Establishment of an efficient pathologic diagnostic platform using core needle biopsy for salivary gland carcinoma

Establishment of an efficient pathologic diagnostic platform using core needle biopsy for salivary gland carcinoma | 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 Establishment of an efficient pathologic diagnostic platform using core needle biopsy for salivary gland carcinoma Kenya Kobayashi, Seiichi Yoshimoto, Go Omura, Yoshifumi Matsumoto, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4703440/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Purpose: A major problem in establishing treatment strategies for salivary gland cancer is the difficulty of preoperative histologic typing. In recent years, genetic evaluation has become essential for salivary gland tumor diagnosis. The core needle biopsy (CNB) allows a small piece of the center of the tumor to be taken intact and analyzed in detail. The purpose of this study was to clarify the efficacy and the limitations of CNB in the preoperative diagnosis, and to establish a generalizable preoperative histologic typing platform. Methods: Paired preoperative 20-gauge core needle biopsy (CNB) and surgical specimens from 41 patients with malignant salivary gland tumors were reviewed. Morphological evaluation, immunohistochemical evaluation, and break-apart fluorescence in situ hybridization (FISH) assay were performed as histologic typing methods for CNB. The quality of biopsy specimens, diagnostic accuracy, and immunostaining concordance rates between biopsy and surgical specimens were analyzed. Results: For CNB, 95% (39/41) of the specimens were of high quality, allowing adequate morphologic, immunohistologic, and genomic analysis. Two patients had unanalyzable specimens due to cystic fluid or tumor firmness. Overall, 75% (31/41) had correct preoperative histologic typing. Compared to other histologic types, carcinoma ex pleomorphic adenoma (CXPA) and salivary duct carcinoma arising from CXPA had a significantly lower concordance rate for histologic typing (50% vs. 89.6%, p=0.016) and poorer HER2 immunostaining concordance rates between CNB and surgical specimens (60% vs. 0%, p=0.001). There were no recurrences due to tumor seeding after CNB. Conclusions: Detailed analysis of CNB specimens allows for highly accurate determination of salivary gland carcinoma histologic type with molecular diagnosis. However, careful histologic typing is necessary in pathologically heterogeneous tumors. core needle biopsy preoperative pathologic diagnosis histologic type Heterogeneity SOX10 Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Because of the diversity in salivary gland carcinoma histopathologic types, pathologic diagnosis is notoriously difficult ( 1 ) . Moreover, in recent years, as the molecular biology of salivary gland carcinoma has become elucidated, genomic information such as fusion genes and gene amplification have been used for pathologic diagnosis in addition to morphologic and immunohistologic information ( 2 , 3 ) . Core needle biopsy (CNB) allows a small piece of the center of the tumor to be taken intact and all of its components to be analyzed in detail ( 4 ) . In addition, immunohistologic status and genomic information such as fusion genes and gene amplification can be extracted, which is expected to dramatically improve diagnostic performance ( 2 , 5 , 6 ) . The histologic heterogeneity of salivary gland carcinoma is due to the complexity of salivary gland structures (2) . Histogenesis-based diagnostic markers are useful for systematic and effective histologic typing. Our previous reports showed that SRY-related HMG-box10 (SOX10) expression can clearly define two distinct subtypes of salivary gland tumors based on their anatomical site of origin ( 7 ) . To make needle biopsies for the diagnosis of salivary gland cancer more widespread, establishing an efficient pathologic diagnostic platform that uses several immunohistologic markers based on the origin and characteristics of the tumor is urgently needed. It is also necessary to identify problems and limitations in pathological diagnosis using needle biopsy specimens. Materials and Methods Study design and setting In this retrospective study, medical records, biopsy specimens, and surgical specimens of 139 patients with parotid gland and submandibular gland tumor who underwent primary surgical treatment at the National Cancer Center Hospital of Japman between 2007 and 2020 were reviewed. Of these, we excluded 51 patients with benign tumor, and of the remaining 88 patients with malignant tumor, 41 patients who underwent surgical treatment after preoperative CNB were considered eligible for the analysis. We have been performing preoperative CNB as a first choice on all patients with suspected malignancy since 2017 to improve the accuracy of preoperative diagnosis. Patient characteristics are shown in Table 1. Median follow-up was 30 (range: 2–169) months. The eighth edition of the tumor–node–metastasis (TNM) classification from the International Union Against Cancer and the American Joint Committee on Cancer was used for clinical staging. Prognostic information was available for all patients. Ethics approval for retrospective biomarker analysis was obtained from our ethics committee (approval number, 2010-77). Ultrasound (US)-guided CNB and FNAC For CNB, we used a semi-automatic biopsy device (FINE CORE ® Needle, 20-gauge, length 55 mm, Dr. Japan Co., Ltd. Tokyo, Japan), which was developed under our advice. This device was developed specifically for performing biopsies of head and neck tumors. It was designed to have a short handle and be compact, lightweight, and easy to maneuver. All biopsies were performed under US guidance by head and neck surgeons (K.K., S.Y., G.O., M.F., or M.Y.). Using US, the surgeon confirmed tumor vascularity and ensured that the biopsy needle penetrated completely into the tumor (Fig. 1 ). In solid-cystic lesions, a biopsy of the solid part was performed. To prevent shrinkage, biopsy specimens were attached to the edges of filter paper cut into 2 x 1 cm pieces and fixed with 10% formalin. To prevent residual tumor from puncture seeding, the surgical skin incision was designed to include the biopsy scar (Fig. 2 ). Regardless of tumor localization, the puncture point was set on the expected skin incision line. Pathologic diagnosis As a preoperative histologic typing platform for CNB specimens of salivary gland carcinoma, in addition to the conventional morphologic evaluation, immunohistochemical evaluation of SOX10, Epithelial membrane antigen (EMA)/Mucin 1 (MUC1), c-kit, p63, Androgen receptor (AR), and Human epidermal growth factor type 2 (HER2); fluorescent in situ hybridization (FISH) analysis of MYB , MAML2 ,and ETV6 ; and dual color in situ hybridization (DISH) analysis of HER2 were performed. SOX10, EMA/MUC1, c-kit, p63, AR, and HER2 were ordered reflexively on all biopsy specimens. Immunostaining for other biomarkers, FISH, and DISH were ordered based on initial pathologic findings within 4 days, with considerations of cost effectiveness. All preoperative diagnoses were performed by 2 of our pathologists (T.M. and Y.Y.) within 8 days of specimen submission. For surgical specimens, the entire tumor was sliced into many sections and thoroughly sampled for detailed evaluation of tumor components and resection margins. Immunostaining for SOX10, EMA/MUC1, c-kit, p63, AR, and HER2 was re-evaluated in surgical specimens and compared as paired samples. Histology and immunohistochemistry (IHC) Histologic examination was performed on 4-µm–thick sections cut from formalin-fixed paraffin-embedded (FFPE) tissue and stained with hematoxylin and eosin. A solid pattern of 30% or more was used to define the cutoff for solid type adenoid cystic carcinoma (AdCC) ( 2 ) . Immunohistochemical staining was performed on FFPE specimens. Antigen retrieval was performed using Target Retrieval Solution pH 9 (Agilent Technologies, Santa Clara, California, USA). All primary antibodies used are summarized in Supplementary Table 1. Nuclear staining of > 10% tumor cells was considered positive for SOX10, p63, and AR, respectively ( 7 ) . Cytoplasmic staining with circumferential membranous accentuation in > 10% tumor cells was considered positive for EMA/MUC1 ( 8 ) . Cytoplasmic or membranous diffuse staining of > 25% tumor cells was considered positive for c-kit ( 9 ) . HER2 status HER2 status was assessed based on IHC and DISH. Staining intensity was classified as 0 (no immunoreactivity), 1+ (incomplete membrane staining in > 10% of tumor cells), 2+ (weak to moderate complete membrane staining in > 10% of tumor cells), or 3+ (strong immunoreactivity in > 10% of tumor cells) based on the guidelines for HER2 assessment in breast cancer ( 10 , 11 ) . The DISH assay for HER2 was performed on a Ventana BenchMark series instrument (Ventana/Roche, Oro Valley, Arizona, USA). HER2 (labeled with spectrum black) and chromosome 17 (CHR17) reference gene signals (labeled with spectrum red) were enumerated with conventional bright-field microscopy after hematoxylin counterstaining. HER2:CHR17 ratio ≥ 2.0 was defined as HER2 DISH +. IHC 3 + or IHC 2+/DISH + was considered to indicate HER2 amplification. FISH FISH was performed on 4-µm–thick FFPE tissue sections from relevant areas for analysis identified by a pathologist (T.M.) and prepared using a standard protocol for probe application. Probes are listed in Supplementary Table 2. FISH images were captured using the Metafer Slide Scanning Platform (MetaSystems, Altlussheim, Germany); 200 non-overlapping tumor nuclei with at least one of each signal were examined. Tumors with > 20% nuclei with fused or split rearrangement signals, were deemed rearrangement positive. Statistical analysis Differences between any two groups were analyzed using Fisher’s exact test. R and its command input platform, ezR, were used for analysis ( 12 ) . A value of P < 0.05 was considered to be statistically significant. Results Details of preoperative histologic typing by CNB In the 41 patients who underwent CNB, 95% (39 patients) had a high-quality specimen that enabled adequate morphologic, immunohistologic, and genomic analysis (Fig. 3 ). In two patients with high-grade mucoepidermoid carcinoma and squamous cell carcinoma, respectively, analyzable specimens were not available due to cystic fluid or tumor firmness. The results of the histologic typing analysis are shown in Fig. 4 . In total, 75% (31/41) had a correct preoperative histologic typing. It was possible to successfully determine the histologic type for even solid type AdCC. However, carcinoma ex pleomorphic adenoma (CXPA) and salivary duct carcinoma arising from CXPA were significantly less able to determine the histologic type than other histologic types (50% vs 89%, p = 0.0164) (Table 2). Concordance of immunostaining in paired CNB and surgical specimens The immunostaining findings in paired CNB and surgical specimens of all 41 patients are shown in Fig. 4 . Concordance between CNB and surgical specimen results for SOX10, EMA/MUC1, c-kit, p63, AR, and HER2 immunostaining were 94% (37/39), 97% (38/39), 94% (37/39), 94% (37/39), 94% (37/39), and 82% (32/39), respectively. CXPA and salivary duct carcinoma arising from CXPA had significantly more HER2 IHC discordance than other histologic types (70% vs 0%, p = 7.80E-06) (Table 2). Tumors with discordant HER2 IHC status had heterogenous HER2 expression (Fig. 3 ). Safety and adverse events of CNB There were no cases of hospitalization or emergency consultation due to hematoma or facial nerve palsy after CNB. The clinicopathologic information of all 41 patients are shown in Fig. 4 . In total, there was one case of local recurrence, however, no recurrence due to tumor seeding by needle puncture was observed. Discussion In salivary gland carcinoma, it is critical to accurately assess preoperative biopsy specimens morphologically, immunohistologically, and genomically to determine histologic type. We performed a retrospective analysis of 41 patients with salivary gland carcinoma treated at a single institution with a uniform treatment strategy and diagnostic method. We found the following: 1) detailed analysis of CNB specimens can determine the histologic type with high accuracy; and 2) it is challenging to determine the histologic type of heterogeneous tumors such as CXPA from small CNB specimens. Most studies of histologic typing with CNB have focused on benign tumors; few studies have evaluated malignant tumors. In general, malignant tumors are more difficult to classify histologically than benign tumors. Walsh et al. reported a 69% histologic typing rate for 29 malignant lesions based on needle biopsy ( 13 ) . Novoa et al. performed CNB on 22 malignant salivary gland tumors and successfully histologically classified 17 (77%) of them ( 14 ) . In this study, we established an efficient preoperative histologic typing platform for salivary gland carcinoma based on CNB. This platform consists of six immunohistologic markers: SOX10, EMA/MUC1, c-kit, p63, AR, and HER2. First, SOX10 clearly differentiated between tumors originating from the acini or intercalated ducts and tumors originating from the striated or excretory ducts ( 7 ) . Second, c-kit and p63 allow for the evaluation of luminal and abluminal cells of biphasic glandular structures, respectively. Third, AR and HER2 are available to evaluate the expression of hormone receptors. Fourth, EMA/MUC1 enables evaluation of glandular differentiation, which is useful for validation of SOX10 status, due to the mutually exclusive relationship between EMA/MUC1 and SOX10. In addition, molecular biology evaluations of gene fusions and amplifications can be performed with FISH and DISH. Due to reliable tumor sampling using US, detailed immunohistologic analysis, and incorporation of genetic information from FISH and DISH, we successfully achieved a favorable histologic typing rate of 75%. This result is consistent with previous reports. It is remarkable that immunohistological and genomic evaluations were possible with a 20-gauge needle. Sample quality and intratumoral heterogeneity affect histologic typing based on CNB. There were two specimens of inadequate quality in this study: a cystic tumor with a predominantly cystic fluid component and a very clinically firm tumor. Histologically, firm tumor had abundant fibrotic tissue with sclerosis. It is difficult to obtain sufficient tumor samples from such cystic or firm tumors. Another factor that interferes with histologic typing is intratumoral heterogeneity ( 15 ) . In this study, CXPA, which is histologically highly heterogenous, had a low concordance rate for HER2 immunostaining and histologic type between CNB and surgical specimens. The impact of intratumor heterogeneity on concordance of biomarker profiles between CNB and surgical specimens has been well examined in the field of breast cancer ( 15 – 17 ) . Concordance between CNB and surgical specimen for estrogen receptor, progesterone receptor, HER2, and Ki67 immunostaining was reported to be 94.1%, 88.1%, 84.5%, and 88.1%, respectively ( 16 , 17 ) . Repeated HER2 testing with immunohistochemical staining or FISH of surgical specimens is considered reasonable, especially when the surgical specimens has tumor characteristics and morphology that differ from those found in CNB specimens ( 18 ) . In salivary gland carcinoma, HER2 assessment is also considered appropriate for both CNB and surgical specimens in highly histologically heterogeneous tumors such as CXPA ( 15 – 17 ) . There are concerns that CNB poses a higher risk of complications than FNAC because it is more invasive. Hematoma is uncommon with both: 0.03% for cytology and 0.6% for CNB. Facial nerve palsy occurred in 0.075% of patients who underwent CNB, but was reported to be temporary due to local anesthesia ( 5 , 19 ) . CNB might pose a greater risk of tumor seeding due to the larger gauge of the needle used ( 20 , 21 ) . Previous reports showed that tumor seeding occurred in 0.01–0.00012% of FNACs and 0.1–0.001% of CNBs ( 21 ) . An increased risk of tumor displacement has been reported with the use of 18-gauge or larger needles ( 5 , 21 ) . In this study, we used a 20-gauge needle to minimize the risk of tumor seeding. We did not see tumor seeding after CNB in our limited number of cases. However, long-term safety for low-grade carcinoma was not fully evaluated. We made every effort to reduce dissemination, including excision of the biopsy scar at the time of surgery or multiple biopsies from the same skin puncture wound. The main limitation of this study is selection bias due to its retrospective nature. Moreover, due to the relatively rarity of salivary gland carcinoma, a large cohort study was not possible. However, the exclusion criteria and treatment strategy were consistent and pathologic information on biopsy or surgical specimens was well annotated with clinical information. All patients with salivary gland carcinoma treated during the study period at a single institution were examined without exception. Notwithstanding, further research is warranted to verify the findings of this study. Conclusion In conclusion, detailed analysis of 20-gauge needle biopsy specimens allows for highly accurate determination of salivary gland carcinoma histologic type. However, careful histologic typing is necessary in pathologically heterogeneous tumors. Declarations Funding This study was founded by Japan Society for the Promotion of Science (JSPS) KAKENHI grants to KK (22K09662) and TM (23K06476). Conflict of Interest The authors declare that they have no conflict of interest. Ethical approval This study was approved by the ethical review board of the National Cancer Center Hospital (approval number, 2010-77). All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Consent to Participate Informed consent was obtained from all individual participants included in the study. Consent for publication Consent for publication was obtained for every individual person’s data included in the study. Availability of data and materials The datasets used and/or analyzed during the current study are available for the corresponding author on reasonable request. Code Availability Not Applicable. Authors’ contribution Conceived and designed the work: K.K. T.M. Provision of study material or patients: K.K., S.Y., G.O., Y.M., A.S., K.E., T.S., Y.H., F.M. Pathological analysis and FISH probe preparation: T.M., E.R., A.Y., Y.Y. Data analysis and interpretation: K.K., T.M, M.K. Statistical analysis: K.K. Manuscript writing: K.K. T.M. Revised the manuscript: T.M., S.Y., G.O., Y.S., M.K. Approved final version: T.M. All authors red and approved the final manuscript. Acknowledgements Not applicable. References Bishop JA. Proceedings of the north America society of head and neck pathology, los angeles, ca march 20, 2022: Emerging entities in salivary gland tumor pathology. Head and Neck Pathol. 2022;16:179 – 89. Justin AB, Lester DRT, Paul EW, Ilan W. Tumors of the salivary glands. AFIP atlas of tumor and non-tumor pathology. Volume 5. Rockville, MD: American Registry of Pathology; 2021. Kobayashi K, Saito Y, Kage H, Fukuoka O, Yamamura K, Mukai T, Oda K, Yamasoba T. CDK12 alterations and ARID1A mutations are predictors of poor prognosis and therapeutic targets in high-grade salivary gland carcinoma: analysis od the National Genomic Profiling Databese. Jpn J Clin Oncol. 2023;53(9):798–807. Birgin E, Yang C, Hetjens S, Reissfelder C, Hohenberger P, Rahbari NN. Core needle biopsy versus incisional biopsy for differentiation of soft-tissue sarcomas: a systematic review and meta-analysis. Cancer. 2020;126(9):1917–28. Hurry KJ, Karunaratene D, Westley S, Booth A, Ramesar KCRB, Zhang TT, Williams M, Howlett DC. Ultrasound-guided core biopsy in the diagnosis of parotid neoplasia: an overview an update with a review of the literature. Br J Radiol. 2022;95(1130):20210972. Orzco JIJ, Chang SC, Matsuba C, Ensenyat-Mendez M, Grunkemeier GL, Marzese DM, et al. Is the 221-Gene Recurrence score on core needle biopsy equivalent to surgical specimen in early-stage breast cancer? A comparison of gene expression between paired core needle biopsy and surgical specimens. Ann Surg Oncol. 2021;28(10):5588–96. Ohtomo R, Mori T, Shibata S, Tsuta K, Maeshima AM, Akazawa C et al. SOX10 is a novel marker of acinus and intercalated duct differentiation in salivary gland tumors: a clue to the histogenesis for tumor diagnosis. Mod Pathol. Hardra-Luca A, Lamas G, Bertrand JC, Fouret P. MUC1, MUC2, MUC4, MUC5AC expression in salivary gland mucoepidermoid carcinoma: diagnostic and prognostic implications. Am J Surg Pathol. 2005;29(7):881–9. Wong SJ, Karrison T, Hayes DN, Kies MS, Cullen KJ, Tanvetyanon T, et al. Phase II trial of dastinib for recurrent or metastatic c-KIT expressing adenoid cystic carcinoma and for nonadenoid cystic malignant salivary tumors. Ann Oncol. 2016;27(2):318–23. Wolff AC, Hammond ME, Hicks DG, et al. Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update. Arch Pathol Lab Med. 2014;138(2):241–56. Takahashi H, Tada Y, Saotome T, Akazawa K, Ojiri H, Fushimi C, et al. Phase II trial of trastuzumab and docetaxel in patients with human epidermal growth factor receptor 2-positive salivary duct carcinoma. J Clin Oncol. 2019;37(2):125–34. Kanda Y. Investigation of the freely available essy-to-use software ‘EZR’ for medical statistics. Bone Marrow Transpl. 2013;48(3):452–8. Walsh E, Allan K, Brennan PA, Tullett M, Gomez RS, Rahimi S. Diagnostic accuracy of ultrasonography-guided core needle biopsy of parotid gland neoplasms: A large, single-institution experience in united kingdom. J Oral Pathol Med. 2022;51(1):1–4. 14Novoa E, Gurtler N, Arnoux A, Kraft M. Diagnostic value of core needle biopsy and fine-needle aspiration in salivary gland lesions. Head Neck. 2016;38(suppl1):E346–52. Apple SK, Lowe AC, Rao PN, Shintaku IP, Moatamed NA. Comparison of fluorescent on situ hybridization HER-2/neu results on core needle biopsy and excisional biopsy in primary breast cancer. Mod Pathol. 2009;22(9):1151–9. Meattini I, Bicchierai G, Saieva C, De Benedetto D, Desideri I, Becherini C et al. Impact of molecular subtypes classification concordance between preoperative core needle biopsy and surgical specimen on early breast cancer management: Single-institution experience and revies of published literature. Eur J Surg Oncol. 2017(4);43:642–48. Slostad JA, Yun NK, Schad AE, Warrior S, Fogg LF, Rao R, et al. Concordance of breast cancer biomarker testing in core needle biopsy and surgical specimens: a single institution experience. Cancer Med. 2022;11(24):4954–65. Apple SK, Lowe AC, Rao PN, Shintaku IP, Moatamed NA. Comparison of fluorescent in situ hybridization HER-2/neu results on core needle biopsy and excisional biopsy in primary breast cancer. Mod Pathol. 2009;22(9):1151–9. Cho J, Kim J, Lee JS, Chee CG, Kim Y, Choi SI. Comparison of core needle biopsy and fine-needle aspiration in diagnosis of malignant salivary gland neoplasm: Systematic review and meta-analysis. Head Neck. 2020;42(10):3041–50. Roussel F, Nouvet G. Evaluation of large-needle biopsy for the diagnosis of cancer. Acta Cytol. 1995;39(3):449–52. Shah KS, Ethunandan M. Tumor seeding after fine-needle aspiration and core biopsy of the head and neck—a systematic review. Br J Oral Maxillofac Surg. 2016;54(3):260–5. Additional Declarations No competing interests reported. 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Moti","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtElEQVRIiWNgGAWjYNACAxsYi42wYh6IljSStTAcJsFJ9uynEx9XFJzP428/wPziAwNfHmFbeHI3G54xuF0scSaBzXIGA1sxEQ7L3SbZYHA7cYMEA5sxDwNbYgNBLfxvQVrOkaJFAmzLAZAW5sfEabnxdrNhg0Fy4owziW2MMwyI8At7f+7Ghw1/7BL72w8f/vCh4hjhEEMCjG0SDAbHEkjRwsD8gYGhhjQto2AUjIJRMCIAAHzDN07bzkayAAAAAElFTkSuQmCC","orcid":"","institution":"National Cancer Center Hospital","correspondingAuthor":true,"prefix":"","firstName":"Taisuke","middleName":"","lastName":"Moti","suffix":""}],"badges":[],"createdAt":"2024-07-08 07:23:44","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4703440/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4703440/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":62156988,"identity":"8be53f44-4fce-46f3-a9c9-bf80378ac092","added_by":"auto","created_at":"2024-08-09 21:14:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1348044,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUltrasound(US)-guided core needle biopsy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. A semi-automatic biopsy device (FINECORE\u003csup\u003eⓇ\u003c/sup\u003e M type, 20-gauge, length 55 mm; Dr. Japan Co., Ltd.) was developed under our advice. To prevent shrinkage, biopsy specimens were attached to the edges of filter paper. B. Tumor located in the superficial lobe of the parotid gland. C. US-guided tumor puncture. D. The biopsy needle completely penetrated the tumor. E. US image after biopsy, with the biopsy scar remaining in the tumor.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4703440/v1/579de520a9a193d7766d0604.png"},{"id":62157728,"identity":"7560f86e-be97-4f89-9151-ded9d5f16fb4","added_by":"auto","created_at":"2024-08-09 21:22:20","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":203063,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSkin incision line including biopsy scar,\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Submandibular gland tumor was located under the supposed skin incision line. The puncture point was set just above the tumor. B. Parotid tumor was located away from the supposed skin incision line. The puncture point was set on the skin incision line. The biopsy scar was resected in a spindle shape and primary sutured. Arrow head: Shape of the tumor from surface. Arrow: Spindle shaped skin resection of biopsy scar.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4703440/v1/b574f8c3e87e0612135057f2.jpg"},{"id":62156990,"identity":"ca26e316-4bc4-40d5-a895-e5f0e2065dcb","added_by":"auto","created_at":"2024-08-09 21:14:20","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":811821,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHE staining, IHC staining, FISH, and DISH of core needle biopsy specimens\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImages from core needle biopsies of adenoid cystic carcinoma (A–D) and salivary duct carcinoma (E–G). A. Typical core needle biopsy specimen of a partially fragmented cribriform lesion under low power. B C. Under high power, the tumor had a biphasic structure of c-kit positive ductal cells and p63 positive myoepithelial cells. D. FISH showed nuclei with positive \u003cem\u003eMYB\u003c/em\u003ebreak-apart. E F. HE staining of a salivary duct carcinoma arising from CXPA with intratumor heterogeneity in HER2 staining in the same section. G. Core needle biopsy specimens showing HER2 gene amplification based on HER2 DISH. Black and red signals represent HER2 and CHR17, respectively.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4703440/v1/6963835fe73cfe017ab15d82.jpg"},{"id":62156993,"identity":"8786a2e2-5a19-44e6-9048-c8468cd3185d","added_by":"auto","created_at":"2024-08-09 21:14:20","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":884771,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eClinicopathologic and genomic landscape of 41 salivary gland carcinomas.\u003c/strong\u003e In 41 patients who underwent CNB, specimens were high-quality with adequate morphologic, immunohistologic, and genomic analysis in all except in 2 patients; histologic typing was possible for 75% (top line, bright blue). SOX10 (5\u003csup\u003eth\u003c/sup\u003e line, burgundy) clearly defined two distinct subtypes of salivary gland carcinoma. Tumors with maintained biphasic structure such as non-solid type adenoid cystic carcinoma were positive for both c-kit and p63. HER2 IHC discordance is prominent in CXPA.\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4703440/v1/bab036b828c5ad52ce9c594b.jpg"},{"id":62157729,"identity":"e290231c-9cbb-468d-b09d-efe766f0c3ae","added_by":"auto","created_at":"2024-08-09 21:22:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3689935,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4703440/v1/91be44ef-82d5-41d2-b084-b437e7e067e6.pdf"},{"id":62157727,"identity":"ad37b327-8eb6-415f-86c4-3f919264fcd1","added_by":"auto","created_at":"2024-08-09 21:22:20","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":15029,"visible":true,"origin":"","legend":"","description":"","filename":"SupplyTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4703440/v1/e0b78b497e0a52376ea44ceb.docx"},{"id":62156991,"identity":"a68326ce-489a-485a-9c39-99c58d76850a","added_by":"auto","created_at":"2024-08-09 21:14:20","extension":"docx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":16544,"visible":true,"origin":"","legend":"","description":"","filename":"SupplyTable2.docx","url":"https://assets-eu.researchsquare.com/files/rs-4703440/v1/a5588c6b945595aff468454a.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Establishment of an efficient pathologic diagnostic platform using core needle biopsy for salivary gland carcinoma","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBecause of the diversity in salivary gland carcinoma histopathologic types, pathologic diagnosis is notoriously difficult \u003csup\u003e(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e)\u003c/sup\u003e. Moreover, in recent years, as the molecular biology of salivary gland carcinoma has become elucidated, genomic information such as fusion genes and gene amplification have been used for pathologic diagnosis in addition to morphologic and immunohistologic information \u003csup\u003e(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCore needle biopsy (CNB) allows a small piece of the center of the tumor to be taken intact and all of its components to be analyzed in detail \u003csup\u003e(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e)\u003c/sup\u003e. In addition, immunohistologic status and genomic information such as fusion genes and gene amplification can be extracted, which is expected to dramatically improve diagnostic performance \u003csup\u003e(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe histologic heterogeneity of salivary gland carcinoma is due to the complexity of salivary gland structures \u003csup\u003e(2)\u003c/sup\u003e. Histogenesis-based diagnostic markers are useful for systematic and effective histologic typing. Our previous reports showed that SRY-related HMG-box10 (SOX10) expression can clearly define two distinct subtypes of salivary gland tumors based on their anatomical site of origin \u003csup\u003e(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e)\u003c/sup\u003e. To make needle biopsies for the diagnosis of salivary gland cancer more widespread, establishing an efficient pathologic diagnostic platform that uses several immunohistologic markers based on the origin and characteristics of the tumor is urgently needed. It is also necessary to identify problems and limitations in pathological diagnosis using needle biopsy specimens.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy design and setting\u003c/h2\u003e \u003cp\u003eIn this retrospective study, medical records, biopsy specimens, and surgical specimens of 139 patients with parotid gland and submandibular gland tumor who underwent primary surgical treatment at the National Cancer Center Hospital of Japman between 2007 and 2020 were reviewed. Of these, we excluded 51 patients with benign tumor, and of the remaining 88 patients with malignant tumor, 41 patients who underwent surgical treatment after preoperative CNB were considered eligible for the analysis. We have been performing preoperative CNB as a first choice on all patients with suspected malignancy since 2017 to improve the accuracy of preoperative diagnosis.\u003c/p\u003e \u003cp\u003ePatient characteristics are shown in Table\u0026nbsp;1. Median follow-up was 30 (range: 2\u0026ndash;169) months. The eighth edition of the tumor\u0026ndash;node\u0026ndash;metastasis (TNM) classification from the International Union Against Cancer and the American Joint Committee on Cancer was used for clinical staging. Prognostic information was available for all patients. Ethics approval for retrospective biomarker analysis was obtained from our ethics committee (approval number, 2010-77).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eUltrasound (US)-guided CNB and FNAC\u003c/h2\u003e \u003cp\u003eFor CNB, we used a semi-automatic biopsy device (FINE CORE\u003csup\u003e\u0026reg;\u003c/sup\u003e Needle, 20-gauge, length 55 mm, Dr. Japan Co., Ltd. Tokyo, Japan), which was developed under our advice. This device was developed specifically for performing biopsies of head and neck tumors. It was designed to have a short handle and be compact, lightweight, and easy to maneuver. All biopsies were performed under US guidance by head and neck surgeons (K.K., S.Y., G.O., M.F., or M.Y.). Using US, the surgeon confirmed tumor vascularity and ensured that the biopsy needle penetrated completely into the tumor (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In solid-cystic lesions, a biopsy of the solid part was performed. To prevent shrinkage, biopsy specimens were attached to the edges of filter paper cut into 2 x 1 cm pieces and fixed with 10% formalin. To prevent residual tumor from puncture seeding, the surgical skin incision was designed to include the biopsy scar (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Regardless of tumor localization, the puncture point was set on the expected skin incision line.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePathologic diagnosis\u003c/h2\u003e \u003cp\u003eAs a preoperative histologic typing platform for CNB specimens of salivary gland carcinoma, in addition to the conventional morphologic evaluation, immunohistochemical evaluation of SOX10, Epithelial membrane antigen (EMA)/Mucin 1 (MUC1), c-kit, p63, Androgen receptor (AR), and Human epidermal growth factor type 2 (HER2); fluorescent in situ hybridization (FISH) analysis of \u003cem\u003eMYB\u003c/em\u003e, \u003cem\u003eMAML2\u003c/em\u003e,and \u003cem\u003eETV6\u003c/em\u003e; and dual color in situ hybridization (DISH) analysis of HER2 were performed. SOX10, EMA/MUC1, c-kit, p63, AR, and HER2 were ordered reflexively on all biopsy specimens. Immunostaining for other biomarkers, FISH, and DISH were ordered based on initial pathologic findings within 4 days, with considerations of cost effectiveness. All preoperative diagnoses were performed by 2 of our pathologists (T.M. and Y.Y.) within 8 days of specimen submission.\u003c/p\u003e \u003cp\u003eFor surgical specimens, the entire tumor was sliced into many sections and thoroughly sampled for detailed evaluation of tumor components and resection margins. Immunostaining for SOX10, EMA/MUC1, c-kit, p63, AR, and HER2 was re-evaluated in surgical specimens and compared as paired samples.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eHistology and immunohistochemistry (IHC)\u003c/h2\u003e \u003cp\u003eHistologic examination was performed on 4-\u0026micro;m\u0026ndash;thick sections cut from formalin-fixed paraffin-embedded (FFPE) tissue and stained with hematoxylin and eosin. A solid pattern of 30% or more was used to define the cutoff for solid type adenoid cystic carcinoma (AdCC) \u003csup\u003e(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e)\u003c/sup\u003e. Immunohistochemical staining was performed on FFPE specimens. Antigen retrieval was performed using Target Retrieval Solution pH 9 (Agilent Technologies, Santa Clara, California, USA). All primary antibodies used are summarized in Supplementary Table\u0026nbsp;1. Nuclear staining of \u0026gt;\u0026thinsp;10% tumor cells was considered positive for SOX10, p63, and AR, respectively \u003csup\u003e(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e)\u003c/sup\u003e. Cytoplasmic staining with circumferential membranous accentuation in \u0026gt;\u0026thinsp;10% tumor cells was considered positive for EMA/MUC1 \u003csup\u003e(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e)\u003c/sup\u003e. Cytoplasmic or membranous diffuse staining of \u0026gt;\u0026thinsp;25% tumor cells was considered positive for c-kit \u003csup\u003e(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eHER2 status\u003c/h2\u003e \u003cp\u003eHER2 status was assessed based on IHC and DISH. Staining intensity was classified as 0 (no immunoreactivity), 1+ (incomplete membrane staining in \u0026gt;\u0026thinsp;10% of tumor cells), 2+ (weak to moderate complete membrane staining in \u0026gt;\u0026thinsp;10% of tumor cells), or 3+ (strong immunoreactivity in \u0026gt;\u0026thinsp;10% of tumor cells) based on the guidelines for HER2 assessment in breast cancer \u003csup\u003e(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e)\u003c/sup\u003e. The DISH assay for HER2 was performed on a Ventana BenchMark series instrument (Ventana/Roche, Oro Valley, Arizona, USA). \u003cem\u003eHER2\u003c/em\u003e (labeled with spectrum black) and chromosome 17 (CHR17) reference gene signals (labeled with spectrum red) were enumerated with conventional bright-field microscopy after hematoxylin counterstaining. HER2:CHR17 ratio\u0026thinsp;\u0026ge;\u0026thinsp;2.0 was defined as HER2 DISH +. IHC 3\u0026thinsp;+\u0026thinsp;or IHC 2+/DISH\u0026thinsp;+\u0026thinsp;was considered to indicate HER2 amplification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eFISH\u003c/h2\u003e \u003cp\u003eFISH was performed on 4-\u0026micro;m\u0026ndash;thick FFPE tissue sections from relevant areas for analysis identified by a pathologist (T.M.) and prepared using a standard protocol for probe application. Probes are listed in Supplementary Table\u0026nbsp;2. FISH images were captured using the Metafer Slide Scanning Platform (MetaSystems, Altlussheim, Germany); 200 non-overlapping tumor nuclei with at least one of each signal were examined. Tumors with \u0026gt;\u0026thinsp;20% nuclei with fused or split rearrangement signals, were deemed rearrangement positive.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eDifferences between any two groups were analyzed using Fisher\u0026rsquo;s exact test. R and its command input platform, ezR, were used for analysis \u003csup\u003e(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e)\u003c/sup\u003e. A value of \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to be statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDetails of preoperative histologic typing by CNB\u003c/h2\u003e \u003cp\u003eIn the 41 patients who underwent CNB, 95% (39 patients) had a high-quality specimen that enabled adequate morphologic, immunohistologic, and genomic analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In two patients with high-grade mucoepidermoid carcinoma and squamous cell carcinoma, respectively, analyzable specimens were not available due to cystic fluid or tumor firmness. The results of the histologic typing analysis are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eIn total, 75% (31/41) had a correct preoperative histologic typing. It was possible to successfully determine the histologic type for even solid type AdCC. However, carcinoma ex pleomorphic adenoma (CXPA) and salivary duct carcinoma arising from CXPA were significantly less able to determine the histologic type than other histologic types (50% vs 89%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0164) (Table\u0026nbsp;2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eConcordance of immunostaining in paired CNB and surgical specimens\u003c/h2\u003e \u003cp\u003eThe immunostaining findings in paired CNB and surgical specimens of all 41 patients are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Concordance between CNB and surgical specimen results for SOX10, EMA/MUC1, c-kit, p63, AR, and HER2 immunostaining were 94% (37/39), 97% (38/39), 94% (37/39), 94% (37/39), 94% (37/39), and 82% (32/39), respectively. CXPA and salivary duct carcinoma arising from CXPA had significantly more HER2 IHC discordance than other histologic types (70% vs 0%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.80E-06) (Table\u0026nbsp;2). Tumors with discordant HER2 IHC status had heterogenous HER2 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSafety and adverse events of CNB\u003c/h2\u003e \u003cp\u003eThere were no cases of hospitalization or emergency consultation due to hematoma or facial nerve palsy after CNB. The clinicopathologic information of all 41 patients are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. In total, there was one case of local recurrence, however, no recurrence due to tumor seeding by needle puncture was observed.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn salivary gland carcinoma, it is critical to accurately assess preoperative biopsy specimens morphologically, immunohistologically, and genomically to determine histologic type. We performed a retrospective analysis of 41 patients with salivary gland carcinoma treated at a single institution with a uniform treatment strategy and diagnostic method. We found the following: 1) detailed analysis of CNB specimens can determine the histologic type with high accuracy; and 2) it is challenging to determine the histologic type of heterogeneous tumors such as CXPA from small CNB specimens.\u003c/p\u003e \u003cp\u003eMost studies of histologic typing with CNB have focused on benign tumors; few studies have evaluated malignant tumors. In general, malignant tumors are more difficult to classify histologically than benign tumors. Walsh et al. reported a 69% histologic typing rate for 29 malignant lesions based on needle biopsy \u003csup\u003e(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e)\u003c/sup\u003e. Novoa et al. performed CNB on 22 malignant salivary gland tumors and successfully histologically classified 17 (77%) of them \u003csup\u003e(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e)\u003c/sup\u003e. In this study, we established an efficient preoperative histologic typing platform for salivary gland carcinoma based on CNB. This platform consists of six immunohistologic markers: SOX10, EMA/MUC1, c-kit, p63, AR, and HER2. First, SOX10 clearly differentiated between tumors originating from the acini or intercalated ducts and tumors originating from the striated or excretory ducts \u003csup\u003e(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e)\u003c/sup\u003e. Second, c-kit and p63 allow for the evaluation of luminal and abluminal cells of biphasic glandular structures, respectively. Third, AR and HER2 are available to evaluate the expression of hormone receptors. Fourth, EMA/MUC1 enables evaluation of glandular differentiation, which is useful for validation of SOX10 status, due to the mutually exclusive relationship between EMA/MUC1 and SOX10. In addition, molecular biology evaluations of gene fusions and amplifications can be performed with FISH and DISH. Due to reliable tumor sampling using US, detailed immunohistologic analysis, and incorporation of genetic information from FISH and DISH, we successfully achieved a favorable histologic typing rate of 75%. This result is consistent with previous reports. It is remarkable that immunohistological and genomic evaluations were possible with a 20-gauge needle.\u003c/p\u003e \u003cp\u003eSample quality and intratumoral heterogeneity affect histologic typing based on CNB. There were two specimens of inadequate quality in this study: a cystic tumor with a predominantly cystic fluid component and a very clinically firm tumor. Histologically, firm tumor had abundant fibrotic tissue with sclerosis. It is difficult to obtain sufficient tumor samples from such cystic or firm tumors.\u003c/p\u003e \u003cp\u003eAnother factor that interferes with histologic typing is intratumoral heterogeneity \u003csup\u003e(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e)\u003c/sup\u003e. In this study, CXPA, which is histologically highly heterogenous, had a low concordance rate for HER2 immunostaining and histologic type between CNB and surgical specimens. The impact of intratumor heterogeneity on concordance of biomarker profiles between CNB and surgical specimens has been well examined in the field of breast cancer \u003csup\u003e(\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e)\u003c/sup\u003e. Concordance between CNB and surgical specimen for estrogen receptor, progesterone receptor, HER2, and Ki67 immunostaining was reported to be 94.1%, 88.1%, 84.5%, and 88.1%, respectively \u003csup\u003e(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e)\u003c/sup\u003e. Repeated HER2 testing with immunohistochemical staining or FISH of surgical specimens is considered reasonable, especially when the surgical specimens has tumor characteristics and morphology that differ from those found in CNB specimens \u003csup\u003e(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e)\u003c/sup\u003e. In salivary gland carcinoma, HER2 assessment is also considered appropriate for both CNB and surgical specimens in highly histologically heterogeneous tumors such as CXPA \u003csup\u003e(\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThere are concerns that CNB poses a higher risk of complications than FNAC because it is more invasive. Hematoma is uncommon with both: 0.03% for cytology and 0.6% for CNB. Facial nerve palsy occurred in 0.075% of patients who underwent CNB, but was reported to be temporary due to local anesthesia \u003csup\u003e(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e)\u003c/sup\u003e. CNB might pose a greater risk of tumor seeding due to the larger gauge of the needle used\u003csup\u003e(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e)\u003c/sup\u003e. Previous reports showed that tumor seeding occurred in 0.01\u0026ndash;0.00012% of FNACs and 0.1\u0026ndash;0.001% of CNBs \u003csup\u003e(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e)\u003c/sup\u003e. An increased risk of tumor displacement has been reported with the use of 18-gauge or larger needles \u003csup\u003e(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e)\u003c/sup\u003e. In this study, we used a 20-gauge needle to minimize the risk of tumor seeding. We did not see tumor seeding after CNB in our limited number of cases. However, long-term safety for low-grade carcinoma was not fully evaluated. We made every effort to reduce dissemination, including excision of the biopsy scar at the time of surgery or multiple biopsies from the same skin puncture wound.\u003c/p\u003e \u003cp\u003eThe main limitation of this study is selection bias due to its retrospective nature. Moreover, due to the relatively rarity of salivary gland carcinoma, a large cohort study was not possible. However, the exclusion criteria and treatment strategy were consistent and pathologic information on biopsy or surgical specimens was well annotated with clinical information. All patients with salivary gland carcinoma treated during the study period at a single institution were examined without exception. Notwithstanding, further research is warranted to verify the findings of this study.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, detailed analysis of 20-gauge needle biopsy specimens allows for highly accurate determination of salivary gland carcinoma histologic type. However, careful histologic typing is necessary in pathologically heterogeneous tumors.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis study was founded by Japan Society for the Promotion of Science (JSPS) KAKENHI grants to KK (22K09662) and TM (23K06476).\u003c/p\u003e\n\u003ch2\u003eConflict of Interest\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003ch2\u003eEthical approval\u003c/h2\u003e\n\u003cp\u003eThis study was approved by the ethical review board of the National Cancer Center Hospital (approval number, 2010-77). All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.\u003c/p\u003e\n\u003ch2\u003eConsent to Participate\u003c/h2\u003e\n\u003cp\u003eInformed consent was obtained from all individual participants included in the study.\u003c/p\u003e\n\u003ch2\u003eConsent for publication\u003c/h2\u003e\n\u003cp\u003eConsent for publication was obtained for every individual person\u0026rsquo;s data included in the study.\u003c/p\u003e\n\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available for the corresponding author on reasonable request.\u003c/p\u003e\n\u003ch2\u003eCode Availability\u003c/h2\u003e\n\u003cp\u003eNot Applicable.\u003c/p\u003e\n\u003ch2\u003eAuthors\u0026rsquo; contribution\u003c/h2\u003e\n\u003cp\u003eConceived and designed the work: K.K. T.M. Provision of study material or patients: K.K., S.Y., G.O., Y.M., A.S., K.E., T.S., Y.H., F.M. Pathological analysis and FISH probe preparation: T.M., E.R., A.Y., Y.Y. Data analysis and interpretation: K.K., T.M, M.K. Statistical analysis: K.K. Manuscript writing: K.K. T.M. Revised the manuscript: T.M., S.Y., G.O., Y.S., M.K. \u0026nbsp;Approved final version: T.M. All authors red and approved the final manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBishop JA. Proceedings of the north America society of head and neck pathology, los angeles, ca march 20, 2022: Emerging entities in salivary gland tumor pathology. Head and Neck Pathol. 2022;16:179\u0026thinsp;\u0026ndash;\u0026thinsp;89.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJustin AB, Lester DRT, Paul EW, Ilan W. Tumors of the salivary glands. AFIP atlas of tumor and non-tumor pathology. Volume 5. Rockville, MD: American Registry of Pathology; 2021.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKobayashi K, Saito Y, Kage H, Fukuoka O, Yamamura K, Mukai T, Oda K, Yamasoba T. CDK12 alterations and ARID1A mutations are predictors of poor prognosis and therapeutic targets in high-grade salivary gland carcinoma: analysis od the National Genomic Profiling Databese. Jpn J Clin Oncol. 2023;53(9):798\u0026ndash;807.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBirgin E, Yang C, Hetjens S, Reissfelder C, Hohenberger P, Rahbari NN. Core needle biopsy versus incisional biopsy for differentiation of soft-tissue sarcomas: a systematic review and meta-analysis. Cancer. 2020;126(9):1917\u0026ndash;28.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHurry KJ, Karunaratene D, Westley S, Booth A, Ramesar KCRB, Zhang TT, Williams M, Howlett DC. Ultrasound-guided core biopsy in the diagnosis of parotid neoplasia: an overview an update with a review of the literature. Br J Radiol. 2022;95(1130):20210972.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOrzco JIJ, Chang SC, Matsuba C, Ensenyat-Mendez M, Grunkemeier GL, Marzese DM, et al. Is the 221-Gene Recurrence score on core needle biopsy equivalent to surgical specimen in early-stage breast cancer? A comparison of gene expression between paired core needle biopsy and surgical specimens. Ann Surg Oncol. 2021;28(10):5588\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOhtomo R, Mori T, Shibata S, Tsuta K, Maeshima AM, Akazawa C et al. SOX10 is a novel marker of acinus and intercalated duct differentiation in salivary gland tumors: a clue to the histogenesis for tumor diagnosis. Mod Pathol.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHardra-Luca A, Lamas G, Bertrand JC, Fouret P. MUC1, MUC2, MUC4, MUC5AC expression in salivary gland mucoepidermoid carcinoma: diagnostic and prognostic implications. Am J Surg Pathol. 2005;29(7):881\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWong SJ, Karrison T, Hayes DN, Kies MS, Cullen KJ, Tanvetyanon T, et al. Phase II trial of dastinib for recurrent or metastatic c-KIT expressing adenoid cystic carcinoma and for nonadenoid cystic malignant salivary tumors. Ann Oncol. 2016;27(2):318\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWolff AC, Hammond ME, Hicks DG, et al. Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update. Arch Pathol Lab Med. 2014;138(2):241\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTakahashi H, Tada Y, Saotome T, Akazawa K, Ojiri H, Fushimi C, et al. Phase II trial of trastuzumab and docetaxel in patients with human epidermal growth factor receptor 2-positive salivary duct carcinoma. J Clin Oncol. 2019;37(2):125\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKanda Y. Investigation of the freely available essy-to-use software \u0026lsquo;EZR\u0026rsquo; for medical statistics. Bone Marrow Transpl. 2013;48(3):452\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWalsh E, Allan K, Brennan PA, Tullett M, Gomez RS, Rahimi S. Diagnostic accuracy of ultrasonography-guided core needle biopsy of parotid gland neoplasms: A large, single-institution experience in united kingdom. J Oral Pathol Med. 2022;51(1):1\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e14Novoa E, Gurtler N, Arnoux A, Kraft M. Diagnostic value of core needle biopsy and fine-needle aspiration in salivary gland lesions. Head Neck. 2016;38(suppl1):E346\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eApple SK, Lowe AC, Rao PN, Shintaku IP, Moatamed NA. Comparison of fluorescent on situ hybridization HER-2/neu results on core needle biopsy and excisional biopsy in primary breast cancer. Mod Pathol. 2009;22(9):1151\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeattini I, Bicchierai G, Saieva C, De Benedetto D, Desideri I, Becherini C et al. Impact of molecular subtypes classification concordance between preoperative core needle biopsy and surgical specimen on early breast cancer management: Single-institution experience and revies of published literature. Eur J Surg Oncol. 2017(4);43:642\u0026ndash;48.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSlostad JA, Yun NK, Schad AE, Warrior S, Fogg LF, Rao R, et al. Concordance of breast cancer biomarker testing in core needle biopsy and surgical specimens: a single institution experience. Cancer Med. 2022;11(24):4954\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eApple SK, Lowe AC, Rao PN, Shintaku IP, Moatamed NA. Comparison of fluorescent in situ hybridization HER-2/neu results on core needle biopsy and excisional biopsy in primary breast cancer. Mod Pathol. 2009;22(9):1151\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCho J, Kim J, Lee JS, Chee CG, Kim Y, Choi SI. Comparison of core needle biopsy and fine-needle aspiration in diagnosis of malignant salivary gland neoplasm: Systematic review and meta-analysis. Head Neck. 2020;42(10):3041\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoussel F, Nouvet G. Evaluation of large-needle biopsy for the diagnosis of cancer. Acta Cytol. 1995;39(3):449\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShah KS, Ethunandan M. Tumor seeding after fine-needle aspiration and core biopsy of the head and neck\u0026mdash;a systematic review. Br J Oral Maxillofac Surg. 2016;54(3):260\u0026ndash;5.\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":"head-and-neck-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Head and Neck Pathology](https://www.springer.com/journal/12105)","snPcode":"12105","submissionUrl":"https://submission.springernature.com/new-submission/12105/3","title":"Head and Neck Pathology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"core needle biopsy, preoperative pathologic diagnosis, histologic type, Heterogeneity, SOX10","lastPublishedDoi":"10.21203/rs.3.rs-4703440/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4703440/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003ePurpose:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA major problem in establishing treatment strategies for salivary gland cancer is the difficulty of preoperative histologic typing. In recent years, genetic evaluation has become essential for salivary gland tumor diagnosis. The core needle biopsy (CNB) allows a small piece of the center of the tumor to be taken intact and analyzed in detail. The purpose of this study was to clarify the efficacy and the limitations of CNB in the preoperative diagnosis, and to establish a generalizable preoperative histologic typing platform.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003ePaired preoperative 20-gauge core needle biopsy (CNB) and surgical specimens from 41 patients with malignant salivary gland tumors were reviewed. Morphological evaluation, immunohistochemical evaluation, and break-apart fluorescence in situ hybridization (FISH) assay were performed as histologic typing methods for CNB. The quality of biopsy specimens, diagnostic accuracy, and immunostaining concordance rates between biopsy and surgical specimens were analyzed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eFor CNB, 95% (39/41) of the specimens were of high quality, allowing adequate morphologic, immunohistologic, and genomic analysis. Two patients had unanalyzable specimens due to cystic fluid or tumor firmness. Overall, 75% (31/41) had correct preoperative histologic typing. Compared to other histologic types, carcinoma ex pleomorphic adenoma (CXPA) and salivary duct carcinoma arising from CXPA had a significantly lower concordance rate for histologic typing (50% vs. 89.6%, p=0.016) and poorer HER2 immunostaining concordance rates between CNB and surgical specimens (60% vs. 0%, p=0.001). There were no recurrences due to tumor seeding after CNB.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eDetailed analysis of CNB specimens allows for highly accurate determination of salivary gland carcinoma histologic type with molecular diagnosis. However, careful histologic typing is necessary in pathologically heterogeneous tumors.\u003c/p\u003e","manuscriptTitle":"Establishment of an efficient pathologic diagnostic platform using core needle biopsy for salivary gland carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-09 21:14:15","doi":"10.21203/rs.3.rs-4703440/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-12T15:05:11+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-12T14:16:33+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-11T16:32:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"240857117922304031491394844048982223109","date":"2024-07-10T13:26:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"69308480652113537107458299555627617736","date":"2024-07-10T13:08:31+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-10T12:48:48+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-10T12:46:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-10T06:57:14+00:00","index":"","fulltext":""},{"type":"submitted","content":"Head and Neck Pathology","date":"2024-07-08T07:22:24+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"head-and-neck-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Head and Neck Pathology](https://www.springer.com/journal/12105)","snPcode":"12105","submissionUrl":"https://submission.springernature.com/new-submission/12105/3","title":"Head and Neck Pathology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e4f61667-ccd9-4304-8581-69d22df5139b","owner":[],"postedDate":"August 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-10-21T18:08:24+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-09 21:14:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4703440","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4703440","identity":"rs-4703440","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}