Introduction: GATA3 is a transcription factor involved in epithelial cell differentiation. GATA3 immunostaining is used as a diagnostic marker for breast and urothelial cancer but can also occur in other neoplasms. Methods: To evaluate GATA3 in normal and tumor tissues, a tissue microarray containing 16,557 samples from 131 different tumor types and subtypes and 608 samples of 76 different normal tissue types was analyzed by immunohistochemistry. Results: GATA3 positivity was found in 69 different tumor types including 23 types (18%) with at least one strongly positive tumor. Highest positivity rates occurred in noninvasive papillary urothelial carcinoma (92–99%), lobular carcinoma (98%), carcinoma of no special type of the breast (92%), basal cell carcinoma of the skin (97%), invasive urothelial carcinoma (73%), T-cell lymphoma (23%), adenocarcinoma of the salivary gland (16%), squamous cell carcinoma of the skin (16%), and colorectal neuroendocrine carcinoma (12%). In breast cancer, low GATA3 staining was linked to high pT stage (p = 0.03), high BRE grade (p < 0.0001), HER2 overexpression (p = 0.0085), estrogen and progesterone receptor negativity (p < 0.0001 each), and reduced survival (p = 0.03). Conclusion: Our data demonstrate that GATA3 positivity can occur in various tumor entities. Low levels of GATA3 reflect cancer progression and poor patient prognosis in breast cancer.

© 2023 S. Karger AG, Basel

Introduction

The GATA3 protein is a transcription factor which is critical for the embryonic development of various tissues including the parathyroid gland and the kidney [1]. GATA3 plays a role in the luminal differentiation of breast epithelium [2, 3], development of the collecting system of the kidney and the urothelium [4-6], and trophoblastic differentiation [7, 8]. In lymphoid cells, GATA3 regulates the expression of a wide range of biologically and clinically important genes which impact inflammatory and humoral immune response [9]. GATA3 is required for the formation of T helper (Th) cells, especially Th2 cells which are pivotal for the development of allergic and humoral immune response [9-11]. GATA3 also plays a role in cancer biology. It is one of the most commonly mutated genes in breast cancer where it plays a role in estrogen and androgen receptor signaling [12-15].

Normal tissue analyses have revealed GATA3 expression in breast, kidney, urothelium, parathyroid, placenta, skin, salivary gland, and lymphatic organs [16-23]. Because of its selective expression in a rather limited number of tissues, GATA3 is used as an immunohistochemical marker in diagnostic pathology, where GATA3 is mainly used for supporting the distinction of breast or urothelial cancer from other diagnostic options [6, 24]. In the literature, the positivity rates range from 70 to 100% for lobular breast cancer [25-28], from 30% to 100% for breast cancer of no special type (NST) [26, 29-33], and from 45–99% for muscle-invasive urothelial carcinoma [34-43]. Discrepant data (also) exist for various other tumor types. For example, GATA3 positivity has been described in 0–44% of gastric adenocarcinoma [44-47], 0–74% of non-small cell lung adenocarcinoma [27, 48-50], 0–76% of clear cell papillary renal cell carcinoma [6, 17, 51], 9–44% of anaplastic thyroid carcinoma [33, 52], and 13–57% of serous endometrial carcinomas [53, 54]. These conflicting data are probably caused by the use of different antibodies, immunostaining protocols, and criteria to determine GATA3 positivity in these studies.

To better understand the prevalence and significance of GATA3 expression in cancer, a comprehensive study analyzing a large number of neoplastic and non-neoplastic tissues under highly standardized conditions is desirable. Therefore, GATA3 expression was analyzed in more than 16,000 tumor tissue samples from 131 different tumor types and subtypes as well as 76 non-neoplastic tissue categories by immunohistochemistry (IHC) in a tissue microarray (TMA) format in this study.

Materials and MethodsTissue Microarrays

Our 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 16,557 primary tumors from 131 tumor types and subtypes. Detailed histopathological and molecular data were available for 1,475 cancers of the breast. Clinical follow-up data were accessible from 877 breast cancer patients with a median follow-up time of 49 months. The composition of normal and tumor TMAs is described 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. The TMA manufacturing process was described earlier in detail [55, 56]. In brief, one tissue spot (diameter: 0.6 mm) was transmitted from a representative tumor containing donor block in an empty recipient paraffin block. The use of archived remnants of diagnostic tissues for TMA manufacturing, their analysis for research purposes, and patient data were according to local laws (HmbKHG, §12), and analysis had been approved 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 cut TMA sections were immunostained on one day and in one experiment. Two different primary antibodies were used for GATA3 detection: MSVA-450M (mouse monoclonal; cat.# 2980-450M; MS Validated Antibodies GmbH, Hamburg, Germany) and clone L50-823 (mouse monoclonal, cat.# MSK100-05, Zytomed Systems, Berlin, Germany). For both antibodies, slides were deparaffinized and exposed to heat-induced antigen retrieval for 5 min in an autoclave at 121°C in a pH 7.8 buffer. MSVA-450M was applied at 37°C for 60 min at a dilution of 1:50. L50-823 was applied at 37°C for 60 min at a dilution of 1:300. Bound antibody was then visualized using the Envision Kit (Dako, Glostrup, Denmark) according to the manufacturer’s directions. All tissues were manually scored by one pathologist (VR). Questionable cases were discussed with the supervisor of the study (FJ). For normal tissues, staining intensity was recorded as 0, 1+, 2+, and 3+. For tumor tissues, the percentage of positive neoplastic cells was estimated, and the staining intensity was recorded as 0, 1+, 2+, and 3+. For statistical analyses, the tumor staining results were categorized into four groups as described before [57]. Tumors without any staining were considered negative. Tumors with 1+ staining intensity in ≤70% of tumor cells or 2+ intensity in ≤30% of tumor cells were considered weakly positive. Tumors with 1+ staining intensity in >70% of tumor cells, or 2+ intensity in 31–70%, or 3+ intensity in ≤30% of tumor cells were considered moderately positive. Tumors with 2+ intensity in >70% or 3+ intensity in >30% of tumor cells were considered strongly positive. Multiplex fluorescence IHC and digital image analysis were performed on prostate cancer tissue to assign GATA3 expression in lymphocytes to specific cell subsets. For details, see online supplementary methods (see www.karger.com/doi/10.1159/000527382 for all online suppl. material).

Statistics

Statistical calculations were performed with JMP 14 software (SAS Institute Inc., NC, USA). Contingency tables and the χ2 test were performed to search for associations between GATA3 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 considered as statistically significant.

ResultsTechnical Issues

A total of 13,093 (79%) of 16,557 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. A sufficient number of samples of each normal tissue type were evaluable.

GATA3 in Normal Tissues

By using MSVA-450M, nuclear GATA3 immunostaining was seen in urothelium (+++), squamous epithelium of the skin (+++; superficial cell layers > basal cell layers), hair follicles (+++), sebaceous glands (++), parathyroid gland (+++), trophoblastic cells (+++), chorion cells (+++), and amnion cells (+) of the placenta, collecting ducts (++; not all) and glomerular podocytes (++) of the kidney, seminal vesicle epithelium (+++), tall columnar cells and basal cells (++) of the epididymis, a fraction of the luminal cells of breast glands (+++), basal cells in the prostate (+; not always visible), glandular cells (especially mucinous) of salivary glands (+ - ++), and a fraction of lymphocytes, most prominently in the thymus (++). Multiplex fluorescence IHC identified the positive lymphocytes as Th cells (online suppl. Fig. 1). A faint cytoplasmic GATA3 staining was seen in gastric glands and goblet cells of respiratory epithelium, most likely representing nonspecific background staining. Representative images of GATA3 in normal tissues analyzed with MSVA-450M are shown in Figure 1. GATA3 immunostaining was absent in gastrointestinal epithelium, gallbladder, liver, pancreas, lung, fallopian tube, endometrium, and endocervical glands of the uterus, ovary, nonkeratinizing squamous epithelium from various sites, mesenchymal tissues, pituitary gland, and the brain. All cell types identified as positive by MSVA-450M were confirmed by L50-823 staining (online suppl. Fig. 2).

Fig. 1.

GATA3 immunostaining of normal tissues. The panels show a strong nuclear GATA3 positivity of predominantly suprabasal cells in the epidermis of the skin (a), urothelial cells of all layers (b), podocytes and collecting duct cells of the kidney (c), luminal cells of the breast (d), trophoblastic cells of the placenta (e), and epithelial cells of the parathyroid gland (f).

GATA3 in Cancer

Positive GATA3 immunostaining was detectable in 2,567 (19.6%) of the 13,093 analyzable tumors, including 315 (2.4%) with weak, 285 (2.2%) with moderate, and 1,967 (15%) with strong immunostaining. Overall, 69 (53%) of 131 tumor categories showed detectable GATA3 expression with 23 (18%) tumor categories including at least one case with strong positivity (Table 1). Representative images of GATA3-positive tumors are shown in Figure 2. The highest rate of positive staining and the highest levels of expression were found in various subtypes of breast and urinary bladder neoplasms as well as in basal cell carcinoma of the skin. At lower frequency and often at lower intensity, GATA3 immunostaining could be seen in various categories of salivary gland tumors, neuroendocrine tumors (NETs) as well as in squamous cell carcinomas or tumors containing squamous cell elements such as endometroid carcinomas. In the cases of phyllodes tumor, myoepithelial tumors, and other biphasic tumors, always the epithelial component was positive. A graphical representation of a ranking order of GATA3-positive and strongly positive tumors is given in Figure 3. In a cohort of 1,079 breast cancers of NST, low or absent GATA3 immunostaining was linked to advanced pT stage (p = 0.032), high BRE grade (p < 0.0001), HER2 overexpression (p = 0.0085), estrogen and progesterone receptor negativity (p < 0.0001 each; Table 2) as well as reduced overall survival (p = 0.0304; Fig. 4).

Table 1.

GATA3 immunostaining in human tumors

Table 2.

GATA3 immunostaining and tumor phenotype in breast carcinoma of NST

Fig. 2.

GATA3 immunostaining in cancer. The panels show a strong GATA3 positivity in cases of noninvasive (pTa) urothelial carcinoma (a), muscle-invasive urothelial carcinoma (b), invasive lobular carcinoma of the breast (c), basal cell carcinoma of the skin (d) as well as a weak to moderate GATA3 staining in a squamous cell carcinoma of the skin (e) and absence of staining in a prostatic adenocarcinoma (Gleason 5 + 5 = 10) (f).

Fig. 3.

Ranking order of GATA3 immunostaining in tumors. Both the frequency of positive cases (blue dots) and the frequency of strongly positive cases (orange dots) are shown.

Fig. 4.

GATA3 immunostaining and overall survival in no special type breast carcinoma.

Discussion

The International Working Group for Antibody Validation (IWGAV) had proposed that antibody validation for IHC on formalin fixed tissues should include a comparison with expression data obtained by another independent method or by a different antibody to the same target [58]. Our normal tissue analysis revealed nuclear GATA3 immunostaining in parathyroid gland, urothelium, squamous epithelium of the skin, placenta, kidney, seminal vesicle, prostate, breast, salivary glands, and lymphatic tissues, most prominently in the thymus. These findings almost completely match data from three independent RNA screening studies including the Human Protein Atlas (HPA) RNA seq tissue dataset [59], the FANTOM5 project [60, 61], and the Genotype-Tissue Expression (GTEx) project [62]. The only discrepancy between our data and these RNA findings is that GATA3 positivity in the vagina as suggested by GTEx and FANTOM5 is not confirmed by our analysis. As we found significant GATA3 staining in keratinizing but not in nonkeratinizing squamous epithelium and because keratinization can occur at the vaginal orifice, we assume that differences in sampling might be responsible for this disagreement. That all GATA3-positive cell types were also detected by a second independent antibody further validates our findings. That multicolor immunofluorescence identified most GATA3-positive lymphocytes in prostate cancer tissue as CD4+ Th cells is well consistent with the literature (reviewed in [9, 63]).

The data obtained by an analysis of 13,093 tumors from 131 different subtypes were largely reflective of the GATA3 expression in corresponding normal tissues. This was to be expected because it has already been shown for many other diagnostic biomarkers that expression of tissue-specific proteins is often retained in neoplasms [64, 65]. That urothelial carcinoma and breast cancer were the most commonly GATA3-positive cancers may be because the urothelium and breast glands are the most cancer susceptible organs among GATA3-positive normal tissues. We found a striking difference in the rate of GATA3 positivity between basal cell carcinoma (97% positive) and squamous cell carcinoma of the skin (16% positive). Previous studies on 14–62 samples per entity reported rather similar overall positivity rates for basal cell (90–100%) and squamous cell carcinomas (81–88%) [19, 33, 66, 67], but found marked differences in the staining intensity or fraction of stained tumor cells. For example, Mertens et al. reported strong staining in 13 of 14 GATA3-positive basal cell carcinomas but in only 4 of 21 GATA3-positive squamous cell cancers [19], and Miettinen et al. [33] mentioned strong and uniform positivity in all cells of basal cell carcinomas but a wide range (5–100%) of positive tumor cells in squamous cell cancers. It is likely that different experimental conditions account for a higher sensitivity to detect (very) low GATA3 expression in these studies. However, GATA3 was more intense in superficial than in basal cell layers of the normal skin in our study, suggesting that GATA3 may belong to these proteins that gradually enrich when cells are pushed from the basal layers through the stratum spinosum toward the outer cell layers of the skin [68]. A general role of GATA3 for a specific growth and maturation status of squamous epithelium – independent from the site of origin – is further supported by our observation that the GATA3 positivity rate was only minimally higher in squamous cell carcinomas derived from the GATA3-positive skin than in squamous cell carcinomas derived from GATA3-negative nonkeratinizing squamous epithelia.

The markedly lower rate and intensity of GATA3 positivity in salivary gland tumors as compared to breast and urothelial cancer fit to the somewhat lower GATA3 expression levels in normal salivary glands as compared to normal breast and urothelial cells. The low rate of GATA3 positivity in kidney and prostate cancer is obviously due to the rarity of malignant transformation of GATA3-positive cell types in these organs. Most renal cell carcinomas develop from the proximal or distal tubules and not from collecting ducts or even podocytes [69], and prostate neoplasms do only very rarely originate from basal cells [70].

Our data support the use of GATA3 IHC for several applications that have been earlier proposed. This includes the distinction of metastatic urothelial and breast carcinomas (often GATA3+) from other metastatic carcinomas (mostly GATA3−) [71, 72], the distinction of urothelial carcinoma (mostly GATA3+) [73] from prostatic carcinoma (mostly GATA3−) [74], and the distinction of metastatic lobular carcinoma of the breast (GATA3+) [75, 76] from gastric signet ring cell carcinoma (GATA3−) [46]. It is of note, however, that a weak or even moderate to strong GATA3 immunostaining can occasionally occur in various further clinically important tumor entities including neuroendocrine cancer (NEC), adenocarcinoma of the lung, malignant mesothelioma, germ cell tumor, renal cell carcinoma, prostate cancer, and pancreatic adenocarcinoma.

The availability of clinical and molecular data enabled us to interrogate the potential clinical significance of GATA3 expression in breast cancer. That a reduced GATA3 expression was linked to unfavorable tumor phenotype, HER2 positivity, ER/PR negativity, and poor prognosis is consistent with the literature [31, 77-81]. These findings are consistent with a model assuming that reduced GATA3 expression in tumors derived from GATA3-positive precursor cells reflects cellular dedifferentiation which is generally linked to unfavorable tumor phenotype and increased cancer aggressiveness. A key result of this study is a ranking order of human tumors according to the prevalence and intensity of GATA3 expression. The comparison with previously published data in the literature demonstrates that this information cannot easily be obtained from the literature given the highly discrepant data on many tumor entities (Fig. 5). While the prevalence levels described in this study are specific to the reagents and the protocol used in our laboratory, it should be expected that the standardized use of other specific antibodies should result in a similar ranking order. In an earlier study, 2,500 cancers from 60 different tumor entities had been analyzed in a study applying a different GATA3 antibody, another protocol, and other scoring criteria [33]. Although these authors found markedly higher GATA3 positivity rates in most tumor types (indicated in Fig. 5) than in our study, the ranking order was indeed highly comparable to the data obtained in our study.

Fig. 5.

GATA3 positivity in the literature. An “X” indicates the fraction of GATA3-positive tumors in the present study, and dots indicate the reported frequencies from the literature for comparison: red dots mark studies with ≤10 tumors, yellow dots mark studies with 11–25 tumors, and black dots mark studies with >25 tumors. The blue rhombus highlights results from the large multi-tumor study by Miettinen et al. [33].

In summary, our data demonstrate that detectable GATA3 expression can occur in various tumor entities including breast, urothelial, salivary gland, squamous cell, and other tumors. Particularly, high frequency and levels of GATA3 occur in breast and urothelial carcinoma. A reduced level of GATA3 reflects cancer progression and poor patient prognosis in breast cancer.

Acknowledgments

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

Statement of Ethics

Consent was not required due to local/national guidelines. 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.

Conflict of Interest Statement

The monoclonal mouse GATA3 antibody, clone MSVA-450M, was provided from MS Validated Antibodies GmbH (owned by a family member of GS).

Funding Sources

No funding was received.

Author Contributions

Viktor Reiswich, Ronald Simon, Guido Sauter, and Frank Jacobsen contributed to conception, design, data collection, data analysis, and manuscript writing. Carol E. Schmidt, Sören Weidemann, Franzisak Büscheck, Claudia Hube-Magg, Doris Höflmayer, Christian Bernreuther, Stefan Steurer, and Sarah Minner participated in pathology data analysis and data interpretation. Katharina Möller, Ria Uhlig, Christoph Fraune, Maximilian Lennartz, Sören Weidemann, Andreas H. Marx, Franziska Büscheck, Doris Höflmayer, Christian Bernreuther, Patrick Lebok, Guido Sauter, Stefan Steurer, Eike Burandt, David Dum, Till Krech, Sarah Minner, Frank Jacobsen, Till S. Clauditz, and Andrea Hinsch: collection of samples. Ronald Simon, Niclas C. Blessin, Elana Bady, Claudia Hube-Magg, Viktor Reiswich, and Frank Jacobsen: data analysis. Viktor Reiswich, Frank Jacobsen, and Guido Sauter: study supervision. All authors agreed to be accountable for the content of the work.

Data Availability Statement

Further inquiries can be directed to the corresponding author. Raw data are available upon reasonable request. All data relevant to the study are included in the article and its online supplementary material.

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