Expression of Annexin A3 in cancer: A tissue microarray study involving more than 17,000 cancers from 148 tumor entities

Expression of Annexin A3 in cancer: A tissue microarray study involving more than 17,000 cancers from 148 tumor entities | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Expression of Annexin A3 in cancer: A tissue microarray study involving more than 17,000 cancers from 148 tumor entities Clara von Bargen, Nayira Hakimi, Fiete Gehrisch, Anne Menz, Florian Lutz, and 28 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9278991/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Background. Annexin A3 (ANXA3) is a member of the annexin family of calcium-dependent phospholipid-binding proteins and plays a major role in various membrane-related processes. Marterial & Methods. ANXA3 expression was analyzed by immunohistochemistry (IHC) on tissue microarrays (TMAs) containing 17,285 samples from 148 different tumor types. Results. ANXA3 staining was seen in 10,223 (75.9%) of the 13,472 interpretable tumors and was considered weak in 16.3%, moderate in 10.1%, and strong in 49.4% of cases. Of 148 tumor categories, 128 showed ANXA3 expression in at least one case, and 110 included at least one case with strong staining. High ANXA3 expression was linked to high grade (p<0.0001), muscle invasive growth (pTa vs pT2-4; p<0.0001) and high pT-stage (p=0.0267) in urothelial carcinoma; lymphatic invasion (p=0.0307) in colorectal adenocarcinoma; high pT-stage (p=0.0211) in gastric adenocarcinoma; high pT-stage (p<0.0001), nodal positivity (p=0.0004) and high grade (p<0.0001) in hepatocellular carcinoma; and high pT-stage (p=0.0410) in papillary thyroid carcinoma. Low ANXA3 expression was linked with lymphatic invasion (p<0.0001) in urothelial carcinoma, high tumor grade in invasive breast carcinoma of no special type (p<0.0001); high UICC- (p=0.0008) and pT-stage (p<0.0001) in clear cell renal cell carcinoma; high ISUP grade (p=0.0135), high UICC- (p=0.0073) and pT-stage (p=0.0003) in papillary renal cell carcinoma; advanced pT-stage (p=0.0464) in pancreatic ductal adenocarcinoma; high pT-stage (p=0.0056) and mismatch repair deficiency (p<0.0001) in colorectal adenocarcinoma; and high pT-stage (p=0.0427) in endometrioid endometrial carcinoma. Conclusion. It is concluded, that ANXA3 is abundantly expressed across a broad spectrum of cancer entities and that it exerts a tissue-dependent oncogenic or tumor-suppressive role. Health sciences/Biomarkers Biological sciences/Cancer Health sciences/Oncology Annexin A3 ANXA3 cancer immunohistochemistry tissue microarray Figures Figure 1 Figure 2 Figure 3 Introduction Annexin A3 (ANXA3) is a member of the annexin family of calcium-dependent phospholipid-binding proteins 1 . Similarly, as other annexin family members, ANXA3 is primarily involved in membrane-related processes including transmembranous signaling, membrane organization and repair, vesicle trafficking, cytoskeletal interaction and regulation of ion channels. Its diverse functions extend across various cellular processes such as apoptosis, cell growth, inflammation, and cell differentiation 2 . Although ANXA3 knockout mice are viable and do not exhibit significant developmental abnormalities, they display altered responses to stress and inflammation, suggesting a role for ANXA3 in cellular homeostasis and disease susceptibility 3,4 . ANXA3 is expressed in numerous different normal cell types 5 . Altered ANXA3 expression has been found in many conditions including immune and inflammatory disease 3,6-11 as well as in cancer 2,12 . Several preclinical models have demonstrated that ANXA3 upregulation can confer a malignant phenotype by inducing cell growth, epithelial-to-mesenchymal transition (EMT), angiogenesis, metastatic spread and apoptosis suppression in cells from breast cancer 13-17 , colorectal carcinoma 18,19 , pancreatic cancer 20 , gastric cancer 21 , hepatocellular carcinoma (HCC) 22-25 , cervical cancer 26 , osteosarcoma 27 , as well as oropharyngeal 28 and esophageal 29 squamous cell carcinoma. However, others have described reduced ANXA3 expression to be associated with malignant cellular properties in renal cell carcinoma 30 , prostate cancer 31-33 , papillary thyroid carcinoma 34 , and lung cancer 35-37 . Fewer than 30 studies so far have used immunohistochemistry (IHC) to assess the role of ANXA3 expression levels in different cancer types and often found associations between aberrant ANXA3 protein levels and unfavorable disease parameters and poor prognosis 16,21-24,32,34,38-49 . However, the role of ANXA3 expression has not been investigated across many other cancer entities, and published data are variable for cancer types analyzed in multiple studies. For example, five studies analyzing 8-60 colorectal adenocarcinomas reported ANXA3 positivity in 65 to 100% of cases 38,39,41,43,46 . Such data variability commonly occurs in IHC studies due to the use of different antibodies, staining protocols, and interpretation criteria 50 . To better comprehend the prevalence and role of ANXA3 in cancer, an extensive and highly standardized survey of ANXA3 protein expression in a broad range of different tumor types is needed. ANXA3 expression was therefore evaluated in more than 17,000 tumor tissue samples from 148 different tumor types and subtypes in a tissue microarray (TMA) format in this study. Materials and Methods Tissue Microarrays. The TMA composition and its manufacturing process have been described previously by our group 51-53 . 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 cancer TMAs contained a total of 17,285 primary tumors from 148 tumor types and subtypes. Detailed histopathological and molecular data were available for cancers of the breast (n=1,110), kidney (n=1,721), bladder (n=1,876), pancreas (n=434), stomach (n=376), liver (n=347), colorectum (n=2,148), endometrium (n=149), ovary (n=266), thyroid (n=299), and the testis (n=442). The composition of both normal and cancer TMAs is described in detail in the results section. All samples were from the archives of the Institutes of Pathology, University Hospital of Hamburg, Germany, the Institute of Pathology, Clinical Center Osnabrueck, Germany, and Department of Pathology, Academic Hospital Fuerth, Germany. Tissues were fixed in 4% buffered formalin and then embedded in paraffin. TMA tissue spot diameter was 0.6 mm. 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). The Ethics commission Hamburg, WF-049/09, waived the need to obtain individual patient informed consent. All work has been carried out in compliance with the Helsinki Declaration. Immunohistochemistry. Our IHC protocol has previously been described 54-56 . Briefly, freshly cut TMA sections were immunostained on one day and in one experiment. Slides were deparaffinized with xylol, rehydrated through a graded alcohol series and exposed to heat-induced antigen retrieval for 5 minutes in an autoclave at 121°C in pH 7.8 Tris-EDTA-Citrat (TEC) puffer. Endogenous peroxidase activity was blocked with Dako REAL Peroxidase-Blocking Solution (Agilent Technologies, Santa Clara, CA, USA; #S2023) for 10 minutes. Primary antibody specific for ANXA3 (recombinant mouse monoclonal antibody (clone-ID ARX-503, ardoci GmbH, Hamburg, Germany; #AO7829-503) was applied at 37°C for 60 minutes at a dilution of 1:150. For the purpose of antibody validation, the normal tissue TMA was also analyzed by the ANXA3 mouse monoclonal antibody 1A11E4 (proteintech, Rosemont, USA; # 66405-1-Ig) at a dilution of 1:450 and an otherwise identical protocol. Bound antibody was then visualized using the 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 tumor tissues, the percentage of positive neoplastic cells was estimated, and the staining intensity was semi-quantitatively recorded (0, 1+, 2+, 3+). In addition, it was recorded whether the staining involved membranes, cytoplasm, and or nuclei. For statistical analyses, the staining results were categorized into four groups. 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, 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. Statistics . Statistical calculations were performed with JMP18 ® (SAS ® , Cary, NC, USA). Contingency tables and the chi²-test were performed to search for associations between ANXA3 immunostaining and tumor phenotype 51-56 . Results Technical issues. A total of 13,472 (77.9%) of 17,285 tumor samples were interpretable in our TMA analysis. Non-interpretable samples demonstrated lack of unequivocal tumor cells or a complete lack of tissue. A sufficient number of samples (≥4) of each normal tissue type was evaluable. ANXA3 immunostaining in normal tissues. ANXA3 staining was found in almost all tissues and its expression level varied markedly between cell types and sometimes between samples. ANXA3 staining was most commonly cytoplasmic but positivity was also seen in nuclei or in membranes. The staining was most intense in granulocytes and their precursors in the bone marrow. A strong staining was also seen in follicular cells of the thyroid, acinar cells of the prostate, amnion cells, chief cells of gastric glands, intercalated ducts and excretory ducts of the pancreas, glandular cells of salivary glands, the parietal layer of the Bowman’s capsule of the kidney, a subset of epithelial cells of the salivary gland, and myoepithelial cells of the breast. A strong staining was also seen in many endothelial cells, but the staining intensity varied between tissues and organs. It was particularly strong in capillaries of the heart and absent in endothelial cells of glomeruli and sinus of the liver. Membranous ANXA3 staining was most prominent in pneumocytes, chorion cells of the placenta, and at apical membranes of excretory ducts of salivary glands. Nuclear ANXA3 staining was most commonly seen in squamous epithelium, umbrella cells of the urothelium, gastric surface epithelium, and in chorion cells of the placenta. ANXA3 staining was lacking in testis, brain, ovary, hepatocytes of the liver, chief cells of the epididymis, lymphocytes, and in adrenocortical cells. Representative images are shown in Figure 1. All these findings were obtained by using the recombinant monoclonal mouse antibody ARX-503 and the monoclonal mouse antibody 1A11E4 and were therefore viewed to be specific. Using the antibody 1A11E4, an additional granular cytoplasmic staining was seen in the mucosa of the small intestine. This staining was considered an antibody specific cross-reactivity of 1A11E4 (Supplementary Figure 1). ANXA3 immunostaining in cancer. ANXA3 immunostaining was predominantly cytoplasmic and/or membranous and was rarely seen in nuclei of cancer cells. ANXA3 immunostaining was found in 10,223 (75.9%) of the 13,472 interpretable tumor samples, including 2,200 (16.3%) with weak, 1,363 (10.1%) with moderate, and 6,660 (49.4%) with strong positivity (Table 1). A total of 128 of 148 tumor entities (86.5%) showed ANXA3 staining in at least one case, and 110 tumor categories (74.3%) included at least one case with strong staining. Among 122 tumor entities with at least 10 evaluable samples, 44 showed a strong ANXA3 staining in more than 50.0% of cases. ANXA3 was always positive in basal cell carcinoma of the skin, adenocarcinoma of the cervix, pleomorphic adenoma of the parotid gland, serous endometrial carcinoma, epithelioid mesothelioma, colorectal adenomatous polyp with high-grade dysplasia, Klatskin tumor and adenocarcinoma of the gallbladder, endometrioid carcinoma of the ovary, Warthin tumor of the parotid gland, prostatic adenocarcinoma (Gleason 3+3) and ampullary and pancreatic adenocarcinoma. Representative images are given in Figure 2. A graphical representation of a ranking order of ANXA3 positive cancers and of strongly positive cancers is provided in Figure 3. The relationship between ANXA3 staining and parameters of cancer aggressiveness is summarized in Table 2. High ANXA3 expression was associated with high grade (p<0.0001), muscle invasive tumor growth (pTa vs pT2-4; p<0.0001) and advanced pT-stage (p=0.0267) in urothelial carcinoma of the urinary bladder; high pT-stage (p=0.0211) in gastric adenocarcinoma; advanced pT-stage (p<0.0001), nodal positivity (p=0.0004) and high tumor grade (p<0.0001) in HCC; lymphatic invasion (p=0.0307) and RAS mutation mutational status (p=0.0127) in colorectal adenocarcinoma, and advanced pT-stage (p=0.0410) in papillary thyroid carcinoma. Low ANXA3 expression was associated with lymphatic invasion (p<0.0001) in urothelial carcinoma of the urinary bladder; high BRE grade in invasive breast carcinoma of no special type (NST; p<0.0001); advanced UICC- (p=0.0008) and pT-stage (p<0.0001) in clear cell renal cell carcinoma (ccRCC); high ISUP (p=0.0135) and Fuhrman grade (p=0.0004), as well as advanced UICC- (p=0.0073) and pT-stage (p=0.0003) in papillary renal cell carcinoma (pRCC); advanced pT-stage (p=0.0464) in pancreatic ductal adenocarcinoma; advanced pT-stage (p=0.0056), right side tumor location (p=0.0295), mismatch repair (MMR) deficiency (p<0.0001), and BRAF (V600E) mutations (p=0.0250) in colorectal adenocarcinoma; and high pT-stage (p=0.0427) in endometrioid endometrial carcinoma. ANXA3 staining was unrelated to tumor phenotype in serous carcinoma of the ovary and testicular seminoma. Discussion Our successful analysis of 13,472 tumors from 148 different tumor categories revealed that ANXA3 is often highly expressed in a large fraction of cancers. Clinically important tumor entities with positivity rates above 90.0% for example included cervical adenocarcinoma, endometrioid, serous, and mucinous carcinoma of the ovary, endometrioid endometrium carcinoma, colorectal, pancreatic ductal, esophageal, gastric, and prostatic adenocarcinoma, cholangiocarcinoma, squamous cell carcinomas of different sites, and papillary renal cell carcinoma while a positivity rate below 50.0% was found in only 39 out of 122 tumor categories with at least 10 evaluable samples. Although only a fraction of cancer entities had previously been analyzed by IHC, most authors described similarly high frequencies of ANXA3 positivity. For example, the fraction of ANXA3 positive cases was 66.4% in 125 pulmonary adenocarcinomas 49 , 72.9% in 1589 prostatic adenocarcinomas 32 , 57.4-61.3% in 80-183 gastric adenocarcinomas 21,45 , 83.3% in 30 invasive breast carcinomas NST 16 , and 74.3% in 101 papillary thyroid carcinomas 34 . Given the high prevalence of ANXA3 expression across most tumor entities, ANXA3 IHC cannot have diagnostic utility for the distinction of different tumor entities. As ANXA3 was markedly overexpressed in testicular seminoma and in HCC as well as in urothelial and squamous cell carcinomas as compared to their corresponding normal tissues, ANXA3 may potentially serve as a diagnostic tumor marker in these tissues. ANXA3 expression in cancer is also of interest because of its potential utility as a therapeutic target. Several studies have demonstrated that elevated ANXA3 expression can induce chemoresistance in various tumors through activation of pro-proliferative pathways 19,26 , inhibition of apoptosis 57 , regulation of drug influx and efflux via exosomes 58 as well as modulation of DNA-damage repair 26 and autophagy 24 . Silencing of ANXA3 via siRNA/shRNA or CRISPR was found to reduce tumor cell viability, impair invasive behavior, and diminish metastatic potential in vitro and in xenograft models, while it restored sensitivity to chemotherapeutics in resistant cell lines 2,13-15,22,24,59,60 . Along these lines, Tong et al. demonstrated that an anti-ANXA3 monoclonal antibody sensitized HCC cells to a treatment with the tyrosine kinase inhibitor sorafenib 24 . Moreover, the effective application of the novel small-molecule inhibitors (R)-SL18 and 18a5, which target ANXA3 degradation, has been demonstrated in vitro and in vivo in triple-negative breast cancer models 59,61 . Our ranking order of cancer entities according to their prevalence of detectable ANXA3 expression identifies cancer types that could benefit most from anti-ANXA3 treatments if these should prove to be safe and efficient. Considering the limited number of tumor entities that were previously analyzed by IHC and the variability of ANXA3 positivity in cancers that were studied by different groups such as colorectal cancer 38,39,41,43,46 , such a ranking order could not have been compiled from the literature. It is of note that the assay used in our study was extensively validated by comparing our IHC findings in normal tissues with data obtained by another independent anti-ANXA3 antibody and RNA data derived from three different publicly accessible databases (https://www.proteinatlas.org/ENSG00000138772-ANXA3/tissue) 62-65 . This approach follows the recommendations of the International Working Group for Antibody Validation (IWGAV) 66 . The identification of cross-reactive antibody binding to other proteins than the target protein represents the most significant challenge for antibody validation which is hard to achieve in cell line experiments. As the tissues from 76 different normal tissue categories (8 donors each) will contain most cell types of adult humans, it is very likely that a near-complete spectrum of human proteins with a broad diversity of posttranslational modifications was evaluated for potential cross-reactivities in our study. The validity of our assay was supported by the preferential detection of ANXA3 staining in bone marrow, lung, salivary glands, stomach, prostate, thyroid, and the placenta which are the tissues with highest ANXA3 RNA expression and the largely absent ANXA3 immunostaining in tissues with very low or absent RNA staining such as testis, brain, ovary, liver, epididymis, lymphocytes, and the adrenal gland. That all cell types identified as ANXA3 positive by ARX-503 were also positive by the independent second antibody 1A11E4 represents further strong support for assay specificity. An additional granular cytoplasmic staining which was only seen by 1A11E4 in the mucosa of the small intestine was considered an antibody specific cross-reactivity of 1A11E4 which was revealed by our comprehensive validation approach (Supplementary Figure 1). The number of cases analyzed was large enough in several tumor entities to study associations with tumor phenotype. Altogether these findings - obtained under highly standardized experimental conditions - are in line with a context dependent dual, oncogenic and tumor suppressive role of ANXA3. The significant association seen between high ANXA3 expression and unfavorable tumor features in urothelial carcinoma, gastric adenocarcinoma, HCC, colorectal adenocarcinoma, and papillary thyroid carcinoma in this study is consistent with previous findings obtained in studies on colorectal adenocarcinoma 38,39,46 , lung adenocarcinoma 38 , breast cancer 16,38,47 , ovarian cancer 44,48 , gastric adenocarcinoma 21,45 and HCC 22-24,40 . For example, Zhou et al. found significant associations between high ANXA3 expression and advanced pT-stage, nodal positivity, high BRE grade and reduced overall as well as disease free survival in a cohort of 309 different breast carcinomas manly consisting of ductal, lobular and medullary carcinomas 42 . Similarly, Wang et al. reported a link between high ANXA3 expression and advanced pT- and UICC-stage, nodal positivity, metastatic disease, and reduced overall survival in a cohort of 183 gastric adenocarcinomas 21 . Suggested mechanisms by which elevated ANXA3 expression can increase tumor cell aggressiveness include sustained proliferative signaling via MAPK/ERG, JNK and PI3K/Akt 19,20,23,57 , reduced apoptosis through suppressed PKCδ/p38 signaling and JNK dependent upregulation of antiapoptotic Bcl-2 24,67 , and promoting invasion, metastasis and angiogenesis via PI3K/Akt, HIF1α and NF-κB 15,20,23 . Our finding of a relationship between low ANXA3 expression and features of cancer aggressiveness in urothelial carcinoma, invasive breast carcinoma of no special type, ccRCC, pRCC, pancreatic ductal adenocarcinoma, colorectal adenocarcinoma and in endometrioid endometrial carcinoma is in agreement with previous data obtained in prostatic adenocarcinoma and papillary thyroid carcinoma 32,34 . For example, Köllermann et al. found a link between reduced ANXA3 expression and advanced pT-stage and Gleason score as well as reduced PSA-free survival and early biochemical recurrence 32 . In summary, our data demonstrate that ANXA3 is abundantly expressed across a broad spectrum of cancer entities. The association between aberrant ANXA3 expression and histopathological features of tumor aggressiveness suggests a tissue-dependent oncogenic or tumor-suppressive role and highlights ANXA3 as a potential diagnostic and prognostic cancer marker. Abbreviations ANXA3 Annexin A3 EMT epithelial-to-mesenchymal transition HCC hepatocellular carcinoma IHC immunohistochemistry TMA tissue microarray TEC Tris-EDTA-Citrat NST no special type ccRCC clear cell renal cell carcinoma pRCC papillary renal cell carcinoma MMR mismatch repair IWGAV International Working Group for Antibody Validation Declarations Acknowledgements We are grateful to Melanie Steurer, Laura Behm, Inge Brandt, and Sünje Seekamp for excellent technical assistance. Authors Contribution Statement CvB, NH, FG, RS, GS and SS: contributed to conception, design, data collection, data analysis and manuscript writing. NH, AM, FL, VC, FV, DD, RSch, AH, CF, CB, SB, MK, CHM, GMF, NS, KM, AML, PL, ML, TSC, AHM, EB, NG, MCT, SM, TK, MF, VR: participated in pathology data analysis, data interpretation, and collection of samples. MK, GMF and CHM: data analysis. SS, RS, GS: study supervision. All authors agree to be accountable for the content of the work. Conflict of Interest The recombinant mouse monoclonal antibody ARX-503 was provided from ardoci GmbH, Hamburg, Germany (owned by a family member of GS). Data Availability Statement All data generated or analyzed during this study are included in this published article. Ethics Approval 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). The Ethics commission Hamburg, WF-049/09, waived the need to obtain individual patient informed consent. 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Hamburg-Eppendorf","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"Christina","lastName":"Tsourlakis","suffix":""},{"id":633610594,"identity":"472f01d9-f4bb-4ccd-a249-a04586fffc86","order_by":28,"name":"Sarah Minner","email":"","orcid":"","institution":"University Medical Center Hamburg-Eppendorf","correspondingAuthor":false,"prefix":"","firstName":"Sarah","middleName":"","lastName":"Minner","suffix":""},{"id":633610595,"identity":"25b3901a-4a10-43c8-b038-ece96c17537c","order_by":29,"name":"Till Krech","email":"","orcid":"","institution":"University Medical Center Hamburg-Eppendorf","correspondingAuthor":false,"prefix":"","firstName":"Till","middleName":"","lastName":"Krech","suffix":""},{"id":633610596,"identity":"5432e411-137b-4d0e-b376-44e18aa56caa","order_by":30,"name":"Morton Freytag","email":"","orcid":"","institution":"University Medical Center Hamburg-Eppendorf","correspondingAuthor":false,"prefix":"","firstName":"Morton","middleName":"","lastName":"Freytag","suffix":""},{"id":633610597,"identity":"7f1ca834-ed79-4e26-9a51-5d999b399d43","order_by":31,"name":"Viktor Reiswich","email":"","orcid":"","institution":"University Medical Center Hamburg-Eppendorf","correspondingAuthor":false,"prefix":"","firstName":"Viktor","middleName":"","lastName":"Reiswich","suffix":""},{"id":633610598,"identity":"1619f7b8-b937-4816-b6e1-aca0fce94e63","order_by":32,"name":"Stefan Steurer","email":"","orcid":"","institution":"University Medical Center Hamburg-Eppendorf","correspondingAuthor":false,"prefix":"","firstName":"Stefan","middleName":"","lastName":"Steurer","suffix":""}],"badges":[],"createdAt":"2026-03-31 11:23:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9278991/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9278991/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108734940,"identity":"ee31ab6f-e6ec-489a-b76e-394c521bd172","added_by":"auto","created_at":"2026-05-07 19:58:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2043750,"visible":true,"origin":"","legend":"\u003cp\u003eANXA3 immunostaining in normal tissues.\u003c/p\u003e\n\u003cp\u003eThe panels show a strong and predominantly cytoplasmic ANXA3 staining in follicular cells of the thyroid gland (A), a subset of precursor cells in the bone marrow (B) and acinar cells of the prostate (C). A strong membranous ANXA3 staining in alveolar pneumocytes (D), a moderate to strong cytoplasmic ANXA3 staining in a subset of tubular cells and of the parietal layer of the Bowman’s membrane of the kidney (E), a weak to moderate ANXA3 staining of subsets of acinar and islet cells and a strong positivity of intercalated and excretory ducts of the pancreas (F), as well as a strong ANXA3 staining of bile ducts in the liver (G), while ANXA3 is not detected in the testis (H).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-9278991/v1/92bd907bd72729708e4bccb7.png"},{"id":108734944,"identity":"60a6e2f7-1883-4717-bc79-e5972b71a62c","added_by":"auto","created_at":"2026-05-07 19:58:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2208908,"visible":true,"origin":"","legend":"\u003cp\u003eANXA3 immunostaining in cancer.\u003c/p\u003e\n\u003cp\u003eThe panels show a strong, predominantly cytoplasmic ANXA3 immunostaining of tumor cells in a colorectal (A) and a prostatic adenocarcinoma (B), a serous high-grade carcinoma of the ovary (C), a pulmonary adenocarcinoma (D), a moderate to strong ANXA3 staining in a muscle-invasive urothelial carcinoma (E) and a testicular seminoma (F), as well as a heterogeneous ANXA3 staining with strongest positivity in peripheral cells of a squamous cell carcinoma of the oral cavity (G) while ANXA3 staining is absent in tumor cells of a renal clear cell carcinoma containing few ANXA3 positive granulocytes (H).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-9278991/v1/6acfe1bae46113cd90386531.png"},{"id":108734943,"identity":"96c48fe0-40c5-4b4c-8047-dbf014a827a6","added_by":"auto","created_at":"2026-05-07 19:58:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":81160,"visible":true,"origin":"","legend":"\u003cp\u003eRanking order of tumors according to ANXA3 positivity.\u003c/p\u003e\n\u003cp\u003eBoth the percentage of positive cases (blue dots) and the percentage of strongly positive cases (orange dots) are shown.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9278991/v1/61bfca78d0ac191bc351a4a0.png"},{"id":109204432,"identity":"6241d322-50df-4ba9-b670-0e1e02c03ca3","added_by":"auto","created_at":"2026-05-13 14:59:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4254389,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9278991/v1/27746169-ef76-4755-ac18-e7b5fb4382bf.pdf"},{"id":109220647,"identity":"a2bcf71d-88ad-430c-9cfe-caa929b7625e","added_by":"auto","created_at":"2026-05-13 20:32:15","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":760456,"visible":true,"origin":"","legend":"","description":"","filename":"Table12.docx","url":"https://assets-eu.researchsquare.com/files/rs-9278991/v1/1bf249dd7b344eb1a37fed47.docx"},{"id":108806934,"identity":"a167ea23-5a26-44ff-9052-717d5ccfc067","added_by":"auto","created_at":"2026-05-08 15:29:39","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":4110757,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryData.docx","url":"https://assets-eu.researchsquare.com/files/rs-9278991/v1/d92ad2c6d25000206083c53c.docx"}],"financialInterests":"Competing interest reported. The recombinant mouse monoclonal antibody ARX-503 was provided from ardoci GmbH, Hamburg, Germany (owned by a family member of GS).","formattedTitle":"Expression of Annexin A3 in cancer: A tissue microarray study involving more than 17,000 cancers from 148 tumor entities","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAnnexin A3 (ANXA3) is a member of the annexin family of calcium-dependent phospholipid-binding proteins\u003csup\u003e1\u003c/sup\u003e. Similarly, as other annexin family members, ANXA3 is primarily involved in membrane-related processes including transmembranous signaling, membrane organization and repair, vesicle trafficking, cytoskeletal interaction and regulation of ion channels. Its diverse functions extend across various cellular processes such as apoptosis, cell growth, inflammation, and cell differentiation\u003csup\u003e2\u003c/sup\u003e. Although ANXA3 knockout mice are viable and do not exhibit significant developmental abnormalities, they display altered responses to stress and inflammation, suggesting a role for ANXA3 in cellular homeostasis and disease susceptibility\u003csup\u003e3,4\u003c/sup\u003e. ANXA3 is expressed in numerous different normal cell types\u003csup\u003e5\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eAltered ANXA3 expression has been found in many conditions including immune and inflammatory disease\u003csup\u003e3,6-11\u003c/sup\u003e as well as in cancer\u003csup\u003e2,12\u003c/sup\u003e. Several preclinical models\u0026nbsp;have demonstrated that\u0026nbsp;ANXA3 upregulation\u0026nbsp;can confer a malignant phenotype by inducing cell growth, epithelial-to-mesenchymal transition (EMT), angiogenesis, metastatic spread and apoptosis suppression in cells from breast cancer\u003csup\u003e13-17\u003c/sup\u003e, colorectal carcinoma\u003csup\u003e18,19\u003c/sup\u003e, pancreatic cancer\u003csup\u003e20\u003c/sup\u003e, gastric cancer\u003csup\u003e21\u003c/sup\u003e, hepatocellular carcinoma (HCC)\u003csup\u003e22-25\u003c/sup\u003e, cervical cancer \u003csup\u003e26\u003c/sup\u003e, osteosarcoma\u003csup\u003e27\u003c/sup\u003e, as well as oropharyngeal\u003csup\u003e28\u003c/sup\u003e and esophageal\u003csup\u003e29\u003c/sup\u003e squamous cell carcinoma. However, others have described reduced ANXA3 expression to be associated with malignant cellular properties in renal cell carcinoma\u003csup\u003e30\u003c/sup\u003e, prostate cancer\u003csup\u003e31-33\u003c/sup\u003e, papillary thyroid carcinoma\u003csup\u003e34\u003c/sup\u003e, and lung cancer\u003csup\u003e35-37\u003c/sup\u003e. Fewer than 30 studies so far have used immunohistochemistry (IHC) to assess the role of ANXA3 expression levels in different cancer types and often found associations between aberrant ANXA3\u0026nbsp;protein levels and unfavorable disease parameters and poor prognosis\u003csup\u003e16,21-24,32,34,38-49\u003c/sup\u003e. However, the role of ANXA3 expression has not been investigated across many other cancer entities, and published data are variable\u0026nbsp;for cancer types analyzed in multiple studies.\u0026nbsp;For example, five studies analyzing 8-60 colorectal adenocarcinomas reported ANXA3 positivity in 65 to 100% of cases\u003csup\u003e38,39,41,43,46\u003c/sup\u003e.\u0026nbsp;Such data variability commonly occurs in IHC studies due to the use of different antibodies, staining protocols, and interpretation criteria\u003csup\u003e50\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eTo better comprehend the prevalence and role of ANXA3 in cancer, an extensive and highly standardized survey of ANXA3 protein expression in a broad range of different tumor types is needed. ANXA3 expression was therefore evaluated in more than 17,000 tumor tissue samples from 148 different tumor types and subtypes in a tissue microarray (TMA) format in this study.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eTissue Microarrays.\u0026nbsp;\u003c/strong\u003eThe TMA composition and its manufacturing process have been described previously by our group\u003csup\u003e51-53\u003c/sup\u003e.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eOur 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 cancer TMAs contained a total of 17,285 primary tumors from 148 tumor types and subtypes. Detailed histopathological and molecular data were available for cancers of the breast (n=1,110), kidney (n=1,721), bladder (n=1,876), pancreas (n=434), stomach (n=376), liver (n=347), colorectum (n=2,148), endometrium (n=149), ovary (n=266), thyroid (n=299), and the testis (n=442). The composition of both normal and cancer TMAs is described in detail in the results section. All samples were from the archives of the Institutes of Pathology, University Hospital of Hamburg, Germany, the Institute of Pathology, Clinical Center Osnabrueck, Germany, and Department of Pathology, Academic Hospital Fuerth, Germany. Tissues were fixed in 4% buffered formalin and then embedded in paraffin. TMA tissue spot diameter was 0.6 mm. 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, \u0026sect;12) and by the local ethics committee (Ethics commission Hamburg). The Ethics commission Hamburg, WF-049/09, waived the need to obtain individual patient informed consent. All work has been carried out in compliance with the Helsinki Declaration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry.\u0026nbsp;\u003c/strong\u003eOur IHC protocol has previously been described\u003csup\u003e54-56\u003c/sup\u003e. Briefly, freshly cut TMA sections were immunostained on one day and in one experiment. Slides were deparaffinized with xylol, rehydrated through a graded alcohol series and exposed to heat-induced antigen retrieval for 5 minutes in an autoclave at 121\u0026deg;C in pH 7.8 Tris-EDTA-Citrat (TEC) puffer. Endogenous peroxidase activity was blocked with Dako REAL Peroxidase-Blocking Solution (Agilent Technologies, Santa Clara, CA, USA; #S2023) for 10 minutes. Primary antibody specific for ANXA3 (recombinant mouse monoclonal antibody (clone-ID ARX-503, ardoci GmbH, Hamburg, Germany; #AO7829-503) was applied at 37\u0026deg;C for 60 minutes at a dilution of 1:150. For the purpose of antibody validation, the normal tissue TMA was also analyzed by the ANXA3 mouse monoclonal antibody 1A11E4 (proteintech, Rosemont, USA; # 66405-1-Ig) at a dilution of 1:450 and an otherwise identical protocol. Bound antibody was then visualized using the Dako REAL EnVision Detection System Peroxidase/DAB+, Rabbit/Mouse kit (Agilent Technologies, Santa Clara, CA, USA; #K5007) according to the manufacturer\u0026rsquo;s directions. The sections were counterstained with hemalaun. For tumor tissues, the percentage of positive neoplastic cells was estimated, and the staining intensity was semi-quantitatively recorded (0, 1+, 2+, 3+). In addition, it was recorded whether the staining involved membranes, cytoplasm, and or nuclei. For statistical analyses, the staining results were categorized into four groups. Tumors without any staining were considered negative. Tumors with 1+ staining intensity in \u0026le;70% of tumor cells or 2+ intensity in \u0026le;30% of tumor cells were considered weakly positive. Tumors with 1+ staining intensity in \u0026gt;70% of tumor cells, 2+ intensity in 31-70%, or 3+ intensity in \u0026le;30% of tumor cells were considered moderately positive. Tumors with 2+ intensity in \u0026gt;70% or 3+ intensity in \u0026gt;30% of tumor cells were considered strongly positive.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistics\u003c/strong\u003e.\u0026nbsp;Statistical calculations were performed with JMP18\u003csup\u003e\u0026reg;\u003c/sup\u003e (SAS\u003csup\u003e\u0026reg;\u003c/sup\u003e, Cary, NC, USA). Contingency tables and the chi\u0026sup2;-test were performed to search for associations between ANXA3 immunostaining and tumor phenotype\u003csup\u003e51-56\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eTechnical issues.\u003c/strong\u003e A total of 13,472 (77.9%) of 17,285 tumor samples were interpretable in our TMA analysis. Non-interpretable samples demonstrated lack of unequivocal tumor cells or a complete lack of tissue. A sufficient number of samples (\u0026ge;4) of each normal tissue type was evaluable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eANXA3 immunostaining in normal tissues.\u0026nbsp;\u003c/strong\u003eANXA3 staining was found in almost all tissues and its expression level varied markedly between cell types and sometimes between samples. ANXA3 staining was most commonly cytoplasmic but positivity was also seen in nuclei or in membranes. The staining was most intense in granulocytes and their precursors in the bone marrow. A strong staining was also seen in follicular cells of the thyroid, acinar cells of the prostate, amnion cells, chief cells of gastric glands, intercalated ducts and excretory ducts of the pancreas, glandular cells of salivary glands, the parietal layer of the Bowman\u0026rsquo;s capsule of the kidney, a subset of epithelial cells of the salivary gland, and myoepithelial cells of the breast. A strong staining was also seen in many endothelial cells, but the staining intensity varied between tissues and organs. It was particularly strong in capillaries of the heart and absent in endothelial cells of glomeruli and sinus of the liver. Membranous ANXA3 staining was most prominent in pneumocytes, chorion cells of the placenta, and at apical membranes of excretory ducts of salivary glands. Nuclear ANXA3 staining was most commonly seen in squamous epithelium, umbrella cells of the urothelium, gastric surface epithelium, and in chorion cells of the placenta. ANXA3 staining was lacking in testis, brain, ovary, hepatocytes of the liver, chief cells of the epididymis, lymphocytes, and in adrenocortical cells. Representative images are shown in Figure 1. All these findings were obtained by using the recombinant monoclonal mouse antibody ARX-503 and the monoclonal mouse antibody 1A11E4 and were therefore viewed to be specific. Using the antibody 1A11E4, an additional granular cytoplasmic staining\u0026nbsp;was seen\u0026nbsp;in the mucosa of the small intestine. This staining was considered an antibody specific cross-reactivity of\u0026nbsp;1A11E4 (Supplementary Figure 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eANXA3 immunostaining\u003c/strong\u003e \u003cstrong\u003ein cancer.\u0026nbsp;\u003c/strong\u003eANXA3 immunostaining was predominantly cytoplasmic and/or membranous and was rarely seen in nuclei of cancer cells. ANXA3 immunostaining was found in 10,223 (75.9%) of the\u0026nbsp;13,472 interpretable tumor samples, including 2,200 (16.3%) with weak, 1,363 (10.1%) with moderate, and 6,660 (49.4%) with strong positivity (Table 1). A total of 128 of 148 tumor entities (86.5%) showed ANXA3 staining in at least one case, and 110 tumor categories (74.3%) included at least one case with strong staining. Among 122 tumor entities with at least 10 evaluable samples, 44 showed a strong ANXA3 staining in more than 50.0% of cases. ANXA3 was always positive in basal cell carcinoma of the skin, adenocarcinoma of the cervix, pleomorphic adenoma of the parotid gland, serous endometrial carcinoma, epithelioid mesothelioma, colorectal adenomatous polyp with high-grade dysplasia, Klatskin tumor and adenocarcinoma of the gallbladder, endometrioid carcinoma of the ovary, Warthin tumor of the parotid gland, prostatic adenocarcinoma (Gleason 3+3) and ampullary and pancreatic adenocarcinoma. Representative images are given in Figure 2. A graphical representation of a ranking order of ANXA3 positive cancers and of strongly positive cancers is provided in Figure 3.\u003c/p\u003e\n\u003cp\u003eThe relationship between ANXA3 staining and parameters of cancer aggressiveness is summarized in Table 2. High ANXA3 expression was associated with high grade (p\u0026lt;0.0001), muscle invasive tumor growth (pTa vs pT2-4; p\u0026lt;0.0001) and advanced pT-stage (p=0.0267) in urothelial carcinoma of the urinary bladder; high pT-stage (p=0.0211) in gastric adenocarcinoma; advanced pT-stage (p\u0026lt;0.0001), nodal positivity (p=0.0004) and high tumor grade (p\u0026lt;0.0001) in HCC; lymphatic invasion (p=0.0307) and RAS mutation mutational status (p=0.0127) in colorectal adenocarcinoma, and advanced pT-stage (p=0.0410) in papillary thyroid carcinoma. Low ANXA3 expression was associated with lymphatic invasion (p\u0026lt;0.0001) in urothelial carcinoma of the urinary bladder; high BRE grade in invasive breast carcinoma of no special type (NST; p\u0026lt;0.0001); advanced UICC- (p=0.0008) and pT-stage (p\u0026lt;0.0001) in clear cell renal cell carcinoma (ccRCC); high ISUP (p=0.0135) and Fuhrman grade (p=0.0004), as well as advanced UICC- (p=0.0073) and pT-stage (p=0.0003) in papillary renal cell carcinoma (pRCC); advanced pT-stage (p=0.0464) in pancreatic ductal adenocarcinoma; advanced pT-stage (p=0.0056), right side tumor location (p=0.0295), mismatch repair (MMR) deficiency (p\u0026lt;0.0001), and BRAF (V600E) mutations (p=0.0250) in colorectal adenocarcinoma; and high pT-stage (p=0.0427) in endometrioid endometrial carcinoma. ANXA3 staining was unrelated to tumor phenotype in serous carcinoma of the ovary and testicular seminoma.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur successful analysis of 13,472 tumors from 148 different tumor categories revealed that ANXA3 is often highly expressed in a large fraction of cancers. Clinically important tumor entities with positivity rates above 90.0% for example included cervical adenocarcinoma, endometrioid, serous, and mucinous carcinoma of the ovary, endometrioid endometrium carcinoma, colorectal, pancreatic ductal, esophageal, gastric, and prostatic adenocarcinoma, cholangiocarcinoma, squamous cell carcinomas of different sites, and papillary renal cell carcinoma while a positivity rate below 50.0% was found in only 39 out of 122 tumor categories with at least 10 evaluable samples. Although only a fraction of cancer entities had previously been analyzed by IHC, most authors described similarly high frequencies of ANXA3 positivity. For example, the fraction of ANXA3 positive cases was 66.4% in 125 pulmonary adenocarcinomas\u003csup\u003e49\u003c/sup\u003e, 72.9% in 1589 prostatic adenocarcinomas\u003csup\u003e32\u003c/sup\u003e, 57.4-61.3% in 80-183 gastric adenocarcinomas\u003csup\u003e21,45\u003c/sup\u003e, 83.3% in 30 invasive breast carcinomas NST\u003csup\u003e16\u003c/sup\u003e, and 74.3% in 101 papillary thyroid carcinomas\u003csup\u003e34\u003c/sup\u003e. Given the high prevalence of ANXA3 expression across most tumor entities, ANXA3 IHC cannot have diagnostic utility for the distinction of different tumor entities. As ANXA3 was markedly overexpressed in testicular seminoma and in HCC as well as in urothelial and squamous cell carcinomas as compared to their corresponding normal tissues, ANXA3 may potentially serve as a diagnostic tumor marker in these tissues.\u003c/p\u003e\n\u003cp\u003eANXA3 expression in cancer is also of interest because of its potential utility as a therapeutic target. Several studies have demonstrated that elevated ANXA3 expression can induce chemoresistance in various tumors through activation of pro-proliferative pathways\u003csup\u003e19,26\u003c/sup\u003e, inhibition of apoptosis\u003csup\u003e57\u003c/sup\u003e, regulation of drug influx and efflux via exosomes\u003csup\u003e58\u003c/sup\u003e as well as modulation of DNA-damage repair\u003csup\u003e26\u003c/sup\u003e and autophagy\u003csup\u003e24\u003c/sup\u003e. Silencing of ANXA3 via siRNA/shRNA or CRISPR was found to reduce tumor cell viability, impair invasive behavior, and diminish metastatic potential \u003cem\u003ein vitro\u003c/em\u003e and in xenograft models, while it restored sensitivity to chemotherapeutics in resistant cell lines\u003csup\u003e2,13-15,22,24,59,60\u003c/sup\u003e. Along these lines, \u003cem\u003eTong et al.\u003c/em\u003e demonstrated that an anti-ANXA3 monoclonal antibody sensitized HCC cells to a treatment with the tyrosine kinase inhibitor sorafenib\u003csup\u003e24\u003c/sup\u003e. Moreover, the effective application of the novel small-molecule inhibitors (R)-SL18 and 18a5, which target ANXA3 degradation, has been demonstrated \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e in triple-negative breast cancer models\u003csup\u003e59,61\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eOur ranking order of cancer entities according to their prevalence of detectable ANXA3 expression identifies cancer types that could benefit most from anti-ANXA3 treatments if these should prove to be safe and efficient. Considering the limited number of tumor entities that were previously analyzed by IHC and the variability of ANXA3 positivity in cancers that were studied by different groups such as colorectal cancer\u003csup\u003e38,39,41,43,46\u003c/sup\u003e, such a ranking order could not have been compiled from the literature.\u0026nbsp;It is of note that the assay used in our study was extensively validated by comparing our IHC findings in normal tissues with data obtained by another independent anti-ANXA3 antibody and RNA data derived from three different publicly accessible databases (https://www.proteinatlas.org/ENSG00000138772-ANXA3/tissue)\u003csup\u003e62-65\u003c/sup\u003e. This approach follows the recommendations of the International Working Group for Antibody Validation (IWGAV)\u003csup\u003e66\u003c/sup\u003e. The identification of cross-reactive antibody binding to other proteins than the target protein represents the most significant challenge for antibody validation which is hard to achieve in cell line experiments. As the tissues from 76 different normal tissue categories (8 donors each) will contain most cell types of adult humans, it is very likely that a near-complete spectrum of human proteins with a broad diversity of posttranslational modifications was evaluated for potential cross-reactivities in our study. The validity of our assay was supported by the preferential detection of ANXA3 staining in bone marrow, lung, salivary glands, stomach, prostate, thyroid, and the placenta which are the tissues with highest ANXA3 RNA expression and the largely absent ANXA3 immunostaining in tissues with very low or absent RNA staining such as testis, brain, ovary, liver, epididymis, lymphocytes, and the adrenal gland. That all cell types identified as ANXA3 positive by ARX-503 were also positive by the independent second antibody 1A11E4 represents further strong support for assay specificity. An additional granular cytoplasmic staining which was only seen by 1A11E4 in the mucosa of the small intestine was considered an antibody specific cross-reactivity of 1A11E4 which was revealed by our comprehensive validation approach (Supplementary Figure 1).\u003c/p\u003e\n\u003cp\u003eThe number of cases analyzed was large enough in several tumor entities to study associations with tumor phenotype. Altogether these findings - obtained under highly standardized experimental conditions - are in line with a context dependent dual, oncogenic and tumor suppressive role of ANXA3. The significant association seen between high ANXA3 expression and unfavorable tumor features in urothelial carcinoma, gastric adenocarcinoma, HCC, colorectal adenocarcinoma, and papillary thyroid carcinoma in this study is consistent with previous findings obtained in studies on colorectal adenocarcinoma\u003csup\u003e38,39,46\u003c/sup\u003e, lung adenocarcinoma\u003csup\u003e38\u003c/sup\u003e, breast cancer\u003csup\u003e16,38,47\u003c/sup\u003e, ovarian cancer\u003csup\u003e44,48\u003c/sup\u003e, gastric adenocarcinoma\u003csup\u003e21,45\u003c/sup\u003e and HCC\u003csup\u003e22-24,40\u003c/sup\u003e. For example, Zhou \u003cem\u003eet al.\u003c/em\u003e found significant associations between high ANXA3 expression and advanced pT-stage, nodal positivity, high BRE grade and reduced overall as well as disease free survival in a cohort of 309 different breast carcinomas manly consisting of ductal, lobular and medullary carcinomas\u003csup\u003e42\u003c/sup\u003e. Similarly, Wang \u003cem\u003eet al.\u003c/em\u003e reported a link between high ANXA3 expression and advanced pT- and UICC-stage, nodal positivity, metastatic disease, and reduced overall survival in a cohort of 183 gastric adenocarcinomas\u003csup\u003e21\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eSuggested mechanisms by which elevated ANXA3 expression can increase tumor cell aggressiveness include sustained proliferative signaling via MAPK/ERG, JNK and PI3K/Akt\u003csup\u003e19,20,23,57\u003c/sup\u003e, reduced apoptosis through suppressed PKC\u0026delta;/p38 signaling and JNK dependent upregulation of antiapoptotic Bcl-2\u003csup\u003e24,67\u003c/sup\u003e, \u0026nbsp;and promoting invasion, metastasis and angiogenesis via PI3K/Akt, HIF1\u0026alpha; and NF-\u0026kappa;B\u003csup\u003e15,20,23\u003c/sup\u003e. Our finding of a relationship between low ANXA3 expression and features of cancer aggressiveness in urothelial carcinoma, invasive breast carcinoma of no special type, ccRCC, pRCC, pancreatic ductal adenocarcinoma, colorectal adenocarcinoma and in endometrioid endometrial carcinoma\u0026nbsp;is in agreement with previous data obtained in prostatic adenocarcinoma and papillary thyroid carcinoma\u003csup\u003e32,34\u003c/sup\u003e. For example, K\u0026ouml;llermann \u003cem\u003eet al.\u003c/em\u003e found a link between reduced ANXA3 expression and advanced pT-stage and Gleason score as well as reduced PSA-free survival and early biochemical recurrence\u003csup\u003e32\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn summary, our data demonstrate that ANXA3 is abundantly expressed across a broad spectrum of cancer entities. The association between aberrant ANXA3 expression and histopathological features of tumor aggressiveness suggests a tissue-dependent oncogenic or tumor-suppressive role and highlights ANXA3 as a potential diagnostic and prognostic cancer marker.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eANXA3 Annexin A3 \u003c/p\u003e\n\u003cp\u003eEMT epithelial-to-mesenchymal transition\u003c/p\u003e\n\u003cp\u003eHCC hepatocellular carcinoma \u003c/p\u003e\n\u003cp\u003eIHC immunohistochemistry\u003c/p\u003e\n\u003cp\u003eTMA tissue microarray\u003c/p\u003e\n\u003cp\u003eTEC Tris-EDTA-Citrat\u003c/p\u003e\n\u003cp\u003eNST no special type \u003c/p\u003e\n\u003cp\u003eccRCC clear cell renal cell carcinoma\u003c/p\u003e\n\u003cp\u003epRCC papillary renal cell carcinoma \u003c/p\u003e\n\u003cp\u003eMMR mismatch repair\u003c/p\u003e\n\u003cp\u003eIWGAV International Working Group for Antibody Validation \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to Melanie Steurer, Laura Behm, Inge Brandt, and S\u0026uuml;nje Seekamp for excellent technical assistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u003c/strong\u003e \u003cstrong\u003eContribution Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCvB, NH, FG, RS, GS and SS: contributed to conception, design, data collection, data analysis and manuscript writing.\u003c/p\u003e\n\u003cp\u003eNH, AM, FL, VC, FV, DD, RSch, AH, CF, CB, SB, MK, CHM, GMF, NS, KM, AML, PL, ML, TSC, AHM, EB, NG, MCT, SM, TK, MF, VR: participated in pathology data analysis, data interpretation, and collection of samples.\u003c/p\u003e\n\u003cp\u003eMK, GMF and CHM: data analysis.\u003c/p\u003e\n\u003cp\u003eSS, RS, GS: study supervision.\u003c/p\u003e\n\u003cp\u003eAll authors agree to be accountable for the content of the work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe recombinant mouse monoclonal antibody ARX-503 was provided from ardoci GmbH, Hamburg, Germany (owned by a family member of GS).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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, \u0026sect;12) and by the local ethics committee (Ethics commission Hamburg). The Ethics commission Hamburg, WF-049/09, waived the need to obtain individual patient informed consent. All work has been carried out in compliance with the Helsinki Declaration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo funding\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZhang, H., Zhang, Z., Guo, T., Chen, G., Liu, G., Song, Q., Li, G., Xu, F., Dong, X., Yang, F., et al. (2023). Annexin A protein family: Focusing on the occurrence, progression and treatment of cancer. Front Cell Dev Biol \u003cem\u003e11\u003c/em\u003e, 1141331. 10.3389/fcell.2023.1141331.\u003c/li\u003e\n\u003cli\u003eYang, L., Lu, P., Yang, X., Li, K., and Qu, S. (2021). Annexin A3, a Calcium-Dependent Phospholipid-Binding Protein: Implication in Cancer. Front Mol Biosci \u003cem\u003e8\u003c/em\u003e, 716415. 10.3389/fmolb.2021.716415.\u003c/li\u003e\n\u003cli\u003eXing, M., Liang, S., Cao, W., Guo, Q., and Zou, W. 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Cancer Manag Res \u003cem\u003e13\u003c/em\u003e, 1767-1776. 10.2147/CMAR.S251679.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 \u0026 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Annexin A3, ANXA3, cancer, immunohistochemistry, tissue microarray","lastPublishedDoi":"10.21203/rs.3.rs-9278991/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9278991/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground.\u003c/strong\u003e Annexin A3 (ANXA3) is a member of the annexin family of calcium-dependent phospholipid-binding proteins and plays a major role in various membrane-related processes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMarterial \u0026amp; Methods.\u003c/strong\u003e ANXA3 expression was analyzed by immunohistochemistry (IHC) on tissue microarrays (TMAs) containing 17,285 samples from 148 different tumor types.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults.\u003c/strong\u003e ANXA3 staining was seen in 10,223 (75.9%) of the 13,472 interpretable tumors and was considered weak in 16.3%, moderate in 10.1%, and strong in 49.4% of cases. Of 148 tumor categories, 128 showed ANXA3 expression in at least one case, and 110 included at least one case with strong staining. High ANXA3 expression was linked to high grade (p\u0026lt;0.0001), muscle invasive growth (pTa vs pT2-4; p\u0026lt;0.0001) and high pT-stage (p=0.0267) in urothelial carcinoma; lymphatic invasion (p=0.0307) in colorectal adenocarcinoma; high pT-stage (p=0.0211) in gastric adenocarcinoma; high pT-stage (p\u0026lt;0.0001), nodal positivity (p=0.0004) and high grade (p\u0026lt;0.0001) in hepatocellular carcinoma; and high pT-stage (p=0.0410) in papillary thyroid carcinoma. Low ANXA3 expression was linked with lymphatic invasion (p\u0026lt;0.0001) in urothelial carcinoma, high tumor grade in invasive breast carcinoma of no special type (p\u0026lt;0.0001); high UICC- (p=0.0008) and pT-stage (p\u0026lt;0.0001) in clear cell renal cell carcinoma; high ISUP grade (p=0.0135), high UICC- (p=0.0073) and pT-stage (p=0.0003) in papillary renal cell carcinoma; advanced pT-stage (p=0.0464) in pancreatic ductal adenocarcinoma; high pT-stage (p=0.0056) and mismatch repair deficiency (p\u0026lt;0.0001) in colorectal adenocarcinoma; and high pT-stage (p=0.0427) in endometrioid endometrial carcinoma.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion.\u003c/strong\u003e It is concluded, that ANXA3 is abundantly expressed across a broad spectrum of cancer entities and that it exerts a tissue-dependent oncogenic or tumor-suppressive role.\u003c/p\u003e","manuscriptTitle":"Expression of Annexin A3 in cancer: A tissue microarray study involving more than 17,000 cancers from 148 tumor entities","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-07 19:57:54","doi":"10.21203/rs.3.rs-9278991/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2026-04-23T12:27:56+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-20T05:20:55+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-17T17:14:54+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-13T12:36:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-04-13T10:06:09+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7e16ee86-7d3f-47fb-825f-bb3c0050a76a","owner":[],"postedDate":"May 7th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":67436836,"name":"Health sciences/Biomarkers"},{"id":67436837,"name":"Biological sciences/Cancer"},{"id":67436838,"name":"Health sciences/Oncology"}],"tags":[],"updatedAt":"2026-05-07T19:57:54+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-07 19:57:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9278991","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9278991","identity":"rs-9278991","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}