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. 2024 Sep 28;14(19):2157. doi: 10.3390/diagnostics14192157

Expression of Trefoil Factor 1 (TFF1) in Cancer: A Tissue Microarray Study Involving 18,878 Tumors

Florian Lutz 1, Soo-Young Han 1, Seyma Büyücek 1, Katharina Möller 1, Florian Viehweger 1, Ria Schlichter 1, Anne Menz 1, Andreas M Luebke 1, Ahmed Abdulwahab Bawahab 2, Viktor Reiswich 1, Martina Kluth 1, Claudia Hube-Magg 1, Andrea Hinsch 1, Sören Weidemann 1, Maximilian Lennartz 1, David Dum 1, Christian Bernreuther 1, Patrick Lebok 1,3, Guido Sauter 1, Andreas H Marx 4, Ronald Simon 1,*, Till Krech 1,3, Christoph Fraune 1,3, Natalia Gorbokon 1, Eike Burandt 1, Sarah Minner 1, Stefan Steurer 1, Till S Clauditz 1, Frank Jacobsen 1
Editor: Ludmilla Morozova-Roche
PMCID: PMC11475926  PMID: 39410561

Abstract

Background/Objectives: Trefoil factor 1 (TFF1) plays a role in the mucus barrier. Methods: To evaluate the prevalence of TFF1 expression in cancer, a tissue microarray containing 18,878 samples from 149 tumor types and 608 samples of 76 normal tissue types was analyzed through immunohistochemistry (IHC). Results: TFF1 staining was detectable in 65 of 149 tumor categories. The highest rates of TFF1 positivity were found in mucinous ovarian carcinomas (76.2%), colorectal adenomas and adenocarcinomas (47.1–75%), breast neoplasms (up to 72.9%), bilio-pancreatic adenocarcinomas (42.1–62.5%), gastro-esophageal adenocarcinomas (40.4–50.0%), neuroendocrine neoplasms (up to 45.5%), cervical adenocarcinomas (39.1%), and urothelial neoplasms (up to 24.3%). High TFF1 expression was related to a low grade of malignancy in non-invasive urothelial carcinomas of the bladder (p = 0.0225), low grade of malignancy (p = 0.0003), estrogen and progesterone receptor expression (p < 0.0001), non-triple negativity (p = 0.0005) in invasive breast cancer of no special type, and right-sided tumor location (p = 0.0021) in colorectal adenocarcinomas. Conclusions: TFF1 IHC has only limited utility for the discrimination of different tumor entities given its expression in many tumor entities. The link between TFF1 expression and parameters of malignancy argues for a relevant biological role of TFF1 in cancer. TFF1 may represent a suitable therapeutic target due to its expression in only a few normal cell types.

Keywords: TFF1, tissue microarray, immunohistochemistry, human cancers

1. Introduction

Trefoil factor 1 (TFF1/pS2) belongs to one of three highly conserved proteins of the trefoil factor family, which are co-expressed with mucins in the gastrointestinal tract. TFF1 is predominantly expressed in gastric mucosa [1], where it plays a role in protection and healing of the mucus barrier (summarized in [2]). Therefore, TFF1 expression is stimulated in response to acute mucosal injury and chronic inflammation (summarized in [3]). In superficial cells of gastric mucosa, TFF1 is co-expressed and bound to Mucin 5AC (MUC5AC), supporting a role for TFF1 in the packaging and function of the gastric mucosa [4]. Based on the identification of recurrent TFF1 deletions, missense mutations, and hypermethylation in gastric cancer, TFF1 has been considered as a tumor suppressor protein (summarized in [5]). In line with this notion, TFF1-deficient mice serve as a popular model for gastric cancer development [6]. All TFF1-deficient mice develop adenomas in the gastric mucosa, and 30% of them eventually progress to invasive carcinomas [7].

Because the TFF1 expression in normal tissue strongly predominates in stomach mucosa, TFF1 immunohistochemistry (IHC) has been suggested as a marker for gastric cancer [8]. However, studies have shown that not all gastric adenocarcinomas produce TFF1 and that various other tumor entities can express TFF1. In several cancer types, elevated TFF1 expression has been linked to either poor [9,10] or improved patient prognosis [11,12,13,14]. However, the reported frequencies of TFF1 positivity vary considerably for most tumor types. For example, the range of reported TFF1-positive cases ranged from 16% to 90% in gastric cancer [13,15,16,17], from 51% to 98% in cholangiocarcinoma [18,19,20], from 0% to 100% in breast cancer [14,21,22,23], from 0% to 100% in colorectal cancer [24,25,26,27], from 0% to 40% in pulmonary adenocarcinoma [28,29,30,31], from 0% to 14% in serous ovarian carcinoma [28,32,33], from 0% to 25% in clear cell renal cell carcinoma [28,32,34], from 0% to 91% in prostate cancer [28,35,36], and from 55% to 95% in pancreatic cancer [10,37,38]. The use of different antibodies, staining protocols, and criteria to determine TFF1 positivity represents the most likely cause for these controversial data.

To better understand the role of TFF1 protein expression in cancer and to elucidate the potential diagnostic role of TFF1 IHC, an extensive survey of TFF1 immunostaining in a broad range of tumor types and under highly standardized conditions is needed. TFF1 expression was, thus, evaluated in more than 18,000 tumor tissue samples from 149 different tumor types and subtypes as well as 76 different non-neoplastic tissue types by IHC in a tissue microarray (TMA) format in this study.

2. Materials and Methods

2.1. Tissue Microarrays (TMAs)

The normal TMA included 8 samples from 8 different donors from 76 different normal tissue types (608 samples on one slide). The cancer TMAs contained 18,878 primary tumor probes from 149 tumor entities and tumor subtypes. Data on histopathological and molecular parameters were available for cancers of the colorectum (n = 2351), stomach (n = 327), pancreas (n = 598), urinary bladder (n = 1073), and the breast (n = 2139). A detailed description of the composition of both normal and cancer TMAs is available in the Results section. All tumor probes were from the archives of the Institute of Pathology, University Hospital of Hamburg, Germany; the Department of Pathology, Academic Hospital Fuerth, Germany; and the Institute of Pathology, Clinical Center Osnabrueck, Germany. A detailed description of the TMA manufacturing process is available in previous publications [39,40]. Per patient/tumor, one tissue spot (diameter: 0.6 mm) was used. The preparation of TMAs from archived diagnostic tissue remnants and their analysis for research purposes as well as the analysis of patient data were approved by local laws (HmbKHG, §12) and the local ethics committee (Ethics Committee Hamburg, WF-049/09). All work was conducted in accordance with the Declaration of Helsinki. Immunohistochemical data on MUC5AC expression were available from a previous study [41].

2.2. Immunohistochemistry (IHC)

Freshly cut TMA sections were immunostained in one experiment on one day. TMA sections were deparaffinized with xylol, rehydrated using a graded alcohol series, and incubated in an autoclave at 121 °C for 5 min in Dako Target Retrieval Solution, pH9 (Agilent Technologies, Santa Clara, CA, USA; #S2367) for heat-induced antigen retrieval. For blocking of endogenous peroxidase activity, TMA sections were incubated in Dako REAL Peroxidase-Blocking Solution (Agilent Technologies, Santa Clara, CA, USA; #S2023) for 10 min. Primary antibody specific for TFF1 (mouse monoclonal, MSVA-482M, MS Validated Antibodies, Hamburg, Germany; #5784-482M) was applied at 37 °C for 60 min at a dilution of 1:150. For antibody validation, the TMA with normal tissue probes was analyzed by the rabbit recombinant monoclonal TFF1 antibody [EPR3972] (Abcam; Cambridge, UK; #ab92377, dilution 1:900) under identical protocol. Bound antibody was visualized with Dako REAL EnVision Detection System Peroxidase/DAB+, Rabbit/Mouse kit (Agilent Technologies, Santa Clara, CA, USA; #K5007) according to the manufacturer’s directions. The sections were counterstained with hemalaun. For all tumor tissues, the staining intensity was recorded (semi-quantitatively as 0, 1+, 2+, 3+), and the fraction of positive tumor cells was estimated as percentage. For statistical analyses, four groups were determined: negative, tumors without any staining; weak, tumors with 1+ staining intensity in ≤70% of neoplastic cells and 2+ intensity in ≤30% of neoplastic cells; moderate, tumors with 1+ staining intensity in >70% of neoplastic cells, 2+ intensity in 31–70%, or 3+ intensity in ≤30% of neoplastic cells; strong, tumors with 2+ intensity in >70% or 3+ intensity in >30% of neoplastic cells.

2.3. Statistics

Statistical calculations were performed with JMP® software (Version 17) SAS®, Cary, NC, USA). Contingency tables, the chi2-test, and Fisher’s exact test were performed to search for associations between TFF1 immunostaining and tumor phenotype and MUC5AC immunostaining.

3. Results

3.1. Technical Issues

A total of 16,817 (89.1%) of 18,878 tumor samples were interpretable in our TMA analysis. Non-interpretable samples demonstrated the absence of unequivocal tumor cells or a complete lack of individual tissue spots. A sufficient number of samples of each normal tissue type was evaluable (≥4).

3.2. TFF1 in Normal Tissues

TFF1 immunostaining was always cytoplasmic. It was strongest in superficial epithelial cells of the stomach, while the deeper glandular cells remained TFF1 negative. Scattered goblet cells with a significant TFF1 positivity were also seen in the small intestine and the colorectum. A weak to moderate TFF1 immunostaining was also observed in some goblet cells of the respiratory epithelium; groups of mucinous glandular cells in bronchial, sublingual, and submandibular glands; and in a fraction of luminal breast epithelial cells. Occasionally, a few cells with a distinct TFF1 positivity were also observed in the urothelium (mostly umbrella cells) or in gall bladder epithelial cells. Representative images are shown in Figure 1.

Figure 1.

Figure 1

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TFF1 immunostaining of normal tissues. The panels show a cytoplasmic staining of surface epithelial cells but not of glands in the stomach (A), subsets of goblet cells in the duodenum (B) and the colon (C), a subset of mucinous cells in the submandibulary gland (D), a subset of luminal epithelial cells and intraluminal mucus in the breast (E), a small subset of urothelial cells (mostly umbrella cells) in the renal pelvis (F), a large subset of urothelial cells in an inflamed urinary bladder (G), and (focally) in epithelial cells of the gallbladder (H).

All these staining patterns were seen by both MSVA-482M and EPR3972 (Supplementary Figure S1). TFF1 staining was absent in skeletal muscle, heart muscle, smooth muscle, myometrium of the uterus, corpus spongiosum of the penis, ovarian stroma, fat, skin (including hair follicle and sebaceous glands), surface epithelium of the tonsil, ectocervix, squamous epithelium of the esophagus, decidua, placenta, bone marrow; lymph node, spleen, thymus, tonsil, liver, pancreas, Brunner gland of the duodenum, cortex and medulla of the kidney, prostate, seminal vesicle, testis, epididymis, lung, endocervix, endometrium, fallopian tube, ovary (including corpus luteum and theca and granulosa cells of follicular cysts) adrenal medulla, thyroid, parathyroid gland, cerebellum, cerebrum and the pituitary gland.

3.3. TFF1 in Tumor Tissues

TFF1 immunostaining was detectable in 3347 (19.9%) of the 16,817 analyzable tumors, including 2093 (12.4%) with weak, 740 (4.4%) with moderate, and 514 (3.1%) with strong positivity. Overall, 65 (43.6%) of 149 tumor categories showed detectable TFF1 expression, with 32 (21.5%) tumor categories containing at least one case with strong positivity (Table 1).

Table 1.

TFF1 immunostaining in human tumors: neg. = negative, mod. = moderate, str. = strong, ca. = carcinoma.

TFF1 Immunostaining
Tumor Category Tumor Entity on TMA (n) Analy-Zable (n) Neg. (%) Weak (%) Mod. (%) Str. (%)
Tumors of the skin Basal cell carcinoma of the skin 89 81 100.0 0.0 0.0 0.0
Benign nevus 29 21 100.0 0.0 0.0 0.0
Squamous cell carcinoma of the skin 145 132 100.0 0.0 0.0 0.0
Malignant melanoma 65 58 100.0 0.0 0.0 0.0
Malignant melanoma lymph node metastasis 86 86 100.0 0.0 0.0 0.0
Merkel cell carcinoma 2 2 100.0 0.0 0.0 0.0
Tumors of the head and neck Squamous cell carcinoma of the larynx 109 98 98.0 2.0 0.0 0.0
Squamous cell ca. of the pharynx 60 60 100.0 0.0 0.0 0.0
Oral squamous cell carcinoma 130 128 100.0 0.0 0.0 0.0
Pleomorphic adenoma of the parotid gland 50 38 100.0 0.0 0.0 0.0
Warthin tumor of the parotid gland 104 91 98.9 1.1 0.0 0.0
Adenocarcinoma, NOS (Papillary Cystadenocarcinoma) 14 10 90.0 0.0 0.0 10.0
Salivary duct carcinoma 15 10 100.0 0.0 0.0 0.0
Acinic cell carcinoma of the salivary gland 181 110 99.1 0.0 0.9 0.0
Adenocarcinoma NOS of the salivary gland 109 54 88.9 1.9 1.9 7.4
Adenoid cystic carcinoma of the salivary gland 180 61 100.0 0.0 0.0 0.0
Basal cell adenocarcinoma of the salivary gland 25 19 100.0 0.0 0.0 0.0
Basal cell adenoma of the salivary gland 101 60 100.0 0.0 0.0 0.0
Epithelial-myoepithelial carcinoma of the salivary gland 53 44 100.0 0.0 0.0 0.0
Mucoepidermoid carcinoma of the salivary gland 343 250 86.0 8.0 4.0 2.0
Myoepithelial carcinoma of the salivary gland 21 15 100.0 0.0 0.0 0.0
Myoepithelioma of the salivary gland 11 9 100.0 0.0 0.0 0.0
Oncocytic carcinoma of the salivary gland 12 6 100.0 0.0 0.0 0.0
Polymorphous adenoca. low grade, of the salivary gland 41 15 100.0 0.0 0.0 0.0
Pleomorphic adenoma of the salivary gland 53 30 93.3 6.7 0.0 0.0
Tumors of the lung, pleura and thymus Adenocarcinoma of the lung 196 188 89.4 6.9 2.1 1.6
Squamous cell carcinoma of the lung 80 74 100.0 0.0 0.0 0.0
Mesothelioma, epithelioid 40 34 100.0 0.0 0.0 0.0
Mesothelioma, biphasic 29 29 100.0 0.0 0.0 0.0
Thymoma 29 24 100.0 0.0 0.0 0.0
Lung, neuroendocrine tumor (NET) 29 27 92.6 0.0 0.0 7.4
Tumors of the female genital tract Squamous cell carcinoma of the vagina 78 71 100.0 0.0 0.0 0.0
Squamous cell carcinoma of the vulva 157 144 100.0 0.0 0.0 0.0
Squamous cell carcinoma of the cervix 136 129 98.4 1.6 0.0 0.0
Adenocarcinoma of the cervix 23 23 60.9 17.4 13.0 8.7
Endometrioid endometrial carcinoma 338 295 95.6 3.7 0.3 0.3
Endometrial serous carcinoma 86 73 98.6 1.4 0.0 0.0
Carcinosarcoma of the uterus 57 53 98.1 0.0 1.9 0.0
Endometrial carcinoma, high grade, G3 13 8 100.0 0.0 0.0 0.0
Endometrial clear cell carcinoma 9 6 100.0 0.0 0.0 0.0
Endometrioid carcinoma of the ovary 130 115 94.8 4.3 0.0 0.9
Serous carcinoma of the ovary 580 507 99.8 0.2 0.0 0.0
Mucinous carcinoma of the ovary 101 84 23.8 21.4 15.5 39.3
Clear cell carcinoma of the ovary 51 45 100.0 0.0 0.0 0.0
Carcinosarcoma of the ovary 47 47 100.0 0.0 0.0 0.0
Granulosa cell tumor of the ovary 44 42 100.0 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 1 100.0 0.0 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 41 92.7 7.3 0.0 0.0
Tumors of the breast Invasive breast carcinoma of no special type 1764 1692 55.5 26.6 10.9 7.0
Lobular carcinoma of the breast 363 346 47.7 27.2 17.1 8.1
Medullary carcinoma of the breast 34 34 88.2 2.9 8.8 0.0
Tubular carcinoma of the breast 29 23 60.9 21.7 13.0 4.3
Mucinous carcinoma of the breast 65 59 27.1 35.6 13.6 23.7
Phyllodes tumor of the breast 50 46 47.8 30.4 21.7 0.0
Tumors of the digestive system Adenomatous polyp, low-grade dysplasia 50 50 32.0 52.0 12.0 4.0
Adenomatous polyp, high-grade dysplasia 50 48 25.0 50.0 16.7 8.3
Adenocarcinoma of the colon 2483 2256 52.9 35.3 7.0 4.7
Gastric adenocarcinoma, diffuse type 215 176 50.0 13.6 17.6 18.8
Gastric adenocarcinoma, intestinal type 215 192 52.1 33.9 7.8 6.3
Gastric adenocarcinoma, mixed type 62 57 59.6 22.8 8.8 8.8
Adenocarcinoma of the esophagus 83 62 62.9 24.2 8.1 4.8
Squamous cell carcinoma of the esophagus 76 55 98.2 0.0 1.8 0.0
Squamous cell carcinoma of the anal canal 91 85 98.8 0.0 1.2 0.0
Cholangiocarcinoma 121 114 63.2 21.1 7.9 7.9
Gallbladder adenocarcinoma 51 50 48.0 22.0 20.0 10.0
Gallbladder Klatskin tumor 42 38 57.9 21.1 15.8 5.3
Hepatocellular carcinoma 312 304 89.5 5.3 2.0 3.3
Ductal adenocarcinoma of the pancreas 659 613 42.6 28.1 15.0 14.4
Pancreatic/Ampullary adenocarcinoma 98 96 37.5 32.3 18.8 11.5
Acinar cell carcinoma of the pancreas 18 18 100.0 0.0 0.0 0.0
Gastrointestinal stromal tumor (GIST) 62 59 100.0 0.0 0.0 0.0
Appendix, neuroendocrine tumor (NET) 25 16 81.3 6.3 6.3 6.3
Colorectal, neuroendocrine tumor (NET) 12 11 54.5 18.2 27.3 0.0
Ileum, neuroendocrine tumor (NET) 53 51 92.2 5.9 2.0 0.0
Pancreas, neuroendocrine tumor (NET) 101 92 83.7 7.6 4.3 4.3
Colorectal, neuroendocrine carcinoma (NEC) 14 12 75.0 16.7 8.3 0.0
Ileum, neuroendocrine carcinoma (NEC) 8 7 85.7 0.0 14.3 0.0
Gallbladder, neuroendocrine carcinoma (NEC) 4 4 100.0 0.0 0.0 0.0
Pancreas, neuroendocrine carcinoma (NEC) 14 14 92.9 0.0 7.1 0.0
Tumors of the urinary system Non-invasive papillary urothelial ca. pTa G2 low grade 177 152 75.7 19.7 3.3 1.3
Non-invasive papillary urothelial ca., pTa G2 high grade 141 116 82.8 13.8 2.6 0.9
Non-invasive papillary urothelial carcinoma, pTa G3 219 131 90.1 7.6 2.3 0.0
Urothelial carcinoma, pT2-4 G3 735 600 94.5 4.0 1.5 0.0
Squamous cell carcinoma of the bladder 22 22 100.0 0.0 0.0 0.0
Small cell neuroendocrine carcinoma of the bladder 5 5 100.0 0.0 0.0 0.0
Sarcomatoid urothelial carcinoma 25 23 100.0 0.0 0.0 0.0
Urothelial carcinoma of the kidney pelvis 62 61 88.5 11.5 0.0 0.0
Clear cell renal cell carcinoma 1286 1230 99.8 0.2 0.0 0.0
Papillary renal cell carcinoma 368 327 100.0 0.0 0.0 0.0
Clear cell (tubulo) papillary renal cell carcinoma 26 23 100.0 0.0 0.0 0.0
Chromophobe renal cell carcinoma 170 151 98.7 1.3 0.0 0.0
Oncocytoma 257 230 80.9 9.6 9.1 0.4
Tumors of the male genital organs Adenocarcinoma of the prostate, Gleason 3 + 3 83 79 92.4 6.3 1.3 0.0
Adenocarcinoma of the prostate, Gleason 4 + 4 80 69 81.2 14.5 4.3 0.0
Adenocarcinoma of the prostate, Gleason 5 + 5 85 79 89.9 8.9 1.3 0.0
Adenocarcinoma of the prostate (recurrence) 258 216 92.6 6.0 1.4 0.0
Small cell neuroendocrine carcinoma of the prostate 2 2 100.0 0.0 0.0 0.0
Seminoma 682 586 100.0 0.0 0.0 0.0
Embryonal carcinoma of the testis 54 41 100.0 0.0 0.0 0.0
Leydig cell tumor of the testis 31 31 100.0 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 53 42 97.6 2.4 0.0 0.0
Teratoma 53 39 100.0 0.0 0.0 0.0
Squamous cell carcinoma of the penis 92 90 100.0 0.0 0.0 0.0
Tumors of endocrine organs Adenoma of the thyroid gland 113 108 99.1 0.9 0.0 0.0
Papillary thyroid carcinoma 391 374 97.3 2.7 0.0 0.0
Follicular thyroid carcinoma 154 146 100.0 0.0 0.0 0.0
Medullary thyroid carcinoma 111 101 77.2 19.8 2.0 1.0
Parathyroid gland adenoma 43 42 100.0 0.0 0.0 0.0
Anaplastic thyroid carcinoma 45 42 100.0 0.0 0.0 0.0
Adrenal cortical adenoma 50 37 100.0 0.0 0.0 0.0
Adrenal cortical carcinoma 28 28 100.0 0.0 0.0 0.0
Pheochromocytoma 50 50 100.0 0.0 0.0 0.0
Tumors of haemotopoetic and lymphoid tissues Hodgkin’s lymphoma 103 90 100.0 0.0 0.0 0.0
Small lymphocytic lymphoma, B-cell type (B-SLL/B-CLL) 50 50 100.0 0.0 0.0 0.0
Diffuse large B cell lymphoma (DLBCL) 113 113 100.0 0.0 0.0 0.0
Follicular lymphoma 88 88 100.0 0.0 0.0 0.0
T-cell non-Hodgkin’s lymphoma 25 25 100.0 0.0 0.0 0.0
Mantle cell lymphoma 18 18 100.0 0.0 0.0 0.0
Marginal zone lymphoma 16 16 100.0 0.0 0.0 0.0
Diffuse large B-cell lymphoma (DLBCL) in the testis 16 16 100.0 0.0 0.0 0.0
Burkitt lymphoma 5 5 100.0 0.0 0.0 0.0
Tumors of soft tissue and bone Granular cell tumor 23 21 76.2 9.5 14.3 0.0
Leiomyoma 50 45 97.8 2.2 0.0 0.0
Leiomyosarcoma 94 88 100.0 0.0 0.0 0.0
Liposarcoma 96 93 100.0 0.0 0.0 0.0
Malignant peripheral nerve sheath tumor (MPNST) 15 13 100.0 0.0 0.0 0.0
Myofibrosarcoma 26 26 100.0 0.0 0.0 0.0
Angiosarcoma 42 40 100.0 0.0 0.0 0.0
Angiomyolipoma 91 89 100.0 0.0 0.0 0.0
Dermatofibrosarcoma protuberans 21 17 100.0 0.0 0.0 0.0
Ganglioneuroma 14 12 100.0 0.0 0.0 0.0
Kaposi sarcoma 8 6 100.0 0.0 0.0 0.0
Neurofibroma 117 116 100.0 0.0 0.0 0.0
Sarcoma, not otherwise specified (NOS) 74 72 100.0 0.0 0.0 0.0
Paraganglioma 41 40 100.0 0.0 0.0 0.0
Ewing sarcoma 23 18 100.0 0.0 0.0 0.0
Rhabdomyosarcoma 7 7 100.0 0.0 0.0 0.0
Schwannoma 122 119 99.2 0.8 0.0 0.0
Synovial sarcoma 12 11 100.0 0.0 0.0 0.0
Osteosarcoma 19 18 100.0 0.0 0.0 0.0
Chondrosarcoma 15 11 100.0 0.0 0.0 0.0
Rhabdoid tumor 5 5 100.0 0.0 0.0 0.0
Solitary fibrous tumor 17 14 100.0 0.0 0.0 0.0
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Representative images of TFF1-positive tumors are shown in Figure 2. The highest rate of TFF1 positivity was found in mucinous carcinomas of the ovary (76.2%), colorectal adenomas and adenocarcinomas (47.1–75.0%), neoplasms of the breast (11.8–72.9%), bilio-pancreatic adenocarcinomas (42.1–62.5%), gastro-esophageal adenocarcinomas (40.4–50.0%), neuroendocrine neoplasms of various sites of origin (7.1–45.5%), adenocarcinomas of the cervix (39.1%), and in urothelial neoplasms (5.5–24.3%).

Figure 2.

Figure 2

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TFF1 immunostaining in cancer. The panels show TFF1 positivity in a mucinous carcinoma of the ovary (A), a colorectal adenocarcinoma (B), a pancreatic adenocarcinoma (C), a gastric adenocarcinoma (D), an adenocarcinoma of the cervix (E), urothelial carcinoma of the bladder (F), and a neuroendocrine tumor of the lung (G). TFF1 staining is absent in a hepatocellular carcinoma (H).

The ranking order of all TFF1-positive entities is shown in Figure 3. The relationship between TFF1 expression and clinically important histopathological and molecular tumor features in colorectal, gastric, pancreatic, urinary bladder, and breast cancer is shown in Table 2.

Figure 3.

Figure 3

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Ranking order of TFF1 immunostaining in tumors. Both the percentage of positive cases (blue dots) and the percentage of strongly positive cases (orange dots) are shown.

Table 2.

TFF1 immunostaining and tumor phenotype.

TFF1 Immunostaining
n Negative (%) Weak (%) Moderate (%) Strong (%) p Remarks
Invasive breast carcinoma of no special type pT1 802 448 (55.9) 203 (25.3) 94 (11.7) 57 (7.1) 0.3766
pT2 635 331 (52.1) 185 (29.1) 67 (10.6) 52 (8.2)
pT3-4 126 71 (56.3) 35 (27.8) 15 (11.9) 5 (4)
G1 202 106 (52.5) 52 (25.7) 32 (15.8) 12 (5.9) <0.0001
G2 845 423 (50.1) 238 (28.2) 110 (13) 74 (8.8)
G3 564 354 (62.8) 146 (25.9) 36 (6.4) 28 (5)
pN0 692 361 (52.2) 174 (25.1) 102 (14.7) 55 (8) 0.3307
pN+ 946 521 (55.1) 241 (25.5) 111 (11.7) 73 (7.7)
pM0 217 121 (55.8) 52 (24) 28 (12.9) 16 (7.4) 0.7665
pM1 112 60 (53.6) 32 (28.6) 14 (12.5) 6 (5.4)
HER2 negative 890 479 (53.8) 258 (29) 97 (10.9) 56 (6.3) 0.9505
HER2 positive 122 69 (56.6) 33 (27.1) 13 (10.7) 7 (5.7)
ER negative 213 184 (86.4) 21 (9.9) 3 (1.4) 5 (2.4) <0.0001
ER positive 745 334 (44.8) 251 (33.7) 105 (14.1) 55 (7.4)
PR negative 408 277 (67.9) 90 (22.1) 27 (6.6) 14 (3.4) <0.0001
PR positive 598 270 (45.2) 197 (32.9) 82 (13.7) 49 (8.2)
non-triple negative 783 366 (46.7) 258 (33) 102 (13) 57 (7.3) <0.0001
triple negative 144 133 (92.4) 7 (4.9) 1 (0.7) 3 (2.1)
Urothelial bladder carcinoma pTa G2 low 152 115 (75.7) 30 (19.7) 5 (3.3) 2 (1.3) <0.0001
pTa G2 high 116 96 (82.8) 16 (13.8) 3 (2.6) 1 (0.9) 0.0072 1
pTa G3 99 91 (91.9) 5 (5.1) 3 (3) 0 (0)
pT2-4 453 429 (94.7) 19 (4.2) 5 (1.1) 0 (0)
pT2 125 122 (97.6) 3 (2.4) 0 (0) 0 (0) 0.3037 2,3,4
pT3 219 206 (94.1) 9 (4.1) 4 (1.8) 0 (0)
pT4 98 91 (92.9) 6 (6.1) 1 (1) 0 (0)
G2 23 20 (87) 3 (13) 0 (0) 0 (0) 0.0995 2,4
G3 429 408 (95.1) 16 (3.7) 5 (1.2) 0 (0)
pN0 271 261 (96.3) 8 (3) 2 (0.7) 0 (0) 0.0414 2,3
pN+ 161 148 (91.9) 11 (6.8) 2 (1.2) 0 (0)
Adenocarcinoma of the pancreas pT1 14 3 (21.4) 6 (42.9) 2 (14.3) 3 (21.4) 0.3769 5
pT2 66 18 (27.3) 22 (33.3) 15 (22.7) 11 (16.7)
pT3 378 133 (35.2) 125 (33.1) 58 (15.3) 62 (16.4)
pT4 27 12 (44.4) 5 (18.5) 8 (29.6) 2 (7.4)
G1 17 5 (29.4) 7 (41.2) 2 (11.8) 3 (17.6) 0.8595 6
G2 343 115 (33.5) 111 (32.4) 61 (17.8) 56 (16.3)
G3 103 39 (37.9) 32 (31.1) 17 (16.5) 15 (14.6)
pN0 101 28 (27.7) 34 (33.7) 18 (17.8) 21 (20.8) 0.3281
pN+ 383 137 (35.8) 125 (32.6) 65 (17) 56 (14.6)
R0 244 80 (32.8) 90 (36.9) 39 (16) 35 (14.3) 0.4142
R1 201 72 (35.8) 59 (29.4) 37 (18.4) 33 (16.4)
Adenocarcinoma of the stomach pT1-2 62 31 (50) 21 (33.9) 7 (11.3) 3 (4.8) 0.8258 3
pT3 128 70 (54.7) 28 (21.9) 13 (10.2) 17 (13.3)
pT4 122 64 (52.5) 26 (21.3) 14 (11.5) 18 (14.8)
pN0 85 42 (49.4) 23 (27.1) 10 (11.8) 10 (11.8)
pN+ 226 123 (54.4) 50 (22.1) 24 (10.6) 29 (12.8)
MMR proficient 257 136 (52.9) 60 (23.3) 34 (13.2) 27 (10.5) 0.6225 3
MMR deficient 41 20 (48.8) 14 (34.1) 1 (2.4) 6 (14.6)
Adenocarcinoma of the colon pT1 75 30 (40) 39 (52) 4 (5.3) 2 (2.7) 0.6503 5
pT2 421 220 (52.3) 141 (33.5) 39 (9.3) 21 (5)
pT3 1200 604 (50.3) 455 (37.9) 86 (7.2) 55 (4.6)
pT4 435 227 (52.2) 153 (35.2) 28 (6.4) 27 (6.2)
pN0 1125 575 (51.1) 413 (36.7) 82 (7.3) 55 (4.9) 0.9973
pN+ 1003 516 (51.4) 364 (36.3) 73 (7.3) 50 (5)
V0 1533 783 (51.1) 555 (36.2) 112 (7.3) 81 (5.3) 0.6924
V1 563 291 (51.7) 208 (36.9) 41 (7.3) 23 (4.1)
L0 701 369 (52.6) 240 (34.2) 54 (7.7) 38 (5.4) 0.4669
L1 1404 706 (50.3) 529 (37.7) 102 (7.3) 67 (4.8)
right side 427 188 (44) 168 (39.3) 47 (11) 24 (5.6) 0.0021
left side 1173 622 (53) 416 (35.5) 75 (6.4) 59 (5)
MMR proficient 1110 586 (52.8) 402 (36.2) 75 (6.8) 47 (4.2) 0.0292 3
MMR deficient 84 34 (40.5) 35 (41.7) 11 (13.1) 4 (4.8)
RAS wildtype 450 274 (60.9) 131 (29.1) 23 (5.1) 22 (4.9) <0.0001
RAS mutation 340 148 (43.5) 138 (40.6) 38 (11.2) 16 (4.7)
BRAF wildtype 111 70 (63.1) 30 (27) 8 (7.2) 3 (2.7) 0.0331 3
BRAF V600E mutation 19 7 (36.8) 6 (31.6) 3 (15.8) 3 (15.8)
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Abbreviations: pT: pathological tumor stage, G: Grade, pN: pathological lymph node status, pM: pathological status of distant metastasis, L: lymphatic infiltration, V: venous infiltration, R: resection margin status, MMR: mismatch repair. Remarks: p-values corespond to 1: pTa only, 2: pT2-4 only, 3: TFF1 negative vs. TFF1 positive (weak, mod. str.), 4: Fisher’s excact test, 5: pT1-2 vs. pT3-4, 6: G1-2 vs. G3.

High TFF1 expression was significantly associated with a low grade of malignancy in non-invasive (pTa) urothelial carcinomas of the urinary bladder (p = 0.0072), low grade of malignancy (p < 0.0001), estrogen (ER) and progesterone receptor (PR) expression and non-triple negativity (p < 0.0001 each) in invasive breast cancer of no special type, as well as with right-sided tumor location (p = 0.0021), MMR deficiency (p = 0.0292), RAS mutations (p < 0.0001), and BRAF mutation (p = 0.0331) in colorectal adenocarcinomas. The extent of TFF1 staining was unrelated to histopathological parameters of malignancy in 312 gastric and 485 pancreatic adenocarcinomas.

3.4. Comparison with MUC5AC

Data on MUC5AC expression were available for 7870 of our tumors, which were evaluated for TFF1. A total of 50 entities showed expression of either TFF1 or MUC5AC. There was a tendency towards a co-expression of TFF1 and MUC5AC, but this relationship was rather weak. Among 7870 tumor samples with available TFF1 and MUC5AC data, 573 (7.3%) showed expression of both TFF1 and MUC5AC, 1391 (17.7%) showed expression of only TFF1, 325 (4.1%) showed expression of only MUC5AC, and 5581 (70.9%) showed expression of none of the two proteins (p < 0.0001). MUC5AC/TFF1 co-expression was particularly common in mucinous carcinoma of the ovary, gastro-intestinal, and bilio-pancreatic neoplasms but also occurred in other entities. A graphical representation of the expression of MUC5AC and TFF1 in 50 tumor entities is given in Figure 4.

Figure 4.

Figure 4

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Comparison of TFF1 and MUC5AC immunostaining.

4. Discussion

The successful analysis of 16,817 tumors from 149 tumor types and subtypes resulted in a comprehensive overview on the expression of TFF1 in human tumors. The ranking order of 65 tumor entities according to their rate of TFF1 positivity is a key result of our study (Figure 3). The comparative representation of our data and previously published data (Figure 5) demonstrates that this information could not be retrieved from literature databases as many tumor entities have, so far, not been analyzed, and data were often highly discordant for entities that have been analyzed in multiple studies. Our data show that TFF1 positivity occurs most commonly in mucinous carcinomas of the ovary, colorectal adenocarcinomas, breast cancer, bilio-pancreatic, and gastro-esophageal adenocarcinomas, neuroendocrine neoplasms, adenocarcinomas of the cervix uteri, and in urothelial neoplasms. Considering that 65 different tumor entities contained TFF1-positive cases and that the rate of positivity did not exceed 80% in any of the most frequently positive cancer entities, we do not believe that TFF1 IHC has a relevant role for the distinction of cancer entities. However, TFF1 IHC may be useful to select patients for potential future therapies using, for example, antibody drug conjugates. Only recently, the de novo expression of TFF1 has been associated with gemcitabine resistance in pancreatic adenocarcinomas, and TFF1 silencing increased sensitivity to gemcitabine in vitro and in vivo [42].

Figure 5.

Figure 5

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Comparison with previous TFF1 literature. An “X” indicates the fraction of TFF1 positive cancers in the present study, dots indicate the reported frequencies from the literature for comparison: red dots mark studies with ≤10 analyzed tumors, yellow dots mark studies with 11 to 25 analyzed tumors, and green dots mark studies with >25 analyzed tumors.

The striking discrepancy between TFF1 expression in only a few normal tissues and TFF1 expression in so many different cancer types demonstrates that TFF1 upregulation is a common phenomenon in cancer, which may have a significant functional role. The increase in TFF1 staining that was seen in colon adenomas as compared to normal colon mucosa further supports this notion. Earlier studies confirmed TFF1 expression in colon adenomas [8,43,44] and also reported TFF1 staining from other preneoplastic lesions, including, for example, intestinal metaplasia [45]. An oncogenic role would be most conceivable for a protein that is often overexpressed in cancer as compared to its cell of origin. In line with this notion, several authors have indeed described evidence for the oncogenic effects of TFF1. For example, TFF1 promoted anchorage-independent growth in colon carcinoma cells [43], phosphoinositide 3-kinases (PI3K) dependent migration and invasion in gastric carcinoma cells [46], and cyclooxygenase-2 (COX-2) and EGF receptor (EGFR)-dependent angiogenesis in kidney and colonic cancer cells [47]. The molecular mechanism underlying a putative oncogenic function is largely unknown. Rodrigues et al. [43] reported that TFF1 upregulates the phosphatase CDC25A, one of the most crucial cell cycle regulators and activator of CDKs [48], suggesting that both factors cooperate with other oncogenic pathways to drive a adenoma–carcinoma transition in colon carcinoma. However, the significant associations found between TFF1 expression and several favorable tumor features in bladder and breast cancer in combination with the absence of significant links with unfavorable prognostic parameters in colorectal, gastric, and pancreatic cancers at least argues against a significant role of TFF1 for driving cancer aggressiveness. TFF1 RNA expression data derived from The Cancer Genome Atlas (TCGA) database also pinpoint a favorable prognosis for breast and endometrium cancer with high TFF1 expression (https://www.proteinatlas.org/ENSG00000160182-TFF1/pathology, accessed on 9 January 2024). In line with these findings, several functional studies have rather supported a tumor-suppressive than oncogenic role for TFF1. For example, the overexpression of TFF1 suppressed nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-kB) signaling and, therefore, pro-tumorigenic and metastatic properties in colon and gastric cancer cells [49,50] as well as ß-catenin signaling in hepatocellular carcinoma cells [51]. TFF1 knockdown enhanced anchorage-independent growth in breast cancer cells in vivo and in vitro [52], reduced apoptosis and induced proliferation in gastric cancer cells [53], and accelerated the development of esophageal adenocarcinoma [54] and hepatocellular carcinoma in vivo [51].

Previous studies analyzing the prognostic role of TFF1 have provided rather controversial results. For breast cancer, gastric adenocarcinoma and pancreatic ampullary adenocarcinoma studies have found associations between high TFF1 expression levels and both favorable [12,13,14,17,55,56] and unfavorable tumor features [10,57,58]. Significant associations of high TFF1 expression with favorable tumor features or favorable prognosis were also found in gallbladder adenocarcinoma [11], while high TFF1 expression was linked to unfavorable tumor parameters or poor patient outcomes in adenocarcinoma of the lung [30,31], colorectal adenocarcinoma [9,59], and in urothelial carcinoma [60]. Another study in renal cell carcinomas did not suggest any relationship between TFF1 expression and parameters of cancer aggressiveness [34].

The availability of data from an earlier study enabled us to compare our TFF1 results with MUC5AC data [41]. Although these proteins are frequently co-expressed and act cooperatively in normal tissues, the relationship between the expression of TFF1 and MUC5AC was rather weak in cancer, since only 7% of 2289 cancer samples with TFF1 and/or MUC5AC expression expressed both proteins. We, therefore, assume that MUC5AC and TFF1 are functionally less dependent on each other in neoplastic than in normal tissues. That mucinous carcinoma of the ovary, gastro-intestinal, and bilio-pancreatic neoplasms showed co-expression of TFF1 and MUC5AC (20% to 53%) more commonly than other tumor entities is in line with some previous studies describing high rates of MUC5AC/TFF1 co-expression in neoplasms of the colon, liver, stomach, and gallbladder [8,18,61,62]. However, other authors found lower co-expression rates in gastric [63] and colorectal adenocarcinomas [25], and markedly higher frequencies of co-expression were reported for cancers from the esophagus, lung, and bladder [31,64,65].

To validate our assay, our IHC results in normal tissue were compared with RNA results from three different publicly available databases [66,67,68,69] and with immunohistochemical data from an independent anti-TFF1 antibody. The antibody comparison study included 76 different categories of normal tissues to ensure the broadest possible range of expressed proteins. The assay validity was supported by the detection of TFF1 strong immunostaining in the stomach, which was the only normal organ for which RNA expression had previously been reported. The additional positive TFF1 stainings in scattered goblet cells in the small intestine, colorectum, and respiratory epithelium, some mucinous glandular cells in salivary and in bronchial glands, a fraction of luminal breast epithelial cells as well as in some cells of the urothelium and gallbladder epithelium were all validated by identical stainings seen by the independent antibody EPR3972. Given that these TFF1-positive cells constituted very small subpopulations of the respective organs, we assume that TFF1 RNA had not been detected due to a massive dilution if RNAs from total organs were analyzed.

5. Conclusions

Our data provide a comprehensive overview on the expression of TFF1 in human cancer. Given that TFF1 is expressed in a broad range of tumor entities, TFF1 IHC may have only limited utility for the discrimination of different tumor entities. Upregulation in multiple tumor entities and the significant link between TFF1 expression and parameters of malignancy in several tumors argue for a relevant biological role of TFF1 in cancer, however. TFF1 may also represent a suitable therapeutic target.

Acknowledgments

We are grateful to Melanie Steurer, Laura Behm, Inge Brandt, and Sünje Seekamp for excellent technical assistance.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/diagnostics14192157/s1. Supplementary Figure S1. Immunohistochemistry validation by comparison of two antibodies. The panels show immunostaining results obtained by two independent TFF1 antibodies. Using MSVA-482M, a distinct cytoplasmic staining was seen in surface epithelial cells (but not of glands) in the stomach (A), subsets of goblet cells in the duodenum (B) and the colon (C), a subset of mucinous cells in the submandibulary gland (D), a subset of luminal epithelial cells and intraluminal mucus in the breast (E), a small subset of urothelial cells (mostly umbrella cells) in the renal pelvis (F), a large subset of urothelial cells in an inflamed urinary bladder (G), and in epithelial cells of the gallbladder (H). Using clone EPR3972, a comparable staining was seen in the stomach (a), the duodenum (b), the colon (c), the submandibulary gland (d), the breast (e), the renal pelvis (f), the bladder (g), and the gallbladder (h). The images A-H and a-h are from consecutive tissue sections. Supplementary Table S1. Comparison of TFF1 staining in normal tissues and corresponding tumor types.

diagnostics-14-02157-s001.zip (723.8KB, zip)

Author Contributions

F.L., S.-Y.H., M.K., R.S. (Ronald Simon) and F.J.: contributed to conception, design, data collection, data analysis and manuscript writing. F.L., S.B., K.M., F.V., R.S. (Ria Schlichter), A.M., A.M.L., A.A.B., V.R., A.H., S.W., M.L., D.D., C.B., P.L., A.H.M., T.K., C.F., N.G., E.B., S.M., S.S., F.J. and T.S.C.: participated in pathology data analysis, data interpretation, and collection of samples. F.L., R.S. (Ria Schlichter), M.K. and C.H.-M.: data analysis. F.J., R.S. (Ronald Simon) and G.S.: study supervision. All authors agree to be accountable for the content of the work. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

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.

Informed Consent Statement

Patient consent was waived due to local laws (HmbKHG, §12,1) that permit research with anonymized diagnostic leftover tissue samples.

Data Availability Statement

Dataset available on request from the authors.

Conflicts of Interest

The mouse monoclonal antibody, clone MSVA-482M, was provided from MS Validated Antibodies GmbH, Hamburg, Germany (owned by a family member of GS).

Funding Statement

This research received no external funding.

Footnotes

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

diagnostics-14-02157-s001.zip (723.8KB, zip)

Data Availability Statement

Dataset available on request from the authors.

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