Negative	457	2.2	13.1	29.5	55.1	0.0305
Positive	654	1.7	7.8	31.5	59
Triple negative
No	858	2	9	31.2	57.8	0.001
Yes	158	1.3	20.3	24.1	54.4
Urinary bladder carcinoma
Primary tumor
pTa G2 low	125	0	1.6	29.6	68.8	<0.0001
pTa G2 high	106	0	1.9	14.2	84
pTa G3	133	0.8	6	26.3	66.9
pT2	122	3.3	0.8	13.9	82
pT3	203	3	3.4	17.2	76.4
pT4	97	8.2	2.1	16.5	73.2
Regional lymph nodes
pN0	242	3.7	2.1	16.1	78.1	0.9068
pN+	170	3.2	3.2	16	77.6
Gastric carcinoma
Laurén type
Diffuse	66	30.3	24.2	43.9	1.5	0.0208
Intestinal	81	22.2	38.3	29.6	9.9
Mixed	57	21.1	22.8	42.1	14
Tumor stage
pT1–2	48	35.4	27.1	29.2	8.3	0.1352
pT3	114	22.8	21.9	42.1	13.2
pT4	111	17.1	27.9	46.8	8.1
Regional lymph nodes
pN0	69	31.9	30.4	30.4	7.2	0.0246
pN1	58	27.6	19	39.7	13.8
pN2	55	16.4	18.2	47.3	18.2
pN3	90	16.7	30	47.8	5.6
Mismatch repair status
MMR defective	32	46.9	18.8	18.8	15.6	0.0002
MMR proficient	233	14.6	24.9	48.9	11.6
Papillary renal cell carcinomas
ISUP stage
1	41	17.1	22	19.5	41.5	0.0005
2	134	14.9	17.2	25.4	42.5
3	81	39.5	14.8	28.4	17.3
4	7	57.1	14.3	14.3	14.3
Fuhrmann grade
1	4	0	50	0	50	<0.0001
2	183	14.2	17.5	25.1	43.2
3	83	41	18.1	24.1	16.9
4	11	45.5	9.1	36.4	9.1
Thoenes grade
1	58	13.8	17.2	22.4	46.6	0.1706
2	157	26.1	17.8	24.2	31.8
3	18	33.3	16.7	33.3	16.7
	n	TROP2 immunostaining result	p value
		negative, %	weak, %	moderate, %	strong, %
UICC stage
1	102	23.5	17.6	22.5	36.3	0.0097
2	15	13.3	13.3	46.7	26.7
3	5	80	0	0	20
4	11	36.4	0	54.5	9.1
Primary tumor
1	208	21.2	17.8	24.5	36.5	0.0009
2	48	10.4	18.8	33.3	37.5
3–4	33	54.5	9.1	21.2	15.2
Regional lymph nodes
0	25	36	4	28	32	0.4776
≥1	15	33.3	6.7	46.7	13.3
Distant metastasis
0	27	25.9	11.1	25.9	37	0.2267
≥1	12	41.7	8.3	41.7	8.3

Discussion

Considering the large scale of our study, emphasis was placed on the appropriate validation of our assay. The International Working Group for Antibody Validation (IWGAV) has proposed that antibody validation for IHC on formalin-fixed tissues should include either a comparison of the findings obtained by two different independent antibodies or a comparison with expression data obtained by another independent method [37]. Seventy-six different normal tissue categories were utilized for assay validation in this study. This broad range of tissues is likely to contain most proteins that are normally expressed at relevant levels in cells of adult humans and should therefore enable the detection of most undesired cross-reactivities of tested antibodies. RNA expression data derived from three independent RNA screening studies, including the Human Protein Atlas (HPA) RNA-seq tissue dataset [38], the FANTOM5 project [39, 40], and the Genotype-Tissue Expression (GTEx) project, had shown particularly high TROP2 expression levels in all tissues covered by squamous epithelium and salivary glands and − at somewhat lower levels − also in breast, prostate, salivary glands, and the urinary bladder [41]. These observations were in concordance with our IHC data. Other cell types that also showed distinct TROP2 immunostaining such as thymic epithelial cells, respiratory epithelium, bronchial glands, amnion and chorion cells of the placenta, superficial epithelial cell layers of the stomach, few scattered epithelial cells in the intestine, gallbladder epithelium, intrahepatic bile ducts as well as of a fraction of cells in the fallopian tube, endometrium, thyroid, and adenohypophysis were all confirmed by the independent second antibody approach (R&D AF650; online suppl. Fig. 1). In all these organs, the TROP2-positive cell populations constitute rather small subsets of the total amount of cells and may thus not have resulted in conspicuous findings in RNA analyses. The widespread expression of TROP2 in a broad range of epithelial cell types is comparable to the expression pattern of its family member TACSTD1 (EpCAM). Antibodies against EpCAM are widely used in routine pathology where they serve as a marker for epithelial cell origin [42]. The main difference between TROP2 and EpCAM is the limitation of TROP2 expression to only few specific cell types in the GIT, whereas EpCAM is abundantly expressed in all epithelial cells of the GIT [43]. The low expression of TROP2 in the GIT may reflect one of the causes for the less severe side effects of anti-TROP2 drugs as compared to various earlier anti-EpCAM drug candidates, many of which earlier failed for systemic therapy [44].

The reported response rates to SG ranged between 19% [13] and 33.3% in clinical trials [15]. Since particularly high levels of TROP2 expression have so far not been required for including patients into clinical trials, it appears possible that absent or very low levels of TROP2 expression might have contributed to some of the therapy failures. Studies performing retrospective analyses of TROP2 expression in cancers treated by SG have indeed described better response rates in strongly TROP2-positive cancers as compared to low expressors [12, 30, 31]. It appears therefore likely that the ranking order of tumor types according to their frequency and intensity of TROP2 immunostaining which was generated in this study might delineate tumor entities that might be particularly suitable for SG therapy. Although our data show that TROP2 expression − often at high level − can be found in virtually all epithelial tumor entities, they do identify several important cancer entities that are particularly prone to high-level TROP2 expression such as squamous cell carcinomas of various origins, urothelial, breast, and ovarian cancers, as well as primary and recurrent adenocarcinomas of the prostate. It is of note that triple negative breast cancer, one of the few cancer types for which SG is currently FDA approved, did not range among the top-ranked TROP2 expressing cancers and only included 54.4% strongly positive cases. Triple negative breast cancer with progression on at least two prior therapies was probably not selected for early SG trials based on their particularly high rate of TROP2 expression but rather because of the lack of further therapeutic options. As compared to triple negative breast cancer, frequency and levels of TROP2 positivity were higher in advanced prostate cancers with recurrence under systemic therapy suggesting that these tumors might also be suitable candidates for SG therapy. Clinical trials applying SG in castration-resistant prostate cancer are ongoing [45]. Other studies investigate the use of SG in urothelial carcinoma [14], nonsmall-cell lung cancer [13], and small-cell lung cancer [12].

The comprehensive analysis of relevant tumor entities for expression of a specific protein also enabled an assessment of the potential diagnostic utility of an antibody in surgical pathology. Based on the marked difference in TROP2 expression between epithelioid mesothelioma (16.1%) and adenocarcinomas of the lung (93.9%), TROP2 IHC could be of use for the difficult distinction of these tumor entities. This differential diagnosis regularly requires the use of diagnostic antibody panels which currently often include BAP-1, WT-1, D2-40, calretinin, EpCAM, TTF-1, and claudin-4 [46]. A low or absent TROP2 expression in an “intestinal-type” adenocarcinoma could favor the diagnosis of colorectal adenocarcinoma (negative or weak TROP2 expression in 84.8%) as morphologically similar tumors such as ductal adenocarcinomas of the pancreas (7% negative/weak), adenocarcinomas of the gall bladder (17.2% negative/weak), and even gastric carcinomas (45–50% negative/weak) typically show low TROP2 expression levels less commonly. Moreover, a high TROP2 expression may favor a diagnosis of papillary over follicular carcinoma of the thyroid. These potential diagnostic applications of TROP2 IHC need to be evaluated in further studies.

The large number of tumors analyzed within several of our tumor categories also enabled an analysis of the potential clinical significance of variable TROP2 expression levels for these tumor entities. The significant association of high TROP2 expression levels with stage progression, nodal metastasis, L1, and V1 status in colorectal carcinomas and with nodal metastasis in papillary thyroid cancers demonstrates that upregulation of TROP2 in a cancer derived from TROP2-negative precursor cells can play a role in cancer progression. Fang et al. [34] and Zhao et al. [47] have also reported an association between high TROP2 expression and unfavorable tumor features in studies on 82–620 colon cancers, and Abdou et al. [48] found a link between TROP2 positivity and lymph node metastasis in 56 papillary thyroid cancers. The significant associations between low TROP2 expression and advanced pT stage and high grade in breast and papillary kidney cancer demonstrate, however, that reduced TROP2 expression in a cancer derived from TROP2-positive precursor cells can also be linked to unfavorable tumor features. Only one study has analyzed the prognostic value of TROP2 in breast cancer as to yet. Ambrogi et al. [33] reported an association between membranous TROP2 expression and poor patient outcome, while merely intracellular TROP2 had a favorable prognostic impact in a study on 702 breast cancers.

Conclusion

Our data show that TROP2 is abundantly expressed in many normal epithelial cell types and in most epithelial neoplasms. Aberrant TROP2 expression including both upregulation and downregulation can be associated with cancer progression in a tumor-type dependent manner. Since anti-TROP2 cancer drugs have demonstrated efficiency and induce tolerable side effects, it can be assumed that these drugs will be applicable to a broad range of tumor entities in the future.

Statement of Ethics

The usage of archived diagnostic leftover tissues for manufacturing of TMAs and their analysis for research purposes as well as patient data analysis has been approved by local laws (HmbKHG, §12.1) and by the Local Ethics Committee (Ethics Commission Hamburg, WF-049/09). All work has been carried out in compliance with the Helsinki Declaration.

Conflict of Interest Statement

The TROP2 antibody clone MSVA-733R was provided from MS Validated Antibodies GmbH (owned by a family member of GS).

Funding Sources

No funding was received.

Author Contributions

D.D., N.T., C.V., W.W., R.S., G.S., A.M., and A.M.L. contributed to conception, design, data collection, data analysis, and manuscript writing. A.H., S.M., M.L., A.M.L., E.B., T.S.C., W.W., C.B., P.L., T.K., and S.S. participated in pathology data analysis and data interpretation. C.F., R.U., N.G., F.J., and S.M.: IHC analysis. A.H.M. and T.K.: conception and design, and collection of samples. C.H.-M. and R.S. performed statistical analysis. D.D., A.M.L., W.W., R.S., and G.S.: study supervision.

Data Availability Statement

Raw data are available upon reasonable request. All data relevant to the study are included in the article. All authors agree to be accountable for the content of the work.

Supplementary data

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Supplementary data

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Supplementary data

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