TTF-1, CDX-2, and PAX-8 Immunoexpression in a Large Serie of Extrapulmonary Small Cell Neuroendocrine Carcinomas: A Study of 138 Cases

TTF-1, CDX-2, and PAX-8 Immunoexpression in a Large Serie of Extrapulmonary Small Cell Neuroendocrine Carcinomas: A Study of 138 Cases | 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 Research Article TTF-1, CDX-2, and PAX-8 Immunoexpression in a Large Serie of Extrapulmonary Small Cell Neuroendocrine Carcinomas: A Study of 138 Cases Klára Pavlíčková, Petr Waldauf, Pavel Dundr, Marián Švajdler, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6821428/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Jan, 2026 Read the published version in Diagnostic Pathology → Version 1 posted 9 You are reading this latest preprint version Abstract Background: Extrapulmonary small cell neuroendocrine carcinoma (EP-SC) is a rare malignancy with a poor prognosis. In our study, one hundred and thirty-eight EP-SCNC tissue samples underwent a complex analysis. Method: We studied the possible benefit of TTF-1, CDX-2, and PAX-8 immunohistochemical analyses for determining the primary site of EP-SCNC origin. Results: The median survival of our cohort was 11.5 months. Patients younger than 70 years had significantly better overall survival (p=0.024). Across the full cohort, CDX-2 positivity was found in 19 tumors (13.9%), TTF-1 in 35 tumors (25.7%), and PAX-8 in 63 tumors (46.3%), regardless of tumor origin. The expression of CDX-2 and PAX-8 did not differ significantly among different organ systems (p=0.2 and 0.3), respectively. The expression of TTF-1 differed significantly in tumors from various sites of origin (p=0.009), expressed more often in breast, pancreatic, and genitourinary tumors. Interestingly, TTF-1 expression proved to have a negative prognostic impact relative to patient survival (age-adjusted Cox regression analysis, HR=1.62, 95%CI:1.06-2.47, p=0.021). Conclusion: As most patients with EP-SCNC had a metastatic presentation, finding the primary tumor origin, which is important for patient prognoses, is both challenging and important at the same time. Only TTF-1 immunohistochemical analysis proved to be helpful with this task. Moreover, a diagnosis of primary lung small cell carcinoma cannot be established based on TTF-1 positivity, since only 25.7% of EP-SCNC in our study displayed TTF-1 positivity. Extrapulmonary small cell neuroendocrine carcinoma TTF-1 CD-X2 PAX-8 Figures Figure 1 Figure 2 Figure 3 Introduction Extrapulmonary small cell neuroendocrine carcinoma (EP-SCNC) is a rare malignancy with a poor prognosis; the average overall patient survival is less than 12 months. ( 1 , 2 ) The incidence of EP-SCNC is less than 0.4% of human malignancies. ( 2 ) Recently, molecular evidence has shown that EP-SCNC can develop late in the progression of various organ-specific carcinoma types. ( 2 ) Therefore, the incidence of extrapulmonary neuroendocrine carcinoma is increasing, which may be related to expanded systemic cancer therapy for organ-specific adenocarcinomas. ( 3 ) EP-SCNC can originate from any organ system, including the genitourinary, gastrointestinal, hepatobiliary systems, the gynecologic tract, pancreas, and the head and neck region. Most patients with EP-SCNC have metastatic disease upon presentation, therefore, the search for the primary origin site of the tumor can be challenging, and some fraction of tumors have undetermined primary sites. CDX-2, TTF-1, and PAX-8 Organ-specific antigens CDX-2, TTF-1, and PAX-8 are routinely used to find the primary sites of metastatic adenocarcinomas. CDX-2 (caudal-related homeobox 2) is essential for intestinal cell fate specification during embryonic development. Its expression begins in the duodenum, gradually increasing through the small intestine, peaking at the ileocecal region, and then declining in the distal colon. (4) TTF-1 (thyroid-transcription factor 1) participates in the differentiation, development, and functional maintenance of the lungs, thyroid gland, and brain structures of diencephalic origin. (5) It is expressed mainly in Clara cells, type II pneumonocytes, thyroid follicular cells, the pituitary gland, and the hypothalamus. ( 6 ) PAX-8 is a transcription factor involved in the organogenesis of the thyroid gland, kidneys, and the Müllerian system. PAX8 is therefore expressed in the thyroid and parathyroid glands, the kidneys, thymus, and female and male genital tracts. ( 7 , 8 ) This study aims to clarify the expression of organ-specific antigens CDX-2, TTF-1, and PAX-8 in a large cohort of EP-SCNC with various primary sites. Materials and methods Sample selection This multicenter study analyzed 138 diagnosed cases of extrapulmonary small cell neuroendocrine carcinoma (EP-SCNC) over 24 years (1999–2023). It included high-grade small cell carcinomas exhibiting neuroendocrine morphology, characterized by scant cytoplasm, finely granular chromatin lacking nucleoli, brisk mitotic activity, frequent apoptotic bodies, and necrosis, but without known pulmonary involvement. Inclusion was independent of neuroendocrine marker expression, though all cases underwent marker analysis. Specimens were sourced from the archives of participating pathology departments. Formalin-fixed, paraffin-embedded (FFPE) tissue samples from biopsies and surgical resections were collected for analysis. Two pathologists (R.M. and K.P.) examined hematoxylin and eosin-stained slides, selecting representative regions from FFPE blocks for tissue microarray (TMA) construction. Additional tumor sections were designated for next-generation sequencing (NGS), with the percentage of vital cells estimated semi-quantitatively. Various clinical data were collected, including sex, age at diagnosis, and primary origin of the tumor. Overall survival (OS), the time between the initial diagnosis and death, was taken from records kept by the Institute of Health Information and Statistics of the Czech Republic. None of the patients received prior oncological therapy before biopsy/surgery. Immunohistochemical analysis Immunohistochemistry was performed on tissue microarrays (TMAs) using 4 µm-thick FFPE tissue sections. For the construction of the TMAs, eligible areas of each tumor were identified, and two tissue cores (each 2.0 mm in diameter) were taken from the donor block using a TMA Master tissue microarray instrument (3DHISTECH Ltd., Budapest, Hungary). Using TMA, immunohistochemistry (IHC) was performed using organ-specific transcription factors CDX-2 (DAKO, ready to use kit DAK-CDX2), TTF-1 (DAKO, clone 8G7G3/1, dilution 1:200), and PAX-8 (Cell Marque, polyclonal, dilution 1:50). Statistical analysis All analyses were performed using R version 4.5.0 and RStudio ( 9 ); survival analyses were carried out using the survival package 3.8-3, survminer 0.5.0 ( 10 ), and adjustedCurves 0.11.2. ( 11 ) Continuous data are expressed as means and standard deviations or medians with 25th and 75th percentiles (or interquartile range, IQR), depending on the normality of the distribution. Binary and categorical data are expressed as counts and percentages. For OS analysis, a Kaplan-Meier analysis was performed with 95% confidence intervals (95% CIs) calculated using the log-log method and p-values from the log-rank test. Univariate and age-adjusted Cox regressions were also performed for age and tumor location, expressed as unadjusted and age-adjusted hazard ratios (HR) with 95% CI. For OS analysis, patients were censored at five years. The Pearson chi-square test and Fisher's exact test were used as appropriate to assess associations between categorized variables. Categorized parameters included primary tumor location and age (≤ 70). Samples were considered CDX-2, TTF-1, and PAX-8 positive if at least 5% of cells displayed moderate to strong nuclear staining, as summarized in detail elsewhere. ( 12 ) P values < 0.05 were considered statistically significant. Results Clinical description of the cohort Of the 138 Caucasian patients included in the analysis, 86 were males (62.8%) and 51 were females (37.2%), with a mean age of 67.0 years (ranged 38.2–94.1 years) at the time of diagnosis. Median survival was 11.5 months (95% CI 8.3–14.5 months), and five-year survival was 16.2%. The primary EP-SCNC tumor location was the genitourinary (male genital and urinary) tract in 71 patients (51.4%), gastrointestinal tract (esophagus, stomach, and intestine) in 20 patients (14.5%), gynecological tract in 16 patients (11.6%), pancreas in 5 patients (3.6%), head and neck in 8 patients (4.4%), breast in 5 patients (3.6%), hepatobiliary system in 3 patients (2.2%), and the skin in 1 patient (0.7%). There were 9 (6.5%) EP-SCNCs with unknown primary sites. In all analyzed cases, a primary pulmonary origin was excluded based on clinical documentation. All specimens were from tumor resections/biopsies (95 biopsies and 43 resections) of primary tumors, apart from the 9 EP-SCNCs with unknown primary sites. The patient's age proved to be an independent prognostic marker (Kaplan-Meier survival analysis comparing patients by age: Patients under 70 years showed a median survival of 12.3 months (95% CI: 9.6–28.2) versus 7.6 months (95% CI: 4.9–13.7) for patients over 70 years (p = 0.024). Immunohistochemical analysis Among the entire cohort, irrespective of tumor origin, 19 (13.9%) tumors were CDX-2 positive, 35 (25.7%) tumors were TTF-1 positive, and 63 (46.3%) tumors were PAX-8 positive (Fig. 1 ). The expressions of CDX-2, TTF-1, and PAX-8 with respect to tumor origin are shown in detail in Fig. 2 . Expression of CDX-2, TTF-1, and PAX-8 in different organs is shown in Supplementary Material 1. The expression of CDX-2 and PAX-8 did not differ significantly among different organ systems (p = 0.2 and 0.3, respectively). At the same time, CDX-2 and PAX-8 expression did not have an impact on patient survival (p = 0.85 and 0.84, respectively). Regarding CDX-2, there were 8 (11.1%) positive genitourinary tumors, 6 (30.0%) positive gastrointestinal tumors, 1 (6.3%) positive gynecological tumor, and 2 (33.3%) positive pancreatic tumors. In PAX-8, there were 34 (46.6%) positive genitourinary tumors, 10 (62.5) positive gynecological tumors, 5 (25.0%) positive gastrointestinal tumors, 5 (62.5%) positive head and neck tumors, 2 (40.0%) positive breast tumors, 2 (66.7%) positive tumors of hepatobiliary origin, and 1 (20.0%) positive pancreatic tumor. Concerning TTF-1 immunohistochemistry, 25 (34.7%) genitourinary tumors displayed TTF-1 positivity, from other locations, there were 4 (20.0%) TTF-1 positive gastrointestinal tumors, 3 (60.0%) TTF-1 positive breast tumors, 2 (40.0%) TTF-1 positive pancreatic tumors, and 1 (12.5%) TTF-1 positive head and neck tumors. Therefore, the expression of TTF-1 differed in tumors of various sites of origin (p = 0.009). Moreover, TTF-1 expression proved to have a prognostic impact on patient survival, as shown by age-adjusted Cox regression analysis (HR = 1.62, 95% CI: 1.06–2.47, p = 0.021) and Kaplan-Meier analysis (p = 0.024; Fig. 3 ). Discussion Our study examined a cohort of 138 extrapulmonary small-cell neuroendocrine carcinomas with respect to organ-specific antigens CDX-2, TTF-1, and PAX-8. An extensive immunohistochemical and molecular analysis of the cohort is summarized in detail elsewhere. ( 13 ) The use of immunohistochemistry in typing tumors of uncertain primary origin is routine in surgical pathology in metastatic disease. Accurate tumor classification is crucial for advancing targeted antitumor therapies. Various recommendations and algorithms for antibody panels have been proposed ( 14 , 15 ), particularly considering the limited tissue available in small biopsy samples, where preservation for molecular examination is crucial. A panel of antibodies is recommended, since the use of a single antibody alone may lead to misleading results. ( 16 , 17 ) TTF-1, CDX-2, and PAX-8 are the most frequently used antibodies for differential diagnostics ( 18 – 20 ) of adenocarcinomas with an unknown primary origin. Extrapulmonary small cell carcinomas are extremely rare tumors. Like small cell lung carcinomas, patients with this condition often present with extensive, disseminated disease. However, the primary sites of origin of EP-SCNCs have proved to be an independent prognostic variable in several large cohort studies ( 1 , 21 , 22 ). This was also supported by our study, where primary tumor sites were an age-independent prognostic marker (p < 0.0001) ); digestive tract EP-SCNCs displayed decreased survival compared to those originating in genitourinary organs. Moreover, in our previous study of 181 EP-SCNCs, we separately analyzed tumors with genitourinary origin (genitourinary tract and gynecological tract tumors; n = 105) and tumors of the digestive system (gastrointestinal tract, pancreas, and hepatobiliary system; n = 49); patients with genitourinary SCNCs had significantly longer survival than those with digestive system SCNCs (HR = 1.73, 95% CI: 1.19–2.52, p = 0.004). ( 13 ) Since few studies have explored this topic, we investigated the utility of TTF-1, CDX-2, and PAX-8 in differentiating EP-SCNCs based on their primary sites, area. Kaufman et al.²² identified TTF-1 expression in 80% of EP-SCNCs, supporting its potential diagnostic role. According to ESMO guidelines¹⁴, CDX-2 positivity is indicative of a neuroendocrine tumor originating in the gastrointestinal tract or pancreas. However, our findings challenge this assumption, since only 30% of gastrointestinal tumors and 33.3% of pancreatic tumors exhibited CDX-2 positivity, while 11.1% of genitourinary tumors and 6.3% of gynecological tumors also demonstrated CDX-2 expression. Consistent with the role of PAX-8 as a transcription factor involved in the organogenesis of the thyroid gland, kidney, and Müllerian system, 62.5% of gynecological tumors, 62.5% of head and neck tumors, and 46.6% of genitourinary tumors showed PAX-8 positivity. On the other hand, PAX-8 positivity was also observed in 66.7% of tumors of hepatobiliary origin, 40% of breast tumors, 25% of gastrointestinal tumors, and 20% of pancreatic tumors. TTF-1 positivity was detected in 60% of breast tumors, 40% of pancreatic tumors, 34.7% of genitourinary tumors, 20% of gastrointestinal tumors, and 12.5% of head and neck tumors, making it the only immunohistochemical marker in our study to reach statistical significance. However, despite its statistical relevance, its practical utility in accurately determining the primary site of EP-SCNC remains limited in routine clinical practice. However, importantly, TTF-1 positivity is not exclusive to lung tumors. A vast majority of small cell lung carcinomas exhibit TTF-1 positivity ( 23 ); however, TTF-1 positivity in small cell neuroendocrine tumors should not be considered definitive evidence of a lung primary origin, since nearly 26% of the EP-SCNCs in our study were TTF-1 positive. In a study of Briski et al. ( 24 ), clone of TTF-1 antibody proved to play an important role in EP-SCNC staining. 83% EP-SCNC were positive for clone SPT24 (Novocastra), whereas 48% EP-SCNC were positive for 8G7G3/1 (DAKO). The differences in staining between 8G7G3/1 and SPT24 in the nonpulmonary cohort were statistically significant ( P < 0.0001). The difference of the clones 8G7G3/1 and SPT24 is discussed in a study of metastatic adenocarcinomas in lungs, where the SPT24 clone seemed to be more sensitive, but less specific for TTF-1 protein. ( 25 ) The biological role of TTF-1 in tumorigenesis remains to be clarified, since few studies explored this topic. In a manuscript of Iso H. et al, TTF-1-negative NSCLC exhibited distinct biological characteristics, such as a higher prevalence of certain genetic mutations (e.g., STK11, KEAP1) and an immunosuppressive tumor microenvironment. According to authors, these factors might contribute to the observed resistance to therapies and poorer prognoses of patients with TTF-1-negative NSCLC in their study. ( 26 ) 4 and 3 tumors had STK11 and KEAP1 mutation in our study and all of them were TTF-1 negative at the same time. ( 13 ) Interestingly, in a study of Iida Y. et al, TTF-1-negative SCLC were different in aspects of neuroendocrine differentiation (negativity of synaptophysin and chromogranin, lower expression of ASCL1) compared to TTF-1-positive SCLC. ( 27 ) Concerning EP-SCNC, ASCL1 negative tumors are known to constitute a larger proportion than in SCLC. ( 13 ) TTF-1 positivity proved to have a negative prognostic value in our study, which can be partly supported by the above-mentioned study of Iso H. et al. ( 26 ) However, this finding is in contrast with results of a robust meta-analysis conducted by Wang Y. et al., who studied 11 manuscripts comprising 1,786 SCLC patients, where TTF-1 expression indicated improved overall survival (OS) (HR = 0.56, 95% CI: 0.45–0.70; P < 0.001) and progression-free survival (PFS) (HR = 0.41, 95% CI: 0.28–0.62; P < 0.001). ( 5 ) Moreover, TTF-1 positivity proved to have a positive prognostic impact in a meta-analysis performed by Qian H.H. et al., who studied 17 manuscripts comprising 2,235 patients with non-small cell lung carcinoma; TTF-1 overexpression had a favorable impact on survival (HR = 0.49, 95% CI: 0.42–0.55). ( 28 ) Although EP-SCNC has been repeatedly shown to be a heterogeneous disease¹² that is partially responsive to platinum-based chemotherapy, no consensus exists on the optimal second-line therapy in cases of progression. ( 3 , 29 ) To advance therapeutic strategies requires a deeper understanding of molecular and immune characteristics. However, our study of TTF-1, CDX-2, and PAX-8 immunohistochemistry did not prove instrumental in identifying the primary site of EP-SCNC origin and, in our opinion, should be interpreted with caution. Limitations The TMA approach, despite its widespread use in research, carries a risk of under- or overestimation of immunohistochemical scoring and may be a limitation due to tumor heterogeneity. Although our cohort of 138 patients with EP-SCNC is one of the largest in literature, the number of patients in each tumor group is limited, which may constrain the statistical interpretation of results. Abbreviations EP-SCNC – Extrapulmonary Small Cell Neuroendocrine Carcinoma TTF-1 – Thyroid Transcription Factor 1 CDX-2 – Caudal-related Homeobox 2 PAX-8 – Paired Box Gene 8 IHC – Immunohistochemistry OS – Overall Survival FFPE – Formalin-Fixed Paraffin-Embedded TMA – Tissue Microarray NGS – Next-Generation Sequencing HR – Hazard Ratio CI – Confidence Interval NSCLC – Non-Small Cell Lung Carcinoma SCLC – Small Cell Lung Carcinoma PFS – Progression-Free Survival ASCL1 – Achaete-Scute Family Transcription Factor 1 Declarations Ethics Approval/Consent to Participate: This study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of the Institute for Clinical and Experimental Medicine and Thomayer University Hospital No G-21-39. Author Contribution Statement (CRediT) Klara Pavlickova: Writing – original draft, Data curation, Resources, Investigation Radoslav Matej: Conceptualization, Writing – review and editing, Supervision, Resources Pavel Dundr: Writing – review and editing, Supervision Jan Hrudka: Writing – review and editing Petr Waldauf: Software Jan Laco, Marian Svajdler, Iva Staniczkova Zambo, Miroslava Fligrova, Helena Hornychova and Jozef Skarda: Data curation Consent for publication: All authors read and approved the paper for publication. Competing interests: The authors declare that they have no competing interests. Data Availability Statement: All data generated or analyzed during this study are included in the published article and its supplementary information files. Funding Statement: This work was supported by the Ministry of Health of the Czech Republic (project Conceptual Development of Research Organization, General University Hospital, Prague (VFN, 00064165) and Thomayer University Hospital (FTN, 00064190)), the Agency for Health Research of the Czech Republic (project NU22-03-00130), by the European Regional Development Fund (BBMRI_CZ LM2023033), and by the Charles University (projects UNCE 24/MED/018 and Cooperatio Medical Diagnostics and Basic Medical Sciences). Acknowledgment We would like to express our special thanks to Mr. Thomas Secrest, MSc, for language editing services and Alena Poláková, MSc, for her excellent technical assistance. References Dasari A, Mehta K, Byers LA, Sorbye H, Yao JC. Comparative study of lung and extrapulmonary poorly differentiated neuroendocrine carcinomas: A SEER database analysis of 162,983 cases. Cancer. 2018;124(4):807–15. Frazier SR, Kaplan PA, Loy TS. 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Supplementary Files SupplementaryMaterial.xlsx Supplementary material 1: Detailed distribution of TTF-1, CDX-2, and PAX-8 expressions in different organs and organ systems. Cite Share Download PDF Status: Published Journal Publication published 26 Jan, 2026 Read the published version in Diagnostic Pathology → Version 1 posted Editorial decision: Revision requested 18 Aug, 2025 Reviews received at journal 17 Aug, 2025 Reviewers agreed at journal 17 Aug, 2025 Reviews received at journal 15 Jul, 2025 Reviewers agreed at journal 15 Jul, 2025 Reviewers invited by journal 13 Jul, 2025 Editor assigned by journal 05 Jun, 2025 Submission checks completed at journal 05 Jun, 2025 First submitted to journal 04 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Kralove","correspondingAuthor":false,"prefix":"","firstName":"Jan","middleName":"","lastName":"Laco","suffix":""},{"id":484665675,"identity":"22c4ed5e-c047-4c88-93c4-c9cecbda23ea","order_by":8,"name":"Helena Hornychová","email":"","orcid":"","institution":"Charles University","correspondingAuthor":false,"prefix":"","firstName":"Helena","middleName":"","lastName":"Hornychová","suffix":""},{"id":484665676,"identity":"3f0f41eb-33e5-4871-88d8-c3b7f0a9ee9f","order_by":9,"name":"Patricie Delongová","email":"","orcid":"","institution":"University Hospital Ostrava, University of Ostrava","correspondingAuthor":false,"prefix":"","firstName":"Patricie","middleName":"","lastName":"Delongová","suffix":""},{"id":484665677,"identity":"abcf25da-2bdc-4ad8-8e2d-2b3325c8aefe","order_by":10,"name":"Jozef Škarda","email":"","orcid":"","institution":"University Hospital Ostrava, University of Ostrava","correspondingAuthor":false,"prefix":"","firstName":"Jozef","middleName":"","lastName":"Škarda","suffix":""},{"id":484665678,"identity":"13cd69f8-90d5-4337-8e15-fc0d8ccfd606","order_by":11,"name":"Jan Hrudka","email":"","orcid":"","institution":"University Hospital Kralovske Vinohrady, Charles University","correspondingAuthor":false,"prefix":"","firstName":"Jan","middleName":"","lastName":"Hrudka","suffix":""},{"id":484665679,"identity":"a9758ada-d461-41b8-be4c-7f3189700633","order_by":12,"name":"Radoslav Matěj","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7UlEQVRIiWNgGAWjYHACAwaGAhibx4afSC0GYAZjAwNPmmQPiVoYDhPWYt5+eOPDHwZ2iQ1ih58/+CBzXsJeuoHx4w88WmTOpBUb8xgkJzZIpxk2zuC5LcEjc4BZQgKPFgmGHDNpBgNmYwbpBMNmHp7bdTwSCWxQl+LQwv/GTPKHQT1QS/rH5j885yTAWhLwaZHIMZPgMTgsxyCdY9jMwHMAouUAXi3PQH45LscmnVM4s4cnWYLnRmKzZANehyUDQ6yimodfOn3Dh589dhLsM5IP4g0xOGADEYzgSGHEZwcGIMr0UTAKRsEoGGkAAKlgQRHYdPpuAAAAAElFTkSuQmCC","orcid":"","institution":"Thomayer University Hospital, Charles University","correspondingAuthor":true,"prefix":"","firstName":"Radoslav","middleName":"","lastName":"Matěj","suffix":""}],"badges":[],"createdAt":"2025-06-04 14:23:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6821428/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6821428/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13000-026-01753-3","type":"published","date":"2026-01-26T15:58:05+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":87032832,"identity":"af09e402-cbe9-4225-add5-644c00df7283","added_by":"auto","created_at":"2025-07-18 13:01:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3870554,"visible":true,"origin":"","legend":"\u003cp\u003eEP-SCNC of colon exhibiting nuclear CDX-2 positivity, with positivity of colon crypts (crypts of Lieberkühn) serving as an internal control, 200×, (A). EP-SCNC with nuclear PAX-8 positivity, 200×, (B). EP-SCNC exhibiting strong nuclear TTF-1 positivity 200×, (C).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6821428/v1/7e03b10406f20ea7d38ded9b.png"},{"id":87032833,"identity":"d6d0d424-4ffa-425a-b233-1b4ea5aa63ae","added_by":"auto","created_at":"2025-07-18 13:01:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":632194,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of TTF-1, CDX-2, and PAX-8 in EP-SCNCs with respect to tumor origin.\u003c/p\u003e","description":"","filename":"OnlineFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6821428/v1/0c608f0e5cc6618f4486f281.png"},{"id":87032827,"identity":"24a8cead-a93c-4fdc-bfcd-a6f72b74446c","added_by":"auto","created_at":"2025-07-18 13:01:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":236913,"visible":true,"origin":"","legend":"\u003cp\u003eKaplan-Meier curve documenting significantly longer OS in those with TTF-1 positive tumors.\u003c/p\u003e","description":"","filename":"OnlineFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6821428/v1/fc81d12ff3ececf4e3ed3d6a.png"},{"id":101690411,"identity":"adbc1e70-bd33-4d07-baeb-c827d4867942","added_by":"auto","created_at":"2026-02-02 16:01:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7026896,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6821428/v1/92203f32-ced1-4da9-9303-f4b10b6d3ae0.pdf"},{"id":87032825,"identity":"fcdf9e74-84a3-4efa-a9af-90d8cad3d1a0","added_by":"auto","created_at":"2025-07-18 13:01:43","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":17098,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary material 1:\u003c/p\u003e\n\u003cp\u003eDetailed distribution of TTF-1, CDX-2, and PAX-8 expressions in different organs and organ systems.\u003c/p\u003e","description":"","filename":"SupplementaryMaterial.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6821428/v1/e6a0a46da2a25d112d3adac5.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"TTF-1, CDX-2, and PAX-8 Immunoexpression in a Large Serie of Extrapulmonary Small Cell Neuroendocrine Carcinomas: A Study of 138 Cases","fulltext":[{"header":"Introduction","content":"\u003cp\u003eExtrapulmonary small cell neuroendocrine carcinoma (EP-SCNC) is a rare malignancy with a poor prognosis; the average overall patient survival is less than 12 months. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) The incidence of EP-SCNC is less than 0.4% of human malignancies. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) Recently, molecular evidence has shown that EP-SCNC can develop late in the progression of various organ-specific carcinoma types. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) Therefore, the incidence of extrapulmonary neuroendocrine carcinoma is increasing, which may be related to expanded systemic cancer therapy for organ-specific adenocarcinomas. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eEP-SCNC can originate from any organ system, including the genitourinary, gastrointestinal, hepatobiliary systems, the gynecologic tract, pancreas, and the head and neck region. Most patients with EP-SCNC have metastatic disease upon presentation, therefore, the search for the primary origin site of the tumor can be challenging, and some fraction of tumors have undetermined primary sites.\u003c/p\u003e\u003cp\u003eCDX-2, TTF-1, and PAX-8\u003c/p\u003e\u003cp\u003eOrgan-specific antigens CDX-2, TTF-1, and PAX-8 are routinely used to find the primary sites of metastatic adenocarcinomas. CDX-2 (caudal-related homeobox 2) is essential for intestinal cell fate specification during embryonic development. Its expression begins in the duodenum, gradually increasing through the small intestine, peaking at the ileocecal region, and then declining in the distal colon. \u003csup\u003e(4)\u003c/sup\u003e TTF-1 (thyroid-transcription factor 1) participates in the differentiation, development, and functional maintenance of the lungs, thyroid gland, and brain structures of diencephalic origin. \u003csup\u003e(5)\u003c/sup\u003e It is expressed mainly in Clara cells, type II pneumonocytes, thyroid follicular cells, the pituitary gland, and the hypothalamus. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e) PAX-8 is a transcription factor involved in the organogenesis of the thyroid gland, kidneys, and the M\u0026uuml;llerian system. PAX8 is therefore expressed in the thyroid and parathyroid glands, the kidneys, thymus, and female and male genital tracts. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e) This study aims to clarify the expression of organ-specific antigens CDX-2, TTF-1, and PAX-8 in a large cohort of EP-SCNC with various primary sites.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eSample selection\u003c/p\u003e\u003cp\u003eThis multicenter study analyzed 138 diagnosed cases of extrapulmonary small cell neuroendocrine carcinoma (EP-SCNC) over 24 years (1999\u0026ndash;2023). It included high-grade small cell carcinomas exhibiting neuroendocrine morphology, characterized by scant cytoplasm, finely granular chromatin lacking nucleoli, brisk mitotic activity, frequent apoptotic bodies, and necrosis, but without known pulmonary involvement. Inclusion was independent of neuroendocrine marker expression, though all cases underwent marker analysis. Specimens were sourced from the archives of participating pathology departments.\u003c/p\u003e\u003cp\u003eFormalin-fixed, paraffin-embedded (FFPE) tissue samples from biopsies and surgical resections were collected for analysis. Two pathologists (R.M. and K.P.) examined hematoxylin and eosin-stained slides, selecting representative regions from FFPE blocks for tissue microarray (TMA) construction. Additional tumor sections were designated for next-generation sequencing (NGS), with the percentage of vital cells estimated semi-quantitatively.\u003c/p\u003e\u003cp\u003eVarious clinical data were collected, including sex, age at diagnosis, and primary origin of the tumor. Overall survival (OS), the time between the initial diagnosis and death, was taken from records kept by the Institute of Health Information and Statistics of the Czech Republic. None of the patients received prior oncological therapy before biopsy/surgery.\u003c/p\u003e\u003cp\u003eImmunohistochemical analysis\u003c/p\u003e\u003cp\u003eImmunohistochemistry was performed on tissue microarrays (TMAs) using 4 \u0026micro;m-thick FFPE tissue sections. For the construction of the TMAs, eligible areas of each tumor were identified, and two tissue cores (each 2.0 mm in diameter) were taken from the donor block using a TMA Master tissue microarray instrument (3DHISTECH Ltd., Budapest, Hungary). Using TMA, immunohistochemistry (IHC) was performed using organ-specific transcription factors CDX-2 (DAKO, ready to use kit DAK-CDX2), TTF-1 (DAKO, clone 8G7G3/1, dilution 1:200), and PAX-8 (Cell Marque, polyclonal, dilution 1:50).\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll analyses were performed using R version 4.5.0 and RStudio (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e); survival analyses were carried out using the survival package 3.8-3, survminer 0.5.0 (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e), and adjustedCurves 0.11.2. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e) Continuous data are expressed as means and standard deviations or medians with 25th and 75th percentiles (or interquartile range, IQR), depending on the normality of the distribution. Binary and categorical data are expressed as counts and percentages.\u003c/p\u003e\u003cp\u003eFor OS analysis, a Kaplan-Meier analysis was performed with 95% confidence intervals (95% CIs) calculated using the log-log method and p-values from the log-rank test. Univariate and age-adjusted Cox regressions were also performed for age and tumor location, expressed as unadjusted and age-adjusted hazard ratios (HR) with 95% CI. For OS analysis, patients were censored at five years.\u003c/p\u003e\u003cp\u003eThe Pearson chi-square test and Fisher's exact test were used as appropriate to assess associations between categorized variables. Categorized parameters included primary tumor location and age (\u0026le;\u0026thinsp;70).\u003c/p\u003e\u003cp\u003eSamples were considered CDX-2, TTF-1, and PAX-8 positive if at least 5% of cells displayed moderate to strong nuclear staining, as summarized in detail elsewhere. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e)\u003c/p\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eClinical description of the cohort\u003c/p\u003e\u003cp\u003eOf the 138 Caucasian patients included in the analysis, 86 were males (62.8%) and 51 were females (37.2%), with a mean age of 67.0 years (ranged 38.2\u0026ndash;94.1 years) at the time of diagnosis. Median survival was 11.5 months (95% CI 8.3\u0026ndash;14.5 months), and five-year survival was 16.2%.\u003c/p\u003e\u003cp\u003eThe primary EP-SCNC tumor location was the genitourinary (male genital and urinary) tract in 71 patients (51.4%), gastrointestinal tract (esophagus, stomach, and intestine) in 20 patients (14.5%), gynecological tract in 16 patients (11.6%), pancreas in 5 patients (3.6%), head and neck in 8 patients (4.4%), breast in 5 patients (3.6%), hepatobiliary system in 3 patients (2.2%), and the skin in 1 patient (0.7%). There were 9 (6.5%) EP-SCNCs with unknown primary sites. In all analyzed cases, a primary pulmonary origin was excluded based on clinical documentation. All specimens were from tumor resections/biopsies (95 biopsies and 43 resections) of primary tumors, apart from the 9 EP-SCNCs with unknown primary sites.\u003c/p\u003e\u003cp\u003eThe patient's age proved to be an independent prognostic marker (Kaplan-Meier survival analysis comparing patients by age: Patients under 70 years showed a median survival of 12.3 months (95% CI: 9.6\u0026ndash;28.2) versus 7.6 months (95% CI: 4.9\u0026ndash;13.7) for patients over 70 years (p\u0026thinsp;=\u0026thinsp;0.024).\u003c/p\u003e\u003cp\u003eImmunohistochemical analysis\u003c/p\u003e\u003cp\u003eAmong the entire cohort, irrespective of tumor origin, 19 (13.9%) tumors were CDX-2 positive, 35 (25.7%) tumors were TTF-1 positive, and 63 (46.3%) tumors were PAX-8 positive (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The expressions of CDX-2, TTF-1, and PAX-8 with respect to tumor origin are shown in detail in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Expression of CDX-2, TTF-1, and PAX-8 in different organs is shown in Supplementary Material 1.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe expression of CDX-2 and PAX-8 did not differ significantly among different organ systems (p\u0026thinsp;=\u0026thinsp;0.2 and 0.3, respectively). At the same time, CDX-2 and PAX-8 expression did not have an impact on patient survival (p\u0026thinsp;=\u0026thinsp;0.85 and 0.84, respectively). Regarding CDX-2, there were 8 (11.1%) positive genitourinary tumors, 6 (30.0%) positive gastrointestinal tumors, 1 (6.3%) positive gynecological tumor, and 2 (33.3%) positive pancreatic tumors. In PAX-8, there were 34 (46.6%) positive genitourinary tumors, 10 (62.5) positive gynecological tumors, 5 (25.0%) positive gastrointestinal tumors, 5 (62.5%) positive head and neck tumors, 2 (40.0%) positive breast tumors, 2 (66.7%) positive tumors of hepatobiliary origin, and 1 (20.0%) positive pancreatic tumor.\u003c/p\u003e\u003cp\u003eConcerning TTF-1 immunohistochemistry, 25 (34.7%) genitourinary tumors displayed TTF-1 positivity, from other locations, there were 4 (20.0%) TTF-1 positive gastrointestinal tumors, 3 (60.0%) TTF-1 positive breast tumors, 2 (40.0%) TTF-1 positive pancreatic tumors, and 1 (12.5%) TTF-1 positive head and neck tumors. Therefore, the expression of TTF-1 differed in tumors of various sites of origin (p\u0026thinsp;=\u0026thinsp;0.009). Moreover, TTF-1 expression proved to have a prognostic impact on patient survival, as shown by age-adjusted Cox regression analysis (HR\u0026thinsp;=\u0026thinsp;1.62, 95% CI: 1.06\u0026ndash;2.47, p\u0026thinsp;=\u0026thinsp;0.021) and Kaplan-Meier analysis (p\u0026thinsp;=\u0026thinsp;0.024; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur study examined a cohort of 138 extrapulmonary small-cell neuroendocrine carcinomas with respect to organ-specific antigens CDX-2, TTF-1, and PAX-8. An extensive immunohistochemical and molecular analysis of the cohort is summarized in detail elsewhere. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eThe use of immunohistochemistry in typing tumors of uncertain primary origin is routine in surgical pathology in metastatic disease. Accurate tumor classification is crucial for advancing targeted antitumor therapies. Various recommendations and algorithms for antibody panels have been proposed (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e), particularly considering the limited tissue available in small biopsy samples, where preservation for molecular examination is crucial. A panel of antibodies is recommended, since the use of a single antibody alone may lead to misleading results. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e) TTF-1, CDX-2, and PAX-8 are the most frequently used antibodies for differential diagnostics (\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e) of adenocarcinomas with an unknown primary origin.\u003c/p\u003e\u003cp\u003eExtrapulmonary small cell carcinomas are extremely rare tumors. Like small cell lung carcinomas, patients with this condition often present with extensive, disseminated disease. However, the primary sites of origin of EP-SCNCs have proved to be an independent prognostic variable in several large cohort studies (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). This was also supported by our study, where primary tumor sites were an age-independent prognostic marker (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) ); digestive tract EP-SCNCs displayed decreased survival compared to those originating in genitourinary organs. Moreover, in our previous study of 181 EP-SCNCs, we separately analyzed tumors with genitourinary origin (genitourinary tract and gynecological tract tumors; n\u0026thinsp;=\u0026thinsp;105) and tumors of the digestive system (gastrointestinal tract, pancreas, and hepatobiliary system; n\u0026thinsp;=\u0026thinsp;49); patients with genitourinary SCNCs had significantly longer survival than those with digestive system SCNCs (HR\u0026thinsp;=\u0026thinsp;1.73, 95% CI: 1.19\u0026ndash;2.52, p\u0026thinsp;=\u0026thinsp;0.004). (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eSince few studies have explored this topic, we investigated the utility of TTF-1, CDX-2, and PAX-8 in differentiating EP-SCNCs based on their primary sites, area. Kaufman et al.\u0026sup2;\u0026sup2; identified TTF-1 expression in 80% of EP-SCNCs, supporting its potential diagnostic role. According to ESMO guidelines\u0026sup1;⁴, CDX-2 positivity is indicative of a neuroendocrine tumor originating in the gastrointestinal tract or pancreas. However, our findings challenge this assumption, since only 30% of gastrointestinal tumors and 33.3% of pancreatic tumors exhibited CDX-2 positivity, while 11.1% of genitourinary tumors and 6.3% of gynecological tumors also demonstrated CDX-2 expression.\u003c/p\u003e\u003cp\u003eConsistent with the role of PAX-8 as a transcription factor involved in the organogenesis of the thyroid gland, kidney, and M\u0026uuml;llerian system, 62.5% of gynecological tumors, 62.5% of head and neck tumors, and 46.6% of genitourinary tumors showed PAX-8 positivity. On the other hand, PAX-8 positivity was also observed in 66.7% of tumors of hepatobiliary origin, 40% of breast tumors, 25% of gastrointestinal tumors, and 20% of pancreatic tumors.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eTTF-1 positivity was detected in 60% of breast tumors, 40% of pancreatic tumors, 34.7% of genitourinary tumors, 20% of gastrointestinal tumors, and 12.5% of head and neck tumors, making it the only immunohistochemical marker in our study to reach statistical significance. However, despite its statistical relevance, its practical utility in accurately determining the primary site of EP-SCNC remains limited in routine clinical practice.\u003c/p\u003e\u003cp\u003eHowever, importantly, TTF-1 positivity is not exclusive to lung tumors. A vast majority of small cell lung carcinomas exhibit TTF-1 positivity (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e); however, TTF-1 positivity in small cell neuroendocrine tumors should not be considered definitive evidence of a lung primary origin, since nearly 26% of the EP-SCNCs in our study were TTF-1 positive.\u003c/p\u003e\u003cp\u003eIn a study of Briski et al. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e), clone of TTF-1 antibody proved to play an important role in EP-SCNC staining. 83% EP-SCNC were positive for clone SPT24 (Novocastra), whereas 48% EP-SCNC were positive for 8G7G3/1 (DAKO). The differences in staining between 8G7G3/1 and SPT24 in the nonpulmonary cohort were statistically significant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). The difference of the clones 8G7G3/1 and SPT24 is discussed in a study of metastatic adenocarcinomas in lungs, where the SPT24 clone seemed to be more sensitive, but less specific for TTF-1 protein. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eThe biological role of TTF-1 in tumorigenesis remains to be clarified, since few studies explored this topic. In a manuscript of Iso H. et al, TTF-1-negative NSCLC exhibited distinct biological characteristics, such as a higher prevalence of certain genetic mutations (e.g., STK11, KEAP1) and an immunosuppressive tumor microenvironment. According to authors, these factors might contribute to the observed resistance to therapies and poorer prognoses of patients with TTF-1-negative NSCLC in their study. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e) 4 and 3 tumors had STK11 and KEAP1 mutation in our study and all of them were TTF-1 negative at the same time. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eInterestingly, in a study of Iida Y. et al, TTF-1-negative SCLC were different in aspects of neuroendocrine differentiation (negativity of synaptophysin and chromogranin, lower expression of ASCL1) compared to TTF-1-positive SCLC. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e) Concerning EP-SCNC, ASCL1 negative tumors are known to constitute a larger proportion than in SCLC. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eTTF-1 positivity proved to have a negative prognostic value in our study, which can be partly supported by the above-mentioned study of Iso H. et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e) However, this finding is in contrast with results of a robust meta-analysis conducted by Wang Y. et al., who studied 11 manuscripts comprising 1,786 SCLC patients, where TTF-1 expression indicated improved overall survival (OS) (HR\u0026thinsp;=\u0026thinsp;0.56, 95% CI: 0.45\u0026ndash;0.70; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and progression-free survival (PFS) (HR\u0026thinsp;=\u0026thinsp;0.41, 95% CI: 0.28\u0026ndash;0.62; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e) Moreover, TTF-1 positivity proved to have a positive prognostic impact in a meta-analysis performed by Qian H.H. et al., who studied 17 manuscripts comprising 2,235 patients with non-small cell lung carcinoma; TTF-1 overexpression had a favorable impact on survival (HR\u0026thinsp;=\u0026thinsp;0.49, 95% CI: 0.42\u0026ndash;0.55). (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e)\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eAlthough EP-SCNC has been repeatedly shown to be a heterogeneous disease\u0026sup1;\u0026sup2; that is partially responsive to platinum-based chemotherapy, no consensus exists on the optimal second-line therapy in cases of progression. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e) To advance therapeutic strategies requires a deeper understanding of molecular and immune characteristics. However, our study of TTF-1, CDX-2, and PAX-8 immunohistochemistry did not prove instrumental in identifying the primary site of EP-SCNC origin and, in our opinion, should be interpreted with caution.\u003c/p\u003e\u003cp\u003eLimitations\u003c/p\u003e\u003cp\u003eThe TMA approach, despite its widespread use in research, carries a risk of under- or overestimation of immunohistochemical scoring and may be a limitation due to tumor heterogeneity.\u003c/p\u003e\u003cp\u003eAlthough our cohort of 138 patients with EP-SCNC is one of the largest in literature, the number of patients in each tumor group is limited, which may constrain the statistical interpretation of results.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eEP-SCNC \u0026ndash; Extrapulmonary Small Cell Neuroendocrine Carcinoma\u003c/p\u003e\n\u003cp\u003eTTF-1 \u0026ndash; Thyroid Transcription Factor 1\u003c/p\u003e\n\u003cp\u003eCDX-2 \u0026ndash; Caudal-related Homeobox 2\u003c/p\u003e\n\u003cp\u003ePAX-8 \u0026ndash; Paired Box Gene 8\u003c/p\u003e\n\u003cp\u003eIHC \u0026ndash; Immunohistochemistry\u003c/p\u003e\n\u003cp\u003eOS \u0026ndash; Overall Survival\u003c/p\u003e\n\u003cp\u003eFFPE \u0026ndash; Formalin-Fixed Paraffin-Embedded\u003c/p\u003e\n\u003cp\u003eTMA \u0026ndash; Tissue Microarray\u003c/p\u003e\n\u003cp\u003eNGS \u0026ndash; Next-Generation Sequencing\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;HR \u0026ndash; Hazard Ratio\u003c/p\u003e\n\u003cp\u003eCI \u0026ndash; Confidence Interval\u003c/p\u003e\n\u003cp\u003eNSCLC \u0026ndash; Non-Small Cell Lung Carcinoma\u003c/p\u003e\n\u003cp\u003eSCLC \u0026ndash; Small Cell Lung Carcinoma\u003c/p\u003e\n\u003cp\u003ePFS \u0026ndash; Progression-Free Survival\u003c/p\u003e\n\u003cp\u003eASCL1 \u0026ndash; Achaete-Scute Family Transcription Factor 1\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics Approval/Consent to Participate:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of the Institute for Clinical and Experimental Medicine and Thomayer University Hospital No G-21-39.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution Statement (CRediT)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKlara Pavlickova: Writing \u0026ndash; original draft, Data curation, Resources, Investigation Radoslav Matej: Conceptualization, Writing \u0026ndash; review and editing, Supervision, Resources Pavel Dundr: Writing \u0026ndash; review and editing, Supervision Jan Hrudka: Writing \u0026ndash; review and editing Petr Waldauf: Software Jan Laco, Marian Svajdler, Iva Staniczkova Zambo, Miroslava Fligrova, Helena Hornychova and Jozef Skarda: Data curation\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the paper for publication.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\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 the published article and its supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Statement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Ministry of Health of the Czech Republic (project Conceptual Development of Research Organization, General University Hospital, Prague (VFN, 00064165) and Thomayer University Hospital (FTN, 00064190)), the Agency for Health Research of the Czech Republic (project NU22-03-00130), by the European Regional Development Fund (BBMRI_CZ LM2023033), and by the Charles University (projects\u0026nbsp;UNCE 24/MED/018 and Cooperatio Medical Diagnostics and Basic Medical Sciences).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to express our special thanks to Mr. Thomas Secrest, MSc, for language editing services and Alena Pol\u0026aacute;kov\u0026aacute;, MSc, for her excellent technical assistance.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDasari A, Mehta K, Byers LA, Sorbye H, Yao JC. 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Cancer Treat Rev. 2023;121:102643.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang Y, Wu Y, Li J, Li J, Che G. Clinicopathological and prognostic significance of thyroid transcription factor-1 expression in small cell lung cancer: A systemic review and meta-analysis. Pathol Res Pract. 2019;215(12):152706.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGuan L, Zhao X, Tang L, Chen J, Zhao J, Guo M, et al. Thyroid Transcription Factor-1: Structure, Expression, Function and Its Relationship with Disease. Biomed Res Int. 2021;2021:9957209.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhou Q, Li H, Cheng Y, Ma X, Tang S, Tang C. Pax-8: Molecular biology, pathophysiology, and potential pathogenesis. BioFactors. 2024;50(3):408\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOrdonez NG. Value of PAX 8 immunostaining in tumor diagnosis: a review and update. Adv Anat Pathol. 2012;19(3):140\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e{{Posit. team}} {Posit Software P. {RStudio: Integrated Development Environment for R}. 2024.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eM TT. A Package for Survival Analysis in R. R package version 3.7-0. 2024.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTimmesfeld} RDaRK-MaN. {A comparison of different methods to adjust survival curves for confounders}. 2023;42:{1461-79}.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKoo J, Mertens RB, Mirocha JM, Wang HL, Dhall D. Value of Islet 1 and PAX8 in identifying metastatic neuroendocrine tumors of pancreatic origin. Mod Pathol. 2012;25(6):893\u0026ndash;901.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePavlickova K, Hojny J, Waldauf P, Svajdler M, Dundr P, Fabian P, et al. 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TTF-1 and Napsin-A are expressed in a subset of cholangiocarcinomas arising from the gallbladder and hepatic ducts: continued caveats for utilization of immunohistochemistry panels. Am J Surg Pathol. 2014;38(2):224\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLaury AR, Perets R, Piao H, Krane JF, Barletta JA, French C, et al. A comprehensive analysis of PAX8 expression in human epithelial tumors. Am J Surg Pathol. 2011;35(6):816\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eErickson LA, Papouchado B, Dimashkieh H, Zhang S, Nakamura N, Lloyd RV. Cdx2 as a marker for neuroendocrine tumors of unknown primary sites. Endocr Pathol. 2004;15(3):247\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWerling RW, Yaziji H, Bacchi CE, Gown AM. CDX2, a highly sensitive and specific marker of adenocarcinomas of intestinal origin: an immunohistochemical survey of 476 primary and metastatic carcinomas. Am J Surg Pathol. 2003;27(3):303\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSorbye H, Welin S, Langer SW, Vestermark LW, Holt N, Osterlund P, et al. Predictive and prognostic factors for treatment and survival in 305 patients with advanced gastrointestinal neuroendocrine carcinoma (WHO G3): the NORDIC NEC study. Ann Oncol. 2013;24(1):152\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eUlanja MB, Beutler BD, Antwi-Amoabeng D, Governor SB, Rahman GA, Djankpa FT, et al. Prognostic Factors and Survival in Gastrointestinal Extrapulmonary Small Cell Carcinoma: A Retrospective Cohort Study. Ann Surg Oncol. 2022;29(13):8250\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang B, Feng N, Shi X, Qi Q, Chi X, Song T, et al. [Analysis of Correlation between TTF-1 and Sensitivity to First-line Chemotherapy and Prognosis in Patients with Small Cell Lung Cancer]. Zhongguo Fei Ai Za Zhi. 2020;23(7):547\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBriski LM, Aron M, Epstein JI, Russell DH, Assarzadegan N, Delma KS, et al. Patterns of Immunoreactivity with TTF-1 Antibodies 8G7G3/1 and SPT24 Suggest Distinct Immunoprofiles Between Most Pulmonary and Nonpulmonary Small Cell Carcinomas. Int J Surg Pathol. 2024;32(2):230\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eComperat E, Zhang F, Perrotin C, Molina T, Magdeleinat P, Marmey B, et al. Variable sensitivity and specificity of TTF-1 antibodies in lung metastatic adenocarcinoma of colorectal origin. Mod Pathol. 2005;18(10):1371\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIso H, Hisakane K, Mikami E, Suzuki T, Matsuki S, Atsumi K, et al. Thyroid transcription factor-1 (TTF-1) expression and the efficacy of combination therapy with immune checkpoint inhibitors and cytotoxic chemotherapy in non-squamous non-small cell lung cancer. Transl Lung Cancer Res. 2023;12(9):1850\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIida Y, Nakanishi NTY, Nishimaki H, Nakagawa Y, Shimizu T, Mizumura K, Maruoka S, Gon Y, Masuda S, Hashimoto S. P2.15-015 Negativity for Thyroid Transcription Factor 1 Was Correlated with Less Neuroendocrine Differentiation in Small Cell Lung Cancers. IASLC 18th World Conference on Lung Cancer: Journal of Thoracic Oncology; 2017. p. Pages S2188-S9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eQian HH, Xu TS, Cai XQ, Ji TL, Guo HX. Prognostic value of TTF-1 expression in patients with non-small cell lung cancer: A meta-analysis. Clin Chim Acta. 2015;451(Pt B):208\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMcNamara MG, Frizziero M, Jacobs T, Lamarca A, Hubner RA, Valle JW, et al. Second-line treatment in patients with advanced extra-pulmonary poorly differentiated neuroendocrine carcinoma: a systematic review and meta-analysis. Ther Adv Med Oncol. 2020;12:1758835920915299.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"diagnostic-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"dpat","sideBox":"Learn more about [Diagnostic Pathology](http://diagnosticpathology.biomedcentral.com)","snPcode":"13000","submissionUrl":"https://submission.nature.com/new-submission/13000/3","title":"Diagnostic Pathology","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Extrapulmonary small cell neuroendocrine carcinoma, TTF-1, CD-X2, PAX-8","lastPublishedDoi":"10.21203/rs.3.rs-6821428/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6821428/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Extrapulmonary small cell neuroendocrine carcinoma (EP-SC) is a rare malignancy with a poor prognosis. In our study, one hundred and thirty-eight EP-SCNC tissue samples underwent a complex analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethod: \u003c/strong\u003eWe studied the possible benefit of TTF-1, CDX-2, and PAX-8 immunohistochemical analyses for determining the primary site of EP-SCNC origin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eThe median survival of our cohort was 11.5 months. Patients younger than 70 years had significantly better overall survival (p=0.024).\u003c/p\u003e\n\u003cp\u003eAcross the full cohort, CDX-2 positivity was found in 19 tumors (13.9%), TTF-1 in 35 tumors (25.7%), and PAX-8 in 63 tumors (46.3%), regardless of tumor origin. The expression of CDX-2 and PAX-8 did not differ significantly among different organ systems (p=0.2 and 0.3), respectively. The expression of TTF-1 differed significantly in tumors from various sites of origin (p=0.009), expressed more often in breast, pancreatic, and genitourinary tumors. Interestingly, TTF-1 expression proved to have a negative prognostic impact relative to patient survival (age-adjusted Cox regression analysis, HR=1.62, 95%CI:1.06-2.47, p=0.021).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e As most patients with EP-SCNC had a metastatic presentation, finding the primary tumor origin, which is important for patient prognoses, is both challenging and important at the same time. Only TTF-1 immunohistochemical analysis proved to be helpful with this task. Moreover, a diagnosis of primary lung small cell carcinoma cannot be established based on TTF-1 positivity, since only 25.7% of EP-SCNC in our study displayed TTF-1 positivity.\u003c/p\u003e","manuscriptTitle":"TTF-1, CDX-2, and PAX-8 Immunoexpression in a Large Serie of Extrapulmonary Small Cell Neuroendocrine Carcinomas: A Study of 138 Cases","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-18 13:01:39","doi":"10.21203/rs.3.rs-6821428/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-18T14:32:34+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-17T16:59:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"174323919370067869047253199002304255909","date":"2025-08-17T08:57:12+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-15T19:16:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"197477739173994009017847865747200611334","date":"2025-07-15T17:30:26+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-13T12:56:41+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-06T01:28:59+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-05T08:59:22+00:00","index":"","fulltext":""},{"type":"submitted","content":"Diagnostic Pathology","date":"2025-06-04T14:13:46+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"diagnostic-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"dpat","sideBox":"Learn more about [Diagnostic Pathology](http://diagnosticpathology.biomedcentral.com)","snPcode":"13000","submissionUrl":"https://submission.nature.com/new-submission/13000/3","title":"Diagnostic Pathology","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5cfa1b7a-b300-46d7-94ec-c8c62efaecc1","owner":[],"postedDate":"July 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-02T15:59:59+00:00","versionOfRecord":{"articleIdentity":"rs-6821428","link":"https://doi.org/10.1186/s13000-026-01753-3","journal":{"identity":"diagnostic-pathology","isVorOnly":false,"title":"Diagnostic Pathology"},"publishedOn":"2026-01-26 15:58:05","publishedOnDateReadable":"January 26th, 2026"},"versionCreatedAt":"2025-07-18 13:01:39","video":"","vorDoi":"10.1186/s13000-026-01753-3","vorDoiUrl":"https://doi.org/10.1186/s13000-026-01753-3","workflowStages":[]},"version":"v1","identity":"rs-6821428","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6821428","identity":"rs-6821428","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}