Sensitivity and specificity of INSM1 compared with Chromogranin and Synaptophysin in neuroendocrine carcinomas of the head and neck region

Sensitivity and specificity of INSM1 compared with Chromogranin and Synaptophysin in neuroendocrine carcinomas of the head and neck region | 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 Sensitivity and specificity of INSM1 compared with Chromogranin and Synaptophysin in neuroendocrine carcinomas of the head and neck region Phatsorn Amattirat, Thirayost Nimmanon, Parsinee Julimasart, Kulachet Wiwatwarayos This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5930915/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background : The head and neck region is an area where neuroendocrine tumors (NETs) and neuroendocrine carcinomas (NECs) can arise. NECs have several mimickers, which make their diagnosis challenging. Accurate diagnosis typically relies on morphology, supported by established markers such as synaptophysin (SYN) or chromogranin A (CGA), each with varying sensitivity and specificity. Insulinoma-associated protein 1 (INSM1) is a newer marker that has shown good sensitivity and specificity in various organs. Despite its promising potential, there are very few studies when compared to other organs. Methods : This case-control study tested INSM1, SYN, and CGA on 14 NEC samples and 109 non-NEC samples to evaluate their sensitivity and specificity. Results: INSM1 demonstrated an overall sensitivity of 92.9%, compared to 78.5% for CGA (P = 0.157) and 100.0% for SYN (P = 0.317). INSM1 exhibited a specificity of 96.3%, compared to 100% for both CGA (P = 0.045) and SYN (P = 0.045). Among non-NEC cases, scattered staining (< 10% of tumor cells) was observed with INSM1, while 4 non-NEC cases showed positive staining for INSM1. All non-NEC cases were negative for CGA and SYN. Conclusion: INSM1 demonstrates good sensitivity, comparable to SYN and CGA, with no statistically significant differences observed between these markers. However, the specificity of INSM1 shows statistically significant differences. Notably, one case of EBV-positive nonkeratinizing squamous cell carcinoma of the nasopharynx exhibited INSM1 positivity in 40% of tumor cells. As such, INSM1 should not be used as a standalone marker for diagnosis. Furthermore, caution is warranted when interpreting results with staining ≤10%, as this may reduce the reliability of a positive finding. head and neck region small cell neuroendocrine carcinoma (SCNC) large cell neuroendocrine carcinoma (LCNC) INSM1 surgical pathology Figures Figure 1 Figure 2 Figure 3 Introduction The most clinically urgent subtype of neuroendocrine neoplasm (NEN) is neuroendocrine carcinoma (NEC), which is characterized by poor differentiation, tumor necrosis, high mitotic activity, and destructive features [23].Morphologically, NEC may mimic several other tumor types, especially in small tissue biopsies. These mimickers include well-differentiated neuroendocrine tumors (NET), Merkel cell carcinoma, NUT carcinoma, sinonasal undifferentiated carcinoma, basaloid squamous cell carcinoma, adenoid cystic carcinoma (solid type), SWI/SNF complex-deficient carcinoma, tumors with neuroectodermal differentiation (such as olfactory neuroblastoma, teratocarcinosarcoma), mucosal melanoma, and round cell sarcomas (including Ewing sarcoma, EWSR1-non-ETS sarcoma, BCOR sarcoma, alveolar rhabdomyosarcoma, and synovial sarcoma) [24]. Research also indicates that other tumor types, such as acinic cell carcinoma[19,20,38], paraganglioma, metastatic medullary thyroid carcinoma, olfactory neuroblastoma, and Ewing’s sarcoma family[26], can exhibit neuroendocrine differentiation [21]. NEC is a rare malignancy, accounting for only 0.3% of all head and neck cancers [27-29]. Similarly, primary NEC of the salivary gland is uncommon, representing less than 2% of salivary gland malignancies, primarily in the parotid and submandibular glands [39,40]. Its clinicopathological and genomic features are not yet fully understood. The FGFR3-TACC3 fusion gene and mutations in genes encoding components of the NOTCH and PI3K/AKT/mTOR pathways, as identified in study, may represent promising therapeutic targets for NEC [25 Small cell neuroendocrine carcinoma (SCNC) is the most common type of NEC in the larynx and salivary gland, while large cell neuroendocrine carcinoma (LCNC) is rare in these locations [31,32]. Common markers used in diagnosing NEC include chromogranin A (CGA), synaptophysin (SYN), and CD56, with reported sensitivities ranging from 50% to 80% [1-3]. However, these markers may also stain positive in other non-neuroendocrine tumors, such as carcinomas, sarcomas, and melanomas [4-6]. Insulinoma-associated protein 1 (INSM1; formerly IA-1), a transcription factor containing five zinc-finger motifs, is predominantly expressed in developing neuroendocrine tissues and the nervous system in mammals[22].INSM1 has shown high sensitivity and specificity for detecting neuroendocrine differentiation in the head and neck region, including middle ear adenoma, pituitary adenoma, paraganglioma, medullary thyroid carcinoma, olfactory neuroblastoma, SCNC, LCNC, sinonasal teratocarcinoma, and laryngeal NEC [7,8]. Additionally, INSM1 demonstrates positivity in squamous cell carcinoma (SCC) of the larynx, pharynx, and floor of the mouth. However, it does not stain positive in pleomorphic adenoma, Warthin tumor, or basal cell adenoma [17]. CD56 has demonstrated the lowest sensitivity and specificity among markers for primary lung neoplasms [9,10], small cell lung carcinoma [11,12], thoracic cavity tumors [13], and urinary tract neoplasms [14]. Certain cases of LCNC and SCNC may be Epstein-Barr virus (EBV)-positive, which can complicate the diagnosis. Similarly, SCNC may show P16 positivity, potentially leading to confusion with other tumors, such as P16-positive oropharyngeal squamous cell carcinoma (OPSCC). These findings highlight the critical importance of selecting appropriate control groups to ensure diagnostic accuracy [15,16,30]. Materials and Methods Ethical statements Our research was approved by the Institutional Review Board at the Institute of Pathology, Department of Medical Services, Ministry of Public Health, prior to its commencement (IOP, No. IOP-KMR66-004). The board exempted the requirement for informed consent documentation, as the study did not involve human subjects. This approval ensures that our research complies with ethical guidelines. Case selection A case-control study identified 329 cases. The inclusion criteria required samples with neoplasms located in the head and neck region, with available paraffin blocks from 2017 to 2024. The exclusion criteria eliminated cases with neoplasms originating from the salivary glands or other tumors that typically exhibit neuroendocrine differentiation. The sample size was determined with 14 cases in the NEC group and 109 cases in the non-NEC group. The NEC group included 10 cases of SCNC and 4 cases of LCNC. The non-NEC group consisted of 109 cases of NK-NPC and 16 cases of P16-positive OPSCC. Due to the rarity of neuroendocrine carcinoma in the head and neck region, any additional cases encountered during the study were also included. The sample size was calculated to ensure reliability using the Finite Population Proportion formula, a commonly used method for determining an appropriate sample size based on prior research values. [37]. Simple random sampling was applied to enroll cases into the study, ensuring unbiased selection from both groups for inclusion in the experiment. Evaluating tumor cells percentage and intensity The percentage (%) of tumor staining was recorded based on 10 consecutive high-power fields (HPFs), starting from the field with the highest tumor presence. The staining was then categorized into the following tiers: 0-10%, 11-20%, 21-30%, 31-40%, 41-50%, 51-60%, 61-70%, 71-80%, 81-90%, and 91-100%. Staining intensity was graded as 0, 1+, 2+, or 3+. Histopathological evaluation was performed using an Olympus BX53 microscope by the researcher, who is a resident physician in anatomical pathology. All diagnoses were verified through a second reading by a supervising pathologist. The results were reviewed, checked, and confirmed, with findings documented in a research record form. INSM1, CGA, and SYN immunohistochemical studies Once the samples are obtained, they will be stained with INSM 1 (MRQ-70). If synaptophysin or chromogranin staining had not been previously performed for a case, Synaptophysin (MRQ-40) and Chromogranin A (LK2H10) were applied. Tissue sections were cut to a thickness of 3 micrometers and underwent antigen retrieval in 10 mM citrate buffer at 100°C for 37 minutes using the LEICA BOND-MAX system. Microscopic evaluation was then conducted. The quality of staining was assessed based on external controls, following the laboratory’s standard procedures, to ensure suitability for microscopic examination. Statistical analysis Statistical Analysis were performed using IBM SPSS Statistics 26 software, and the R programming language. Results were considered significant when the p-value was less than 0.05. The optimal range of tumor cell percentages in relation to INSM1 positivity was determined using the Receiver Operating Characteristic (ROC) curve. The ROC curve serves as a tool for assessing the effectiveness of a diagnostic test [36]. Sensitivity and specificity of all stains were compared using the McNemar x 2 test. Results Table 1: Sensitivity and Specificity at INSM1 percentage INSM1 percentage Sensitivity Specificity 0-10 100.0% 0.0% 11-20 92.9% 96.3% 21-30 92.9% 98.2% 31-40 92.9% 99.1% 71-80 92.9% 100.0% 81-90 64.3% 100.0% 91-100 57.1% 100.0% Figure 1 and Table 1 illustrate that the optimal range of 11–20% tumor cells was determined based on INSM1 positivity using the Receiver Operating Characteristic (ROC) curve. The area under the curve (AUC) was 0.952, indicating excellent accuracy in distinguishing between NEC and non-NEC cases. Tumor cell proportions greater than 10% demonstrated optimal sensitivity and specificity. Table 2 shows that INSM1 had an overall sensitivity of 92.9%, compared to 78.6% for CGA (P = 0.157) and 100.0% for SYN (P = 0.317). INSM1 also had an overall specificity of 96.3%, compared to 100.0% specificity for both CGA and SYN (P = 0.045). Figure 2 demonstrates that INSM1 exhibits positive nuclear staining, which is easier to interpret. The proportion of tumor staining for INSM1 averaged 91.9%, compared to 91.4% for SYN and 82.2% for CGA. Additionally, INSM1 had a mean staining intensity of 2.8, compared to 2.1 for SYN and 2.0 for CGA. In p16-positive OPSCC, all cases were negative for INSM1, SYN, and CGA. Figure 3 illustrates that, in NK-NPC, INSM1 was positive in four cases, while SYN and CGA were negative in all cases. Table 2 clarifies that the positive cases had tumor staining proportions of 20%, 20%, 30%, and 40%, respectively. The remaining cases showed a scattered tumor staining proportion of < 10%. Discussion In this study, we evaluated the performance of INSM1 in detecting SCNC and LCNC in the head and neck region. The results indicated that INSM1 did not demonstrate a statistically significant difference in sensitivity compared to SYN and CGA. These findings are consistent with previous research, which found that SYN had the highest sensitivity (93.3%), followed by INSM1 (89.2%) and CGA (87.5%). That study analyzed a total of 471 NEN samples from various organs, further supporting the effectiveness of INSM1 as a diagnostic tool for neuroendocrine tumors across different tissues [17]. Our findings also reveal differences compared to other research. For example, previous studies reported INSM1 exhibiting a high sensitivity of 99%, compared to SYN (92.3%) and CGA (77.3%) [7]. This higher sensitivity highlights INSM1's potential as a more reliable marker for diagnosing NET. Moreover, in areas with significant crushing artifacts and difficult morphology, INSM1 still demonstrates good staining. In our study, INSM1 demonstrated a specificity of 96.3%, which was significantly lower than SYN and CGA. This value differs from previous research that reported a specificity of 97.6% for INSM1 at a cutoff point of 10%. These discrepancies suggest that INSM1's specificity may vary depending on the study population or the sample type analyzed. Further investigations are necessary to better understand the conditions under which INSM1's specificity is most reliable [7]. A study investigating NE differentiation in SCC of the head and neck reported sparsely scattered CGA expression in 41% of cases, which correlated with high CGA messenger RNA expression. Additionally, SYN expression was observed in 18% of cases [18]. In contrast, our study found that all non-NEC cases were negative for CGA and SYN. However, four of these cases showed positive INSM1 staining, while some others exhibited sparsely scattered staining in less than 10% of the tumor cells. This highlights the need for caution when interpreting INSM1 results in the nasopharynx region. Furthermore, all cases of P16-positive OPSCC in our study were negative for INSM1, suggesting that INSM1 can be used to rule out NEC in the oropharynx. This finding aligns with previous research that reported 129 out of 130 oral SCC samples were negative for INSM [17]. Such consistency with prior findings supports the specificity of INSM1 in identifying neuroendocrine carcinomas and underscores its utility in distinguishing these tumors from other types in specific anatomical regions. TABLE 2. Expression of INSM1, Synaptophysin, and Chromogranin in NEC and non-NEC Diagnosis (n/N [%]) INSM1 Synaptophysin Chromogranin positive negative positive negative positive negative SCNC * 10/10 (100) 0/10 (0) 10/10 (100) 0/10 (0) 9/10 (90) 1/10 (10) LCNC µ 3/4 (75) 1/4 (25) 4/4 (100) 0/4 (0) 2/4 (50) 2/4(50) Total NEC 13/14 (92.9) 1/14 (7.1) 14/14 (100) 0/14 (0) 11/14 (78.6) 3/14 (21.4) P16+OPSCC 0/16 (0) 16/16 (100) 0/16 (0) 16/16 (100) 0/16 (0) 16/16 (100) NK-NPC π 4 α /93 (4.3) 89/93 (95.7) 0/93 (0) 93/93 (100) 0/93 (0) 93/93 (100) Total Non-NEC 4/109 (3.7) 105/109 (96.3) 0/109 (0) 109/109 (100) 0/109 (0) 109/109 (100) *Including base of tongue (n=2), nasal cavity (n=3), tonsil (n=1), pyriform (n=2), cricoid (n=1), neck (n=1) µ Including nasal cavity (n=1), base of tongue (n=1), gum (n=1), mandibular (n=1) α Tumor cells percentage 20% (n=2), 30% (n=1), 40% (n=1) π EBV-associated NPC (n=49), non-EBV-associated NPC (n=5), and NPC with unknown EBV status (n=39) were categorized based on EBV status, while differentiation subtype (n=19) and undefined subtype (n=74) were categorized based on differentiated morphology. In our study, four cases exhibited scattered tumor cell staining of 10% or more. Therefore, cases with already established differentiation should not undergo INSM1 staining, as a positive result in such cases would not alter the diagnosis of neuroendocrine carcinoma (NEC). Additionally, in cases with limited tumor samples from biopsy and undifferentiated morphology, if INSM1 is positive but no other markers are present, obtaining a larger tissue sample is crucial for accurate interpretation. A small biopsy may not provide sufficient evidence to definitively diagnose NEC. We defined positivity as >10% based on the ROC curve. While raising the cutoff value can increase both sensitivity and specificity, a higher threshold is not always more appropriate. INSM1 already demonstrates strong staining in NEC, and increasing the cutoff does not significantly improve sensitivity. This approach aligns with other studies that have used similar cutoff values. For example, a study reported INSM1 positivity in adenocarcinoma from lung bronchial brushing samples in less than 10% of tumor cells. The optimal cutoff value for INSM1 to differentiate between small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC) was determined to be 8.68%, with a sensitivity of 95.8% and a specificity of 100% [35]. Compared to previous research on lung samples, which found that any percentage could serve as the optimal cutoff for CD56, CGA, and INSM1 in differentiating NET from NSCLC cases, a cutoff of at least 10% positive tumor cells was identified as the best threshold for SYN [33]. This is similar to another study on NE differentiation in breast carcinoma, which found that INSM1 is a sensitive marker and should be included with SYN and CGA in the panel to evaluate NE differentiation, using a cutoff point of 50% [34]. Since NEC in the head and neck region is quite rare, the researchers believe that future studies, which continue to collect data over time, will likely provide sufficient information for more definitive conclusions. However, the nuclear staining pattern of INSM1 offers an advantage in interpretation, as it simplifies the process compared to the cytoplasmic staining patterns commonly observed with other markers. This can be particularly helpful in challenging cases where other markers provide less clarity. While many studies have demonstrated that INSM1 has high sensitivity and specificity, suggesting its potential as a standalone marker, our findings indicate that it is a reliable marker. However, it should not be used as the sole diagnostic tool. Given that SCNC and LCNC in the head and neck region are rare and infrequently encountered at our pathology institution, future studies with larger sample sizes may provide insights that refine or modify the current conclusions. Additionally, further investigation into combining INSM1 with other markers or utilizing advanced techniques, such as molecular profiling, could enhance its overall diagnostic accuracy. Conclusion INSM1 demonstrates good sensitivity, comparable to SYN and CGA, without showing a statistically significant difference compared to traditional markers. Its nuclear staining pattern enhances its value, making it easier to interpret in clinical practice. When tumor staining is ≤ 10%, the confidence in interpretation decreases. As such, INSM1 should not be used as a standalone marker. In such cases, it is recommended to consider the morphology and additional immunohistochemical markers, such as chromogranin A (CGA) and synaptophysin (SYN), for an accurate assessment. Declarations Acknowledgments I would like to express my heartfelt gratitude to the Institute of Pathology Foundation for their financial support, which made this research possible. Fundings This research was funded by the Institute of Pathology Foundation (grant number 7/2567), which supports research activities within the Institute of Pathology. The funder had no involvement in the study design, data collection, analysis, or interpretation, and did not influence the writing of this manuscript. Conflict of interest The authors declare that they have no conflict of interest. Ethical approval For this type of study formal consent is not required. Consent to participate For this type of study formal consent is not required. Consent for publication For this type of study consent for publication is not required. Availability of data and materials The data analyzed in this study is stored within the Institute of Pathology, Ministry of Public Health, Thailand. Due to patient confidentiality and institutional regulations, the data is not publicly available. However, researchers may request access from the corresponding author, subject to approval by the Institute of Pathology’s ethics committee. Code availability This study utilized proprietary software, IBM SPSS Statistics (version 26, IBM Corp.), and the R programming language (version 4.2, R Foundation for Statistical Computing). References A. Ferlito, I. Friedmann; Contribution of Immunohistochemistry in the Diagnosis of Neuroendocrine Neoplasms of the Larynx. ORL 1 April 1991; 53 (4): 235–244. https://doi.org/10.1159/000276222 Rinaldo, A., Devaney, K. O., & Ferlito, A. (2004). Immunohistochemical studies in support of a diagnosis of small cell neuroendocrine carcinoma of the larynx. Acta Oto-Laryngologica , 124 (5), 638–641. https://doi.org/10.1080/00016480410016540 Serrano MF, El-Mofty SK, Gnepp DR, Lewis JS Jr. Utility of high molecular weight cytokeratins, but not p63, in the differential diagnosis of neuroendocrine and basaloid carcinomas of the head and neck. Hum Pathol. 2008 Apr;39(4):591-8. https://doi.org/10.1016/j.humpath.2007.08.019 Bahrami A, Gown AM, Baird GS, Hicks MJ, Folpe AL. Aberrant expression of epithelial and neuroendocrine markers in alveolar rhabdomyosarcoma: a potentially serious diagnostic pitfall. Mod Pathol. 2008 Jul;21(7):795-806. https://doi.org/10.1038/modpathol.2008.8 Lee H, Torres FX, McLean SA, Chen R, Lee MW. Immunophenotypic heterogeneity of primary sinonasal melanoma with aberrant expression of neuroendocrine markers and calponin. Appl Immunohistochem Mol Morphol. 2011 Jan;19(1):48-53. https://doi.org/10.1097/PAI.0b013e3181ee8dcb Schartinger VH, Falkeis C, Laimer K, Sprinzl GM, Riechelmann H, Rasse M, Virgolini I, Dudás J. Neuroendocrine differentiation in head and neck squamous cell carcinoma. J Laryngol Otol. 2012 Dec;126(12):1261-70. https://doi.org/10.1017/S0022215112002265 Rooper LM, Bishop JA, Westra WH. INSM1 is a Sensitive and Specific Marker of Neuroendocrine Differentiation in Head and Neck Tumors. Am J Surg Pathol. 2018 May;42(5):665-671. https://doi.org/10.1097/PAS.0000000000001037 Yuan C, Jiao F, Zhai C, Zhang J, Wang S, Zhu L. Application of INSM1 in Diagnosis and Grading of Laryngeal Neuroendocrine Carcinoma. Laryngoscope. 2021 Oct;131(10): E2662-E2668. https://doi.org/10.1002/lary.29554 Mukhopadhyay S, Dermawan JK, Lanigan CP, Farver CF. Insulinoma-associated protein 1 (INSM1) is a sensitive and highly specific marker of neuroendocrine differentiation in primary lung neoplasms: an immunohistochemical study of 345 cases, including 292 whole-tissue sections. Mod Pathol. 2019 Jan;32(1):100-109. https://doi.org/10.1038/s41379-018-0122-7 Kriegsmann K, Zgorzelski C, Kazdal D, Cremer M, Muley T, Winter H, et al. Insulinoma-associated Protein 1 (INSM1) in Thoracic Tumors is Less Sensitive but More Specific Compared with Synaptophysin, Chromogranin A, and CD56. Applied Immunohistochemistry & Molecular Morphology. 2018 Oct 24;28(3):237–42. https://doi.org/10.1097/PAI.0000000000000715 ‌[11] Sakakibara R, Kobayashi M, Takahashi N, Inamura K, Ninomiya H, Wakejima R, et al. Insulinoma-associated Protein 1 (INSM1) Is a Better Marker for the Diagnosis and Prognosis Estimation of Small Cell Lung Carcinoma Than Neuroendocrine Phenotype Markers Such as Chromogranin A, Synaptophysin, and CD56. American Journal of Surgical Pathology. 2020 Feb 28;44(6):757–64. https://doi.org/10.1097/PAS.0000000000001444 Rocha R, Henrique R. Insulinoma-Associated Protein 1 (INSM1): Diagnostic, Prognostic, and Therapeutic Use in Small Cell Lung Cancer. Journal of Molecular Pathology. 2022 Aug 5;3(3):140–69. https://doi.org/10.3390/jmp3030013 ‌[13] Rooper LM, Sharma R, Li QK, Illei PB, Westra WH. INSM1 Demonstrates Superior Performance to the Individual and Combined Use of Synaptophysin, Chromogranin and CD56 for Diagnosing Neuroendocrine Tumors of the Thoracic Cavity. Am J Surg Pathol. 2017 Nov;41(11):1561-1569. https://doi.org/10.1097/PAS.0000000000001003 Kim IE Jr, Amin A, Wang LJ, Cheng L, Perrino CM. Insulinoma-associated Protein 1 (INSM1) Expression in Small Cell Neuroendocrine Carcinoma of the Urinary Tract. Appl Immunohistochem Mol Morphol. 2020 Oct;28(9):687-693. https://doi.org/10.1097/PAI.0000000000000824 Nada Al Masalmeh, Kukreja G, Feras Zaiem, S. Naweed Raza, Kim H, Misako Nagasaka, et al. p16 positive oropharyngeal small cell cancer: A case report. Oral Oncology. 2021 Oct 1; 121:105391–1. https://doi.org/10.1016/j.oraloncology.2021.105391 Su Z, Yan H, Zou G. EBV‑positive large‑cell and small‑cell neuroendocrine carcinoma of the nasopharynx: A case report and review of the literature. Oncology Letters. 2022 Nov 7;24(6). https://doi.org/10.3892/ol.2022.13584 Möller K, Gorbokon N, Hube-Magg C, Fraune C, Lennartz M, Blessin NC, et al. Comparison of INSM1 immunostaining with established neuroendocrine markers Synaptophysin and Chromogranin A in over 14,000 neuroendocrine and non-neuroendocrine tumors. American Journal of Clinical Pathology. 2023 Nov 1;160(Supplement_1): S36–7. https://doi.org/10.1093/ajcp/aqad086 Schartinger VH, C Falkeis, K Laimer, Sprinzl GM, H Riechelmann, M Rasse, et al. Neuroendocrine differentiation in head and neck squamous cell carcinoma. The Journal of Laryngology & Otology. 2012 Oct 11;126(12):1261–70. https://doi.org/10.1017/S0022215112002265 Ito K, Kakudo K, Mori I, et al. Neuroendocrine differentiation in a case of acinic cell carcinoma of the parotid gland. Acta Pathol Jpn. 1990; 40:279–287. https://doi.org/10.1111/j.1440-1827.1990.tb01562.x Roy S, Dhingra KK, Gupta P, et al. Acinic cell carcinoma with extensive neuroendocrine differentiation: a diagnostic challenge. Head Neck Pathol. 2009; 3:163–168. https://doi.org/10.1007/s12105-009-0114-5 Mills SE. Neuroectodermal Neoplasms of the Head and Neck with Emphasis on Neuroendocrine Carcinomas. Modern Pathology. 2002 Mar 1;15(3):264–78. https://doi.org/10.1038/modpathol.3880522 Lan MS, Breslin MB. Structure, expression, and biological function of INSM1 transcription factor in neuroendocrine differentiation. FASEB J. 2009; 23:2024–2033. https://doi.org/10.1096/fj.08-125971 Rindi G, Mete O, Uccella S, Basturk O, La Rosa S, Brosens LAA, et al. Overview of the 2022 WHO Classification of Neuroendocrine Neoplasms. Endocrine Pathology. 2022 Mar;33(1):115–54. https://doi.org/10.1007/s12022-022-09708-2 C. Christofer Juhlin, Bal M. Top 10 Histological Mimics of Neuroendocrine Carcinoma You Should Not Miss in the Head and Neck. Head and Neck Pathology [Internet]. 2023 Mar 20 [cited 2023 Nov 30];17(1):66–84. https://doi.org/10.1007/s12105-023-01647-0 Akihiro Ohmoto, Sato Y, Reimi Asaka, Fukuda N, Wang X, Tetsuya Urasaki, et al. Clinicopathological and genomic features in patients with head and neck neuroendocrine carcinoma. Modern Pathology. 2021 Nov 1;34(11):1979–89. https://doi.org/10.1038/s41379-021-00869-9 Machado I, Navarro S, López-Guerrero JA, Verdini L, Piero Picci, Giner F, et al. Neuroendocrine differentiation in a large series of genetically-confirmed Ewing’s sarcoma family tumor: Does it provide any diagnostic or prognostic information? Pathology - Research and Practice. 2021 Feb 7; 219:153362–2. https://doi.org/10.1016/j.prp.2021.153362 Anas Bouzbouz, Bushra Abdulhakeem, Rabii Laababsi, Sami Rouadi, Abada R, Roubal M, et al. rare cases of head and neck’s neuroendocrine carcinomas disease: Case series of 4 patients and review of the literature. International journal of surgery case reports. 2020 Jan 1; 66:270–6. https://doi.org/10.1016/j.ijscr.2019.12.003 Subedi N, Prestwich R, Chowdhury F, Patel C, Scarsbrook A. Neuroendocrine tumours of the head and neck: anatomical, functional and molecular imaging and contemporary management. Cancer Imaging. 2013;407–22. https://doi.org/10.1186/1477-7819-11-66 Perez-Ordoñez B. Neuroendocrine Carcinomas of the Larynx and Head and Neck: Challenges in Classification and Grading. Head and Neck Pathology. 2018 Mar;12(1):1–8. https://doi.org/10.1007/s12105-018-0894-6 Chen Y, Zhou N, Huang C, He X, Wang X, Tang H, et al. EBV-positive small cell neuroendocrine carcinoma of nasopharynx as a probably unique subtype of neuroendocrine carcinoma: a clinicopathologic study of three cases and literature review. Diagnostic Pathology. 2024 Jul 24;19(1). https://doi.org/10.1186/s13000-024-01526-w Tom, Boudewijn E. C. Plaat, F.A.M B, Halmos GB. Clinical recommendations on the treatment of neuroendocrine carcinoma of the larynx: A meta-analysis of 436 reported cases. Head & Neck. 2014 Jun 18;37(5):707–15. https://doi.org/10.1002/hed.23456 Bellahammou K, Lakhdissi A, Akkar O, Kouhen F, Rais F, Dahraoui S, et al. SMALL-CELL NEUROENDOCRINE CARCINOMA OF NASOPHARYNX: A CASE REPORT. International Journal of Surgery and Medicine. 2017;3(1):1. https://doi.org/10.13107/ijscr.2017.v03i01.101 Staaf J, Tran L, Söderlund L, Nodin B, Jirström K, Vidarsdottir H, et al. Diagnostic Value of Insulinoma-Associated Protein 1 (INSM1) and Comparison with Established Neuroendocrine Markers in Pulmonary Cancers. Archives of Pathology & Laboratory Medicine [Internet]. 2020 Sep 1 [cited 2024 Jun 17];144(9):1075–85. https://doi.org/10.5858/arpa.2019-0291-OA Zhong E, Pareja F, Hanna MG, Jungbluth AA, Rekhtman N, Brogi E. Expression of novel neuroendocrine markers in breast carcinomas: a study of INSM1, ASCL1, and POU2F3. Human Pathology. 2022 Sep; 127:102–11. https://doi.org/10.1016/j.humpath.2022.06.003 Abe H, Takase Y, Sadashima E, Fukumitsu C, Murata K, Ito T, et al. Insulinoma‐associated protein 1 is a novel diagnostic marker of small cell lung cancer in bronchial brushing and cell block cytology from pleural effusions: Validity and reliability with cutoff value. Cancer Cytopathology. 2019 Aug 22;127(9):598–605. https://doi.org/10.1002/cncy.22177 Nahm FS. Receiver operating characteristic curve: overview and practical use for clinicians. Korean Journal of Anesthesiology [Internet]. 2022 Feb 1;75(1):25–36. https://doi.org/10.4097/kja.21209 Daniel WW, Cross CL. Biostatistics: a foundation for analysis in the health sciences. 10th ed. Hoboken, NJ: Wiley; 2013. Rath A, Tomar R, Agarwal R, Singh M, Jain S, Khurana N, et al. Acinic cell carcinoma of the parotid gland with neuroendocrine differentiation. Journal of Cancer Research and Therapeutics [Internet]. 2021 Jul 1 [cited 2023 Nov 11];17(4):1115. https://doi.org/10.4103/jcrt.JCRT_1050_20 LIU M, ZHONG M, SUN C. Primary neuroendocrine small cell carcinoma of the parotid gland: A case report and review of the literature. Oncology Letters. 2014 Jun 17;8(3):1275–8. https://doi.org/10.3892/ol.2014.2340 Petrone G, Santoro A, Angrisani B, Novello M, Scarano E, Guido Rindi, et al. Neuroendocrine Tumors of the Submandibular Gland. International Journal of Surgical Pathology. 2012 May 21;21(1):85–8. https://doi.org/10.1177/1066896912446747 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5930915","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":409751905,"identity":"339f5b1d-c6d7-42c8-a193-06801897fafe","order_by":0,"name":"Phatsorn Amattirat","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4ElEQVRIiWNgGAWjYJACZijN+ABI8PARqcUATINIHjZStLBJgElCys0l0p89Lqj5Y88vffZY5dccOxk2BuaHj27g0WI5I8fceMYxg8SZfXlpt2W3JQMdxmZsnINHi8GNHDZpHjaDBIMzPGa3JbcxA7XwsEnj15L+TJrnn4E9SEux5LZ6YrQkmEnzthkwbgBqYfy47TBhLZY9b8yNefuME2f28BhLM247zsPGTMAv5uzAEOP5JmfPz8Nj+PHntmp7fvbmh4/xOgw5Iph5wCQe5RhaGH8QUD0KRsEoGAUjEwAAgOw9OV/BvxgAAAAASUVORK5CYII=","orcid":"","institution":"Institute of Pathology, Department of Medical Services, Ministry of Public Health, Thailand","correspondingAuthor":true,"prefix":"","firstName":"Phatsorn","middleName":"","lastName":"Amattirat","suffix":""},{"id":409751907,"identity":"607c7264-e90b-4751-8aa8-bded75fa10e2","order_by":1,"name":"Thirayost Nimmanon","email":"","orcid":"","institution":"Department of Pathology, Phramongkutklao College of Medicine, Bangkok, Thailand","correspondingAuthor":false,"prefix":"","firstName":"Thirayost","middleName":"","lastName":"Nimmanon","suffix":""},{"id":409751908,"identity":"316c5916-832c-4cd4-b7da-bdb150484c1f","order_by":2,"name":"Parsinee Julimasart","email":"","orcid":"","institution":"Institute of Pathology, Department of Medical Services, Ministry of Public Health, Thailand","correspondingAuthor":false,"prefix":"","firstName":"Parsinee","middleName":"","lastName":"Julimasart","suffix":""},{"id":409751910,"identity":"6de64116-e20c-4d2d-80bd-a7198740f5d8","order_by":3,"name":"Kulachet Wiwatwarayos","email":"","orcid":"","institution":"Institute of Pathology, Department of Medical Services, Ministry of Public Health, Thailand","correspondingAuthor":false,"prefix":"","firstName":"Kulachet","middleName":"","lastName":"Wiwatwarayos","suffix":""}],"badges":[],"createdAt":"2025-01-30 14:23:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5930915/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5930915/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":75409834,"identity":"97be4266-0caf-4c50-ac16-08bcf2c4043f","added_by":"auto","created_at":"2025-02-04 09:05:29","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":52305,"visible":true,"origin":"","legend":"\u003cp\u003eThe receiver operating characteristic (ROC) curve generated using the data from Table 1.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5930915/v1/293cd9e1e469738db3a0bf27.png"},{"id":75409831,"identity":"edf967da-9e10-42f9-b25b-d8cdaa9dcfe1","added_by":"auto","created_at":"2025-02-04 09:05:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":610706,"visible":true,"origin":"","legend":"\u003cp\u003eSmall cell neuroendocrine carcinoma with with H\u0026amp;E staining (A), showing positive staining for INSM1 (B), synaptophysin (C), and chromogranin A (D).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5930915/v1/52eb1c0190d635b3843fbbc6.png"},{"id":75409827,"identity":"b4bf2426-6f05-4c9a-802e-1f42ed80f49b","added_by":"auto","created_at":"2025-02-04 09:05:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":574023,"visible":true,"origin":"","legend":"\u003cp\u003eINSM1 shows strong staining in non-keratinizing nasopharyngeal carcinoma (NK-NPC) (A, H\u0026amp;E; B, INSM1), in contrast to the lack of staining observed for synaptophysin (C) and chromogranin A (D).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5930915/v1/a38aea74b8c5c516e171ec6a.png"},{"id":77708591,"identity":"bfa7f952-fb4e-471a-8914-8aa169b8a244","added_by":"auto","created_at":"2025-03-04 12:32:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1742014,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5930915/v1/0f78236c-7de3-48f5-8705-aa79c719e79e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Sensitivity and specificity of INSM1 compared with Chromogranin and Synaptophysin in neuroendocrine carcinomas of the head and neck region","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe most clinically urgent subtype of neuroendocrine neoplasm (NEN) is neuroendocrine carcinoma (NEC), which is characterized by poor differentiation, tumor necrosis, high mitotic activity, and destructive features [23].Morphologically, NEC may mimic several other tumor types, especially in small tissue biopsies. These mimickers include well-differentiated neuroendocrine tumors (NET), Merkel cell carcinoma, NUT carcinoma, sinonasal undifferentiated carcinoma, basaloid squamous cell carcinoma, adenoid cystic carcinoma (solid type), SWI/SNF complex-deficient carcinoma, tumors with neuroectodermal differentiation (such as olfactory neuroblastoma, teratocarcinosarcoma), mucosal melanoma, and round cell sarcomas (including Ewing sarcoma, EWSR1-non-ETS sarcoma, BCOR sarcoma, alveolar rhabdomyosarcoma, and synovial sarcoma) [24].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eResearch also indicates that other tumor types, such as acinic cell carcinoma[19,20,38], paraganglioma, metastatic medullary thyroid carcinoma, olfactory neuroblastoma, and Ewing\u0026rsquo;s sarcoma family[26], can exhibit neuroendocrine differentiation [21]. NEC is a rare malignancy, accounting for only 0.3% of all head and neck cancers [27-29]. Similarly, primary NEC of the salivary gland is uncommon, representing less than 2% of salivary gland malignancies, primarily in the parotid and submandibular glands [39,40]. \u0026nbsp;Its clinicopathological and genomic features are not yet fully understood. The FGFR3-TACC3 fusion gene and mutations in genes encoding components of the NOTCH and PI3K/AKT/mTOR pathways, as identified in study, may represent promising therapeutic targets for NEC [25\u003c/p\u003e\n\u003cp\u003eSmall cell neuroendocrine carcinoma (SCNC) is the most common type of NEC in the larynx and salivary gland, while large cell neuroendocrine carcinoma (LCNC) is rare in these locations [31,32]. Common markers used in diagnosing NEC include chromogranin A (CGA), synaptophysin (SYN), and CD56, with reported sensitivities ranging from 50% to 80% [1-3]. However, these markers may also stain positive in other non-neuroendocrine tumors, such as carcinomas, sarcomas, and melanomas [4-6].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInsulinoma-associated protein 1 (INSM1; formerly IA-1), a transcription factor containing five zinc-finger motifs, is predominantly expressed in developing neuroendocrine tissues and the nervous system in mammals[22].INSM1 has shown high sensitivity and specificity for detecting neuroendocrine differentiation in the head and neck region, including middle ear adenoma, pituitary adenoma, paraganglioma, medullary thyroid carcinoma, olfactory neuroblastoma, SCNC, LCNC, sinonasal teratocarcinoma, and laryngeal NEC [7,8]. Additionally, INSM1 demonstrates positivity in squamous cell carcinoma (SCC) of the larynx, pharynx, and floor of the mouth. However, it does not stain positive in pleomorphic adenoma, Warthin tumor, or basal cell adenoma [17].\u003c/p\u003e\n\u003cp\u003eCD56 has demonstrated the lowest sensitivity and specificity among markers for primary lung neoplasms [9,10], small cell lung carcinoma [11,12], thoracic cavity tumors [13], and urinary tract neoplasms [14]. Certain cases of LCNC and SCNC may be Epstein-Barr virus (EBV)-positive, which can complicate the diagnosis. Similarly, SCNC may show P16 positivity, potentially leading to confusion with other tumors, such as P16-positive oropharyngeal squamous cell carcinoma (OPSCC). These findings highlight the critical importance of selecting appropriate control groups to ensure diagnostic accuracy [15,16,30]. \u0026nbsp; \u0026nbsp;\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cem\u003eEthical statements\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eOur research was approved by the Institutional Review Board at the Institute of Pathology, Department of Medical Services, Ministry of Public Health, prior to its commencement (IOP, No. IOP-KMR66-004). The board exempted the requirement for informed consent documentation, as the study did not involve human subjects. This approval ensures that our research complies with ethical guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCase selection\u003c/em\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA case-control study identified 329 cases. The inclusion criteria required samples with neoplasms located in the head and neck region, with available paraffin blocks from 2017 to 2024. The exclusion criteria eliminated cases with neoplasms originating from the salivary glands or other tumors that typically exhibit neuroendocrine differentiation.\u003c/p\u003e\n\u003cp\u003eThe sample size was determined with 14 cases in the NEC group and 109 cases in the non-NEC group. The NEC group included 10 cases of SCNC and 4 cases of LCNC. The non-NEC group consisted of 109 cases of NK-NPC and 16 cases of P16-positive OPSCC. Due to the rarity of neuroendocrine carcinoma in the head and neck region, any additional cases encountered during the study were also included.\u003c/p\u003e\n\u003cp\u003eThe sample size was calculated to ensure reliability using the Finite Population Proportion formula, a commonly used method for determining an appropriate sample size based on prior research values. [37]. Simple random sampling was applied to enroll cases into the study, ensuring unbiased selection from both groups for inclusion in the experiment.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cem\u003eEvaluating tumor cells percentage and intensity\u003c/em\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe percentage (%) of tumor staining was recorded based on 10 consecutive high-power fields (HPFs), starting from the field with the highest tumor presence. The staining was then categorized into the following tiers: 0-10%, 11-20%, 21-30%, 31-40%, 41-50%, 51-60%, 61-70%, 71-80%, 81-90%, and 91-100%.\u003c/p\u003e\n\u003cp\u003eStaining intensity was graded as 0, 1+, 2+, or 3+. Histopathological evaluation was performed using an Olympus BX53 microscope by the researcher, who is a resident physician in anatomical pathology. All diagnoses were verified through a second reading by a supervising pathologist. The results were reviewed, checked, and confirmed, with findings documented in a research record form.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eINSM1, CGA, and SYN immunohistochemical studies\u003c/em\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOnce the samples are obtained, they will be stained with INSM 1 (MRQ-70). If synaptophysin or chromogranin staining had not been previously performed for a case, Synaptophysin (MRQ-40) and Chromogranin A (LK2H10) were applied.\u003c/p\u003e\n\u003cp\u003eTissue sections were cut to a thickness of 3 micrometers and underwent antigen retrieval in 10 mM citrate buffer at 100\u0026deg;C for 37 minutes using the LEICA BOND-MAX system. Microscopic evaluation was then conducted. The quality of staining was assessed based on external controls, following the laboratory\u0026rsquo;s standard procedures, to ensure suitability for microscopic examination.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStatistical analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eStatistical Analysis were performed using IBM SPSS Statistics 26 software, and the R programming language. Results were considered significant when the p-value was less than 0.05. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; The optimal range of tumor cell percentages in relation to INSM1 positivity was determined using the Receiver Operating Characteristic (ROC) curve. The ROC curve serves as a tool for assessing the effectiveness of a diagnostic test [36]. Sensitivity and specificity of all stains were compared using the McNemar x\u003csup\u003e2\u003c/sup\u003e test.\u003c/p\u003e"},{"header":"Results","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"296\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" style=\"width: 296px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTable 1: Sensitivity and Specificity at INSM1 percentage\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 129px;\"\u003e\n \u003cp\u003eINSM1 percentage\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003eSensitivity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003eSpecificity\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003e0-10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e0.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e11-20\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e92.9%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e96.3%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003e21-30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e92.9%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e98.2%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003e31-40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e92.9%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e99.1%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003e71-80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e92.9%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003e81-90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e64.3%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003e91-100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e57.1%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eFigure 1 and Table 1 illustrate that the optimal range of 11\u0026ndash;20% tumor cells was determined based on INSM1 positivity using the Receiver Operating Characteristic (ROC) curve. The area under the curve (AUC) was 0.952, indicating excellent accuracy in distinguishing between NEC and non-NEC cases. Tumor cell proportions greater than 10% demonstrated optimal sensitivity and specificity.\u003c/p\u003e\n\u003cp\u003eTable 2 shows that INSM1 had an overall sensitivity of 92.9%, compared to 78.6% for CGA (P = 0.157) and 100.0% for SYN (P = 0.317). INSM1 also had an overall specificity of 96.3%, compared to 100.0% specificity for both CGA and SYN (P = 0.045).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFigure 2 demonstrates that INSM1 exhibits positive nuclear staining, which is easier to interpret. The proportion of tumor staining for INSM1 averaged 91.9%, compared to 91.4% for SYN and 82.2% for CGA. Additionally, INSM1 had a mean staining intensity of 2.8, compared to 2.1 for SYN and 2.0 for CGA.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn p16-positive OPSCC, all cases were negative for INSM1, SYN, and CGA. Figure 3 illustrates that, in NK-NPC, INSM1 was positive in four cases, while SYN and CGA were negative in all cases. Table 2 clarifies that the positive cases had tumor staining proportions of 20%, 20%, 30%, and 40%, respectively. The remaining cases showed a scattered tumor staining proportion of \u0026lt; 10%.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we evaluated the performance of INSM1 in detecting SCNC and LCNC in the head and neck region.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe results indicated that INSM1 did not demonstrate a statistically significant difference in sensitivity compared to SYN and CGA. These findings are consistent with previous research, which found that SYN had the highest sensitivity (93.3%), followed by INSM1 (89.2%) and CGA (87.5%). That study analyzed a total of 471 NEN samples from various organs, further supporting the effectiveness of INSM1 as a diagnostic tool for neuroendocrine tumors across different tissues [17].\u003csup\u003e\u0026nbsp;\u003c/sup\u003eOur findings also reveal differences compared to other research. For example, previous studies reported INSM1 exhibiting a high sensitivity of 99%, compared to SYN (92.3%) and CGA (77.3%) [7]. This higher sensitivity highlights INSM1\u0026apos;s potential as a more reliable marker for diagnosing NET. Moreover, in areas with significant crushing artifacts and difficult morphology, INSM1 still demonstrates good staining.\u003c/p\u003e\n\u003cp\u003eIn our study, INSM1 demonstrated a specificity of 96.3%, which was significantly lower than SYN and CGA. This value differs from previous research that reported a specificity of 97.6% for INSM1 at a cutoff point of 10%. These discrepancies suggest that INSM1\u0026apos;s specificity may vary depending on the study population or the sample type analyzed. Further investigations are necessary to better understand the conditions under which INSM1\u0026apos;s specificity is most reliable [7].\u003c/p\u003e\n\u003cp\u003eA study investigating NE differentiation in SCC of the head and neck reported sparsely scattered CGA expression in 41% of cases, which correlated with high CGA messenger RNA expression. Additionally, SYN expression was observed in 18% of cases [18]. In contrast, our study found that all non-NEC cases were negative for CGA and SYN. However, four of these cases showed positive INSM1 staining, while some others exhibited sparsely scattered staining in less than 10% of the tumor cells. This highlights the need for caution when interpreting INSM1 results in the nasopharynx region.\u003c/p\u003e\n\u003cp\u003eFurthermore, all cases of P16-positive OPSCC in our study were negative for INSM1, suggesting that INSM1 can be used to rule out NEC in the oropharynx. This finding aligns with previous research that reported 129 out of 130 oral SCC samples were negative for INSM [17]. Such consistency with prior findings supports the specificity of INSM1 in identifying neuroendocrine carcinomas and underscores its utility in distinguishing these tumors from other types in specific anatomical regions.\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"604\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\" valign=\"top\" style=\"width: 604px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTABLE 2. Expression of INSM1, Synaptophysin, and Chromogranin in NEC and non-NEC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eDiagnosis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"6\" valign=\"top\" style=\"width: 510px;\"\u003e\n \u003cp\u003e(n/N [%])\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003eINSM1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003eSynaptophysin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003eChromogranin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003epositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003enegative\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eSCNC\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e10/10 (100)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0/10 (0)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e10/10 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0/10 (0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e9/10 (90)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e1/10 (10)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eLCNC\u003csup\u003e\u0026micro;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e3/4 (75)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e1/4 (25)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e4/4 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0/4 (0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e2/4 (50)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e2/4(50)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eTotal NEC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e13/14 (92.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e1/14 (7.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e14/14 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0/14 (0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e11/14 (78.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e3/14 (21.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eP16+OPSCC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0/16 (0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e16/16 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0/16 (0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e16/16 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0/16 (0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e16/16 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eNK-NPC\u003csup\u003e\u0026pi;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e4\u003csup\u003e\u0026alpha;\u003c/sup\u003e/93 (4.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e89/93 (95.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0/93 (0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e93/93 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0/93 (0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e93/93 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eTotal Non-NEC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e4/109\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(3.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e105/109 (96.3)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0/109\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e109/109 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0/109\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e109/109 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\" valign=\"top\" style=\"width: 604px;\"\u003e\n \u003cp\u003e*Including base of tongue (n=2), nasal cavity (n=3), tonsil (n=1), pyriform (n=2), cricoid (n=1), neck (n=1)\u003c/p\u003e\n \u003cp\u003e\u003csup\u003e\u0026micro;\u003c/sup\u003eIncluding nasal cavity (n=1), base of tongue (n=1), gum (n=1), mandibular (n=1)\u003c/p\u003e\n \u003cp\u003e\u003csup\u003e\u0026alpha;\u003c/sup\u003e Tumor cells percentage 20% (n=2), 30% (n=1), 40% (n=1)\u003c/p\u003e\n \u003cp\u003e\u003csup\u003e\u0026pi;\u003c/sup\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003eEBV-associated NPC (n=49), non-EBV-associated NPC (n=5), and NPC with unknown EBV status (n=39) were categorized based on EBV status, while differentiation subtype (n=19) and undefined subtype (n=74) were categorized based on differentiated morphology.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eIn our study, four cases exhibited scattered tumor cell staining of 10% or more. Therefore, cases with already established differentiation should not undergo INSM1 staining, as a positive result in such cases would not alter the diagnosis of neuroendocrine carcinoma (NEC). Additionally, in cases with limited tumor samples from biopsy and undifferentiated morphology, if INSM1 is positive but no other markers are present, obtaining a larger tissue sample is crucial for accurate interpretation. A small biopsy may not provide sufficient evidence to definitively diagnose NEC. We defined positivity as \u0026gt;10% based on the ROC curve. While raising the cutoff value can increase both sensitivity and specificity, a higher threshold is not always more appropriate. INSM1 already demonstrates strong staining in NEC, and increasing the cutoff does not significantly improve sensitivity. This approach aligns with other studies that have used similar cutoff values.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor example, a study reported INSM1 positivity in adenocarcinoma from lung bronchial brushing samples in less than 10% of tumor cells. The optimal cutoff value for INSM1 to differentiate between small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC) was determined to be 8.68%, with a sensitivity of 95.8% and a specificity of 100% [35].\u0026nbsp;Compared to previous research on lung samples, which found that any percentage could serve as the optimal cutoff for CD56, CGA, and INSM1 in differentiating NET from NSCLC cases, a cutoff of at least 10% positive tumor cells was identified as the best threshold for SYN\u003csup\u003e\u0026nbsp;\u003c/sup\u003e[33]. This is similar to another study on NE differentiation in breast carcinoma, which found that INSM1 is a sensitive marker and should be included with SYN and CGA in the panel to evaluate NE differentiation, using a cutoff point of 50%\u003csup\u003e\u0026nbsp;\u003c/sup\u003e[34].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSince NEC in the head and neck region is quite rare, the researchers believe that future studies, which continue to collect data over time, will likely provide sufficient information for more definitive conclusions. However, the nuclear staining pattern of INSM1 offers an advantage in interpretation, as it simplifies the process compared to the cytoplasmic staining patterns commonly observed with other markers. This can be particularly helpful in challenging cases where other markers provide less clarity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhile many studies have demonstrated that INSM1 has high sensitivity and specificity, suggesting its potential as a standalone marker, our findings indicate that it is a reliable marker. However, it should not be used as the sole diagnostic tool. Given that SCNC and LCNC in the head and neck region are rare and infrequently encountered at our pathology institution, future studies with larger sample sizes may provide insights that refine or modify the current conclusions. Additionally, further investigation into combining INSM1 with other markers or utilizing advanced techniques, such as molecular profiling, could enhance its overall diagnostic accuracy.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eINSM1 demonstrates good sensitivity, comparable to SYN and CGA, without showing a statistically significant difference compared to traditional markers. Its nuclear staining pattern enhances its value, making it easier to interpret in clinical practice. When tumor staining is \u0026le;\u0026thinsp;10%, the confidence in interpretation decreases. As such, INSM1 should not be used as a standalone marker. In such cases, it is recommended to consider the morphology and additional immunohistochemical markers, such as chromogranin A (CGA) and synaptophysin (SYN), for an accurate assessment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eI would like to express my heartfelt gratitude to the Institute of Pathology Foundation for their financial support, which made this research possible.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eFundings\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the Institute of Pathology Foundation (grant number 7/2567), which supports research activities within the Institute of Pathology. The funder had no involvement in the study design, data collection, analysis, or interpretation, and did not influence the writing of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eConflict of interest \u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFor this type of study formal consent is not required.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eConsent to\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003cem\u003eparticipate\u003c/em\u003e \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor this type of study formal consent is not required.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFor this type of study consent for publication is not required.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe data analyzed in this study is stored within the Institute of Pathology, Ministry of Public Health, Thailand. Due to patient confidentiality and institutional regulations, the data is not publicly available. However, researchers may request access from the corresponding author, subject to approval by the Institute of Pathology\u0026rsquo;s ethics committee.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eCode availability\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis study utilized proprietary software, IBM SPSS Statistics (version 26, IBM Corp.), and the R programming language (version 4.2, R Foundation for Statistical Computing).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eA. Ferlito, I. Friedmann; Contribution of Immunohistochemistry in the Diagnosis of Neuroendocrine Neoplasms of the Larynx. ORL 1 April 1991; 53 (4): 235\u0026ndash;244. https://doi.org/10.1159/000276222\u003c/li\u003e\n\u003cli\u003eRinaldo, A., Devaney, K. O., \u0026amp; Ferlito, A. (2004). Immunohistochemical studies in support of a diagnosis of small cell neuroendocrine carcinoma of the larynx. \u003cem\u003eActa Oto-Laryngologica\u003c/em\u003e, \u003cem\u003e124\u003c/em\u003e(5), 638\u0026ndash;641. https://doi.org/10.1080/00016480410016540\u003c/li\u003e\n\u003cli\u003eSerrano MF, El-Mofty SK, Gnepp DR, Lewis JS Jr. Utility of high molecular weight cytokeratins, but not p63, in the differential diagnosis of neuroendocrine and basaloid carcinomas of the head and neck. Hum Pathol. 2008 Apr;39(4):591-8. https://doi.org/10.1016/j.humpath.2007.08.019\u003c/li\u003e\n\u003cli\u003eBahrami A, Gown AM, Baird GS, Hicks MJ, Folpe AL. Aberrant expression of epithelial and neuroendocrine markers in alveolar rhabdomyosarcoma: a potentially serious diagnostic pitfall. Mod Pathol. 2008 Jul;21(7):795-806. https://doi.org/10.1038/modpathol.2008.8\u003c/li\u003e\n\u003cli\u003eLee H, Torres FX, McLean SA, Chen R, Lee MW. Immunophenotypic heterogeneity of primary sinonasal melanoma with aberrant expression of neuroendocrine markers and calponin. Appl Immunohistochem Mol Morphol. 2011 Jan;19(1):48-53. https://doi.org/10.1097/PAI.0b013e3181ee8dcb\u003c/li\u003e\n\u003cli\u003eSchartinger VH, Falkeis C, Laimer K, Sprinzl GM, Riechelmann H, Rasse M, Virgolini I, Dud\u0026aacute;s J. Neuroendocrine differentiation in head and neck squamous cell carcinoma. J Laryngol Otol. 2012 Dec;126(12):1261-70. https://doi.org/10.1017/S0022215112002265\u003c/li\u003e\n\u003cli\u003eRooper LM, Bishop JA, Westra WH. INSM1 is a Sensitive and Specific Marker of Neuroendocrine Differentiation in Head and Neck Tumors. Am J Surg Pathol. 2018 May;42(5):665-671. https://doi.org/10.1097/PAS.0000000000001037\u003c/li\u003e\n\u003cli\u003eYuan C, Jiao F, Zhai C, Zhang J, Wang S, Zhu L. Application of INSM1 in Diagnosis and Grading of Laryngeal Neuroendocrine Carcinoma. Laryngoscope. 2021 Oct;131(10): E2662-E2668. https://doi.org/10.1002/lary.29554\u003c/li\u003e\n\u003cli\u003eMukhopadhyay S, Dermawan JK, Lanigan CP, Farver CF. Insulinoma-associated protein 1 (INSM1) is a sensitive and highly specific marker of neuroendocrine differentiation in primary lung neoplasms: an immunohistochemical study of 345 cases, including 292 whole-tissue sections. Mod Pathol. 2019 Jan;32(1):100-109. https://doi.org/10.1038/s41379-018-0122-7\u003c/li\u003e\n\u003cli\u003eKriegsmann K, Zgorzelski C, Kazdal D, Cremer M, Muley T, Winter H, et al. Insulinoma-associated Protein 1 (INSM1) in Thoracic Tumors is Less Sensitive but More Specific Compared with Synaptophysin, Chromogranin A, and CD56. Applied Immunohistochemistry \u0026amp; Molecular Morphology. 2018 Oct 24;28(3):237\u0026ndash;42. https://doi.org/10.1097/PAI.0000000000000715\u003c/li\u003e\n\u003cli\u003e\u0026zwnj;[11] Sakakibara R, Kobayashi M, Takahashi N, Inamura K, Ninomiya H, Wakejima R, et al. Insulinoma-associated Protein 1 (INSM1) Is a Better Marker for the Diagnosis and Prognosis Estimation of Small Cell Lung Carcinoma Than Neuroendocrine Phenotype Markers Such as Chromogranin A, Synaptophysin, and CD56. American Journal of Surgical Pathology. 2020 Feb 28;44(6):757\u0026ndash;64. https://doi.org/10.1097/PAS.0000000000001444\u003c/li\u003e\n\u003cli\u003eRocha R, Henrique R. Insulinoma-Associated Protein 1 (INSM1): Diagnostic, Prognostic, and Therapeutic Use in Small Cell Lung Cancer. Journal of Molecular Pathology. 2022 Aug 5;3(3):140\u0026ndash;69. https://doi.org/10.3390/jmp3030013\u003c/li\u003e\n\u003cli\u003e\u0026zwnj;[13] Rooper LM, Sharma R, Li QK, Illei PB, Westra WH. INSM1 Demonstrates Superior Performance to the Individual and Combined Use of Synaptophysin, Chromogranin and CD56 for Diagnosing Neuroendocrine Tumors of the Thoracic Cavity. Am J Surg Pathol. 2017 Nov;41(11):1561-1569. https://doi.org/10.1097/PAS.0000000000001003\u003c/li\u003e\n\u003cli\u003eKim IE Jr, Amin A, Wang LJ, Cheng L, Perrino CM. Insulinoma-associated Protein 1 (INSM1) Expression in Small Cell Neuroendocrine Carcinoma of the Urinary Tract. Appl Immunohistochem Mol Morphol. 2020 Oct;28(9):687-693. https://doi.org/10.1097/PAI.0000000000000824\u003c/li\u003e\n\u003cli\u003eNada Al Masalmeh, Kukreja G, Feras Zaiem, S. Naweed Raza, Kim H, Misako Nagasaka, et al. p16 positive oropharyngeal small cell cancer: A case report. Oral Oncology. 2021 Oct 1; 121:105391\u0026ndash;1. https://doi.org/10.1016/j.oraloncology.2021.105391\u003c/li\u003e\n\u003cli\u003eSu Z, Yan H, Zou G. EBV‑positive large‑cell and small‑cell neuroendocrine carcinoma of the nasopharynx: A case report and review of the literature. Oncology Letters. 2022 Nov 7;24(6). https://doi.org/10.3892/ol.2022.13584\u003c/li\u003e\n\u003cli\u003eM\u0026ouml;ller K, Gorbokon N, Hube-Magg C, Fraune C, Lennartz M, Blessin NC, et al. Comparison of INSM1 immunostaining with established neuroendocrine markers Synaptophysin and Chromogranin A in over 14,000 neuroendocrine and non-neuroendocrine tumors. American Journal of Clinical Pathology. 2023 Nov 1;160(Supplement_1): S36\u0026ndash;7. https://doi.org/10.1093/ajcp/aqad086\u003c/li\u003e\n\u003cli\u003eSchartinger VH, C Falkeis, K Laimer, Sprinzl GM, H Riechelmann, M Rasse, et al. Neuroendocrine differentiation in head and neck squamous cell carcinoma. The Journal of Laryngology \u0026amp; Otology. 2012 Oct 11;126(12):1261\u0026ndash;70. https://doi.org/10.1017/S0022215112002265\u003c/li\u003e\n\u003cli\u003eIto K, Kakudo K, Mori I, et al. Neuroendocrine differentiation in a case of acinic cell carcinoma of the parotid gland. Acta Pathol Jpn. 1990; 40:279\u0026ndash;287. https://doi.org/10.1111/j.1440-1827.1990.tb01562.x\u003c/li\u003e\n\u003cli\u003eRoy S, Dhingra KK, Gupta P, et al. Acinic cell carcinoma with extensive neuroendocrine differentiation: a diagnostic challenge. Head Neck Pathol. 2009; 3:163\u0026ndash;168. https://doi.org/10.1007/s12105-009-0114-5\u003c/li\u003e\n\u003cli\u003eMills SE. Neuroectodermal Neoplasms of the Head and Neck with Emphasis on Neuroendocrine Carcinomas. Modern Pathology. 2002 Mar 1;15(3):264\u0026ndash;78. https://doi.org/10.1038/modpathol.3880522 \u003c/li\u003e\n\u003cli\u003eLan MS, Breslin MB. Structure, expression, and biological function of INSM1 transcription factor in neuroendocrine differentiation. FASEB J. 2009; 23:2024\u0026ndash;2033. https://doi.org/10.1096/fj.08-125971\u003c/li\u003e\n\u003cli\u003eRindi G, Mete O, Uccella S, Basturk O, La Rosa S, Brosens LAA, et al. Overview of the 2022 WHO Classification of Neuroendocrine Neoplasms. Endocrine Pathology. 2022 Mar;33(1):115\u0026ndash;54. https://doi.org/10.1007/s12022-022-09708-2\u003c/li\u003e\n\u003cli\u003eC. Christofer Juhlin, Bal M. Top 10 Histological Mimics of Neuroendocrine Carcinoma You Should Not Miss in the Head and Neck. Head and Neck Pathology [Internet]. 2023 Mar 20 [cited 2023 Nov 30];17(1):66\u0026ndash;84. https://doi.org/10.1007/s12105-023-01647-0\u003c/li\u003e\n\u003cli\u003eAkihiro Ohmoto, Sato Y, Reimi Asaka, Fukuda N, Wang X, Tetsuya Urasaki, et al. Clinicopathological and genomic features in patients with head and neck neuroendocrine carcinoma. Modern Pathology. 2021 Nov 1;34(11):1979\u0026ndash;89. https://doi.org/10.1038/s41379-021-00869-9\u003c/li\u003e\n\u003cli\u003eMachado I, Navarro S, L\u0026oacute;pez-Guerrero JA, Verdini L, Piero Picci, Giner F, et al. Neuroendocrine differentiation in a large series of genetically-confirmed Ewing\u0026rsquo;s sarcoma family tumor: Does it provide any diagnostic or prognostic information? Pathology - Research and Practice. 2021 Feb 7; 219:153362\u0026ndash;2. https://doi.org/10.1016/j.prp.2021.153362 \u003c/li\u003e\n\u003cli\u003eAnas Bouzbouz, Bushra Abdulhakeem, Rabii Laababsi, Sami Rouadi, Abada R, Roubal M, et al. rare cases of head and neck\u0026rsquo;s neuroendocrine carcinomas disease: Case series of 4 patients and review of the literature. International journal of surgery case reports. 2020 Jan 1; 66:270\u0026ndash;6. https://doi.org/10.1016/j.ijscr.2019.12.003\u003c/li\u003e\n\u003cli\u003eSubedi N, Prestwich R, Chowdhury F, Patel C, Scarsbrook A. Neuroendocrine tumours of the head and neck: anatomical, functional and molecular imaging and contemporary management. Cancer Imaging. 2013;407\u0026ndash;22. https://doi.org/10.1186/1477-7819-11-66\u003c/li\u003e\n\u003cli\u003ePerez-Ordo\u0026ntilde;ez B. Neuroendocrine Carcinomas of the Larynx and Head and Neck: Challenges in Classification and Grading. Head and Neck Pathology. 2018 Mar;12(1):1\u0026ndash;8. https://doi.org/10.1007/s12105-018-0894-6\u003c/li\u003e\n\u003cli\u003eChen Y, Zhou N, Huang C, He X, Wang X, Tang H, et al. EBV-positive small cell neuroendocrine carcinoma of nasopharynx as a probably unique subtype of neuroendocrine carcinoma: a clinicopathologic study of three cases and literature review. Diagnostic Pathology. 2024 Jul 24;19(1). https://doi.org/10.1186/s13000-024-01526-w\u003c/li\u003e\n\u003cli\u003eTom, Boudewijn E. C. Plaat, F.A.M B, Halmos GB. Clinical recommendations on the treatment of neuroendocrine carcinoma of the larynx: A meta-analysis of 436 reported cases. Head \u0026amp; Neck. 2014 Jun 18;37(5):707\u0026ndash;15. https://doi.org/10.1002/hed.23456\u003c/li\u003e\n\u003cli\u003eBellahammou K, Lakhdissi A, Akkar O, Kouhen F, Rais F, Dahraoui S, et al. SMALL-CELL NEUROENDOCRINE CARCINOMA OF NASOPHARYNX: A CASE REPORT. International Journal of Surgery and Medicine. 2017;3(1):1. https://doi.org/10.13107/ijscr.2017.v03i01.101\u003c/li\u003e\n\u003cli\u003eStaaf J, Tran L, S\u0026ouml;derlund L, Nodin B, Jirstr\u0026ouml;m K, Vidarsdottir H, et al. Diagnostic Value of Insulinoma-Associated Protein 1 (INSM1) and Comparison with Established Neuroendocrine Markers in Pulmonary Cancers. Archives of Pathology \u0026amp; Laboratory Medicine [Internet]. 2020 Sep 1 [cited 2024 Jun 17];144(9):1075\u0026ndash;85. https://doi.org/10.5858/arpa.2019-0291-OA\u003c/li\u003e\n\u003cli\u003eZhong E, Pareja F, Hanna MG, Jungbluth AA, Rekhtman N, Brogi E. Expression of novel neuroendocrine markers in breast carcinomas: a study of INSM1, ASCL1, and POU2F3. Human Pathology. 2022 Sep; 127:102\u0026ndash;11. https://doi.org/10.1016/j.humpath.2022.06.003\u003c/li\u003e\n\u003cli\u003eAbe H, Takase Y, Sadashima E, Fukumitsu C, Murata K, Ito T, et al. Insulinoma‐associated protein 1 is a novel diagnostic marker of small cell lung cancer in bronchial brushing and cell block cytology from pleural effusions: Validity and reliability with cutoff value. Cancer Cytopathology. 2019 Aug 22;127(9):598\u0026ndash;605. https://doi.org/10.1002/cncy.22177\u003c/li\u003e\n\u003cli\u003eNahm FS. Receiver operating characteristic curve: overview and practical use for clinicians. Korean Journal of Anesthesiology [Internet]. 2022 Feb 1;75(1):25\u0026ndash;36. https://doi.org/10.4097/kja.21209\u003c/li\u003e\n\u003cli\u003eDaniel WW, Cross CL. Biostatistics: a foundation for analysis in the health sciences. 10th ed. Hoboken, NJ: Wiley; 2013.\u003c/li\u003e\n\u003cli\u003eRath A, Tomar R, Agarwal R, Singh M, Jain S, Khurana N, et al. Acinic cell carcinoma of the parotid gland with neuroendocrine differentiation. Journal of Cancer Research and Therapeutics [Internet]. 2021 Jul 1 [cited 2023 Nov 11];17(4):1115. https://doi.org/10.4103/jcrt.JCRT_1050_20\u003c/li\u003e\n\u003cli\u003eLIU M, ZHONG M, SUN C. Primary neuroendocrine small cell carcinoma of the parotid gland: A case report and review of the literature. Oncology Letters. 2014 Jun 17;8(3):1275\u0026ndash;8. https://doi.org/10.3892/ol.2014.2340\u003c/li\u003e\n\u003cli\u003ePetrone G, Santoro A, Angrisani B, Novello M, Scarano E, Guido Rindi, et al. Neuroendocrine Tumors of the Submandibular Gland. International Journal of Surgical Pathology. 2012 May 21;21(1):85\u0026ndash;8. https://doi.org/10.1177/1066896912446747\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"head and neck region, small cell neuroendocrine carcinoma (SCNC), large cell neuroendocrine carcinoma (LCNC), INSM1, surgical pathology ","lastPublishedDoi":"10.21203/rs.3.rs-5930915/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5930915/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e:\u003c/strong\u003e The head and neck region is an area where neuroendocrine tumors (NETs) and neuroendocrine carcinomas (NECs) can arise. NECs have several mimickers, which make their diagnosis challenging. Accurate diagnosis typically relies on morphology, supported by established markers such as synaptophysin (SYN) or chromogranin A (CGA), each with varying sensitivity and specificity. Insulinoma-associated protein 1 (INSM1) is a newer marker that has shown good sensitivity and specificity in various organs. Despite its promising potential, there are very few studies when compared to other organs.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e: \u003c/strong\u003eThis case-control study tested INSM1, SYN, and CGA on 14 NEC samples and 109 non-NEC samples to evaluate their sensitivity and specificity.\u003cbr\u003e\n\u003cem\u003e\u003cstrong\u003eResults: \u003c/strong\u003e\u003c/em\u003eINSM1 demonstrated an overall sensitivity of 92.9%, compared to 78.5% for CGA (P = 0.157) and 100.0% for SYN (P = 0.317). INSM1 exhibited a specificity of 96.3%, compared to 100% for both CGA (P = 0.045) and SYN (P = 0.045). Among non-NEC cases, scattered staining (\u0026lt; 10% of tumor cells) was observed with INSM1, while 4 non-NEC cases showed positive staining for INSM1. All non-NEC cases were negative for CGA and SYN.\u003cstrong\u003e \u003cbr\u003e\n\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e\u003c/em\u003e INSM1 demonstrates good sensitivity, comparable to SYN and CGA, with no statistically significant differences observed between these markers. However, the specificity of INSM1 shows statistically significant differences. Notably, one case of EBV-positive nonkeratinizing squamous cell carcinoma of the nasopharynx exhibited INSM1 positivity in 40% of tumor cells. As such, INSM1 should not be used as a standalone marker for diagnosis. Furthermore, caution is warranted when interpreting results with staining ≤10%, as this may reduce the reliability of a positive finding.\u003c/p\u003e","manuscriptTitle":"Sensitivity and specificity of INSM1 compared with Chromogranin and Synaptophysin in neuroendocrine carcinomas of the head and neck region","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-04 09:05:22","doi":"10.21203/rs.3.rs-5930915/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ff5159d1-f047-4c64-9980-42d8c9627601","owner":[],"postedDate":"February 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-03-04T12:24:39+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-04 09:05:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5930915","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5930915","identity":"rs-5930915","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}