Metastatic Pheochromocytoma and Paraganglioma: Clinicopathological Analysis of 35 Cases

Metastatic Pheochromocytoma and Paraganglioma: Clinicopathological Analysis of 35 Cases | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Metastatic Pheochromocytoma and Paraganglioma: Clinicopathological Analysis of 35 Cases Noriko Kimura, TAKUYUKI KATABAMI, Tomoaki Tanaka, Kazuhiro Takekoshi, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9585500/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 Pheochromocytoma (PCC) and sympathetic paraganglioma (PGL), together PPGL are rare tumors of the adrenal medulla and extra-adrenal paraganglion, and 10–15% of PCC and 30–50% of PGL metastases. Histopathological analysis of metastatic PPGL has not been well defined. We analyzed 35 metastatic PPGLs based on clinicopathological data and the grading of adrenal pheochromocytoma and paraganglioma (GAPP) which is one of the tools for evaluating malignancy. We added succinate dehydrogenase type B (SDHB) immunohistochemistry with partly SDHB gene analysis and compared them to the patient prognosis. The mean age of patients was 34-year-old (range: 17–81). Mean tumor size was 8.6 cm. Synchronous metastasis was 18 (51.4%) cases, and metachronous metastasis occurred in 17 cases from 1 to 19 years (mean duration: 5 years) after first operation. Catecholamine types were 8 epinephrine, 16 norepinephrine, 6 norepinephrine plus dopamine, and 5 nonfunctional. Loss of SDHB-immunohistochemistry was observed in 43%. Ki67 labeling index was 11% in synchronous and 7% in metachronous metastasis (P < 0.05). Mean Ki67 was 6.0 in all and 14% in expired patients. GAPP score was 6 in all and 8.5 in expired patients. GAPP risk stratification was high in 47%, intermediate in 41%, and low in 12% in all patients, and high in 73%, intermediate in 27%, and low in 0% in expired patients. In conclusion, metastasis occurred in any type of PPGL. SDHB mutation was not confirmed as a major prognostic parameter in metastatic PPGL. Both higher Ki67 labeling index and higher GAPP score were histological indicators for poor prognosis. Biological sciences/Cancer Health sciences/Diseases Health sciences/Oncology pheochromocytoma and paraganglioma Ki67 GAPP SDHB prognosis mortality Figures Figure 1 Figure 2 Figure 3 Introduction Pheochromocytoma (PCC) and sympathetic paraganglioma (PGL), together with PPGL, are adrenal and extra-adrenal paraganglion derived rare tumors. The incidence of PPGL is approximately 4.6–6.6 cases per one million person-years [ 1 , 2 , 3 ]. Approximately 10 to 15% of PCC and up to 50% of PGL will exhibit metastatic behavior [ 4 ]. The 5-year survival among patients with metastatic PPGL is approximately 60% [ 4 , 5 ]. Mornex et al. reported that patients with metastatic PPGL are divided into two different groups; the one group seemed benign with complete recovery after the operation, but patients recurred on average 7.8 year later (range 1–22 years). The other group had simultaneous metastasis and aggressively progressive type [ 6 ]. Hamidi and Young, Jr et al. reported large series of patients with metastatic PPGLs and divided them into two groups as rapidly progressive (n = 29) and indolent disease (n = 188), and resulted in shorter survival correlated with male sex, older age at the time of primary tumor, synchronous metastases, larger primary tumor size, elevated dopamine, and not undergoing primary tumor resection. There was no difference in the type of primary tumor or presence of SDHB mutation in their report [ 7 ]. Despite the rather high accuracy of biochemical and imaging analyses in pinpointing PPGLs in the clinical setting, pathology remains the gold standard for diagnostic purposes [ 7 ]. A postoperative histological analysis of the resected specimen is standard practice [ 8 ]. The utility of tumor proliferative index (MIB-1/Ki-67) was 3% [ 9 ] and 5% [ 10 ] for differentiating malignant and benign PPGL. van der Harst et al. compared large series composed of 110 patients operated on for a total of 99 benign and 37 malignant PPGLs and emphasized the utility of tumor proliferative index (MIB-1/Ki-67) as 3.0% in malignant PPGL vs 0.2% in benign PPGLs (p < 0.0005), and Ki67 index can predict the occurrence of metastases [ 11 ]. The other factors such as depletion of S100-positive sustentacular cells (p < 0.0005), tumor size, extra-adrenal location and intratumor necrosis were also confirmed to be useful for differentiating between benign and malignant PPGLs [ 11 ]. Although there were many investigations for differentiating malignant and benign PCC, such as telomerase reverse transcriptase, heat-shock protein 90, vascular endothelial growth factor (VEGF), hypoxia-inducible transcription factor 1a (HIF1a), cyclooxygenase-2, N-cadherin, tenascin C, and specific cleavage products of chromogranin and secretogranin proteins, absence of labeling for cell adhesion molecule CD44-S, however, it was still uncertain whether these were useful tool to predict metastatic PPGLs [ 12 ]. Despite lots of efforts, it was still difficult to decide a single and direct marker for indicating malignant PPGL, then scoring system such as Pheochromocytoma of the Adrenal gland Scaled Score (PASS) [ 13 ] and Pathological grading for predicting metastasis in phaeochromocytoma and paraganglioma [ 14 ] and Grading of Adrenal Pheochromocytoma and Paraganglioma (GAPP) [ 15 ] were made. Then, WHO Endocrine Tumour Classification 4th (2017) declared that all PCC have some metastatic potential. Therefore, the previous categories of benign and malignant PCC have been eliminated in favor of an approach based on risk stratification [ 16 , 17 ]. After that, the term malignant PPGL was changed with metastatic PPGL. The GAPP has good predictive performance; a higher GAPP score was significantly associated with higher risk of both recurrent disease overall and distant metastasis and was inversely associated with risk of new primary tumors, and GAPP was validated by the groups composed of endocrinologists and pathologists with some comments [ 18 , 19 , 21 , 22 ]. On the other hand, PPGL have the highest rate of heritability among all tumors with around 30% to 35% of Caucasian patients showing germline mutations in susceptibility genes and further 35% to 40% of patients are affected by somatic driver mutations. Thus, around 70% of all patients with PPGL can be assigned to 1 of 3 main molecular clusters with different phenotypes and clinical behavior [ 23 ]. Based on driving genes, PPGLs can be categorized into 3 distinct clusters. Tumors in cluster 1, (pseudohypoxia pathway) are typically less differentiated, often extra-adrenal, and lack the complete catecholamine synthesis pathway, resulting in elevated dopamine and norepinephrine, along with their metabolites 3-methoxytyramine and normetanephrine. Cluster 2 (kinase cluster) tumors are generally more differentiated, adrenal in origin, express PNMT, secrete epinephrine, and have a lower risk of metastasis [ 23 ]. Cluster 3 tumors are currently poorly described, but aggressive behavior seems likely. In a subsequent study, 60.5% of all metastatic cases were in the cluster 1 group and only 2.3% in the cluster 2 group [ 23 ]. In a retrospective study investigating 169 patients, 50% of all patients with metastatic disease had cluster 1 tumors (42% SDHB-related tumors), only 4% had cluster 2 tumors, and 46% had apparently sporadic disease [ 24 ]. Thus, histopathological diagnosis should be related with cluster classification as possible. Here, we analyzed 35 metastatic PPGL using clinicopathological data such as catecholamine, mutation of SDHB, VHL, MAX, and GAPP score with Ki67 labeling index, and compared them with patients’ clinical course and outcome. Materials and Methods This is a retrospective study of 35 metastatic PPGL of 20 male and 15 female from 17 to 81-year-old age from the list of about 300 cases of PPGL for histopathology examination during 2011–2025, in Japan. The number and location of the tumors were 20 in the adrenal gland (PCC) and 14 in the retroperitoneum and 1 urinary bladder, total 15 PGL. Surgically operated tissues were fixed in 10% buffered formalin and embedded in paraffin, and three-micron thick sections were sent to Department of Diagnostic Pathology, National Hospital Organization, Hakodate Medical Center for pathological consultation. The sections were stained with hematoxylin eosin (H&E), and elastica-Masson trichrome stain (EM). Immunohistochemistry (IHC) was performed using a VENTANA Bench Mark ULTRA Slide Staining System (Roche Diagnostics, Indianapolis, IN, USA) with primary antibodies against chromogranin A (CgA) (DAKO; DAK-A3, 1:100), synaptophysin (Nichirei, Tokyo, Japan; clone 27G12, prediluted), CD56 (Nichirei clone MRQ42, prediluted), tyrosine hydroxylase (TH) (Chemicon, Temecula, CA, USA; rabbit polyclonal, 1:500), dopamine beta- hydroxylase (DBH) (Abcam, clone EPR209487, 1:3000), Ki67 (MIB1) (Ventana; clone 30 − 9, prediluted), S100 (Nichirei, rabbit polyclonal, prediluted), 1:3000), Ki67 (MIB1) (Ventana; clone 30 − 9, prediluted), carboxyl anhydrase 9 (CA9) (Abcam,clone15086, 1:100), succinate dehydrogenase B (SDHB) (Sigma, clone HPA002868, 1:200), MYC-associated factor X (MAX ) (Santacruz, SC-197, 1:600), and somatostatin receptor type 2 (SSTR2) (Nichirei, clone EP149, prediluted). Appropriate positive controls, such as human adrenal medulla for CgA, synaptophysin, CD56, S100, TH, and DBH [ 25 ] pancreatic tissue for SSTR2 [ 26 , 27 ], and duodenal mucosa for CA9 [ 28 ] were added. IHC-positive locations were on the cell membrane for CA9 [ 29 , 30 ] and SSTR2 [ 31 ], in the cytoplasm for SDHB [ 32 , 33 ] and in the nucleus for MAX [ 34 , 35 ]. PBS was used as a negative control. Tissue localizations for identifying VHL, SDHB, and MAX mutations using IHC were as follows: CA9-IHC positivity localized on the cell membrane, SDHB-IHC negativity in the cytoplasm and MAX-IHC negativity in the nucleus provided supportive evidence for mutations in VHL, SDHB, and MAX, respectively. Genomic types were defined by the presence of gene mutations and/or immunohistochemistry. Gene analysis was performed in each institute retrospectively. Statistical analyses were performed using StatMate VI Software (Takahashi Y, ATMS, Tokyo, Japan). Ethics Approval: The study was carried out in accordance with the Declaration of Helsinki and with the approval of the Ethics committee of the National Hospital Organization Hakodate Medical Center R7-1210001. Results Thirty-five patients with metastatic PPGL are summarized in Table 1. Tumor types were 20 PCC and 15 PGL of 20 male and 15 female, and patients mean age was 34 with range from 17 to 81-year-old. The mean tumor size was 8.6 cm varied from 2.5–19 cm. The frequent metastatic organs were bones (50%) followed by lymph node (26%), liver (26%) and lung (23%). Metastatic types were 18 synchronous and 17 metachronous. Mean duration for postoperative metastasis was 5 years with range from 4 month to 19 years. The patient with extraordinarily long survival of 37 years after 1st operation was excluded from mean duration in Table 1 and included in Table 3 of long survival patients. Catecholamine types examined at pretreated state were 46% of norepinephrine (NE), 23% of epinephrine (E), 17% of NE plus dopamine (DA), and 14% of nonfunctional. There was no case of DA only in this study of metastatic PPGL. Gene analysis was performed in 5 cases in this series including 4 SDHB; SDHB (c424-delA), SDHB (c.G268 > C/T), SDHB (c.137G > A (p.R46), SDHB (c201-2A > C), and one case with no gene mutations of SDHB, VHL, and MAX. There were no suspicious cases of VHL by CA9-immunoreactivity and MAX by MAX-immunonegativity in the examined cases. Immunohistochemical study of SDHB revealed that 43% of the 32 PPGL was negative, which was compatible with SDH-deficient PPGL [ 32 , 33 ], and four of them examined had SDHB gene mutations too. Ki67 labeling index was 7% (mean) in all patients and 11% in synchronous and 7% in metachronous types (P < 0.05). The mean GAPP score was 6 in entire cases. The ratio of patients in GAPP grade was 12% in low grade, 41% in moderate grade, and 47% in high grade [Table 1]. In this series, we had 10 expired patients with metastatic PPGL. Mean tumor size was 10.6cm. Metastatic type was all metachronous type (100%). Mean survival duration after 1st operation was very short, as 21 months. Catecholamine types were 40% of NE, 30% of NE plus DA, 20% of nonfunctional, and 10% of E type. SDHB mutation was observed in 50%. Mean Ki67 labeling index was 14% and mean GAPP score was 8.5. GAPP grade of this type was 0% in low grade, 27% in intermediate grade, and 73% in high grade [Table 2]. Although the number of patients with long survival, more than 10 years, is small as 4 cases in our study. Mean tumor size of this type was 7 cm in diameter. Metastatic organs were limited in bone and lungs. Metastatic types were one synchronous, and three metachronous types. Two patients had multiple tumor metastases in only bone and survived 37 years in one patient and mor than 19 years in another patient. Both patients had a histologically typical pseudorosette pattern which was negative for SDHB-immunohistochemistry and SDHB mutations (SDHB c201-2A > C) in one patient. One of them was previously reported to have been 26 years long survival after operation for PCC [ 36 ]. The other one patient of long survival group had multiple small metastatic foci in the lung five year after operation of PCC, the patient underwent operation for all pulmonary metastatic foci, and then he is still healthy without recurrence of tumors. The rest patient had paraaortic PGL with metastases in the lymph node and lung at 9 years and 11 years after the first operation, however, the patient became complete remission by CVD therapy after that mean Ki67 labeling index was 5.4%, and mean GAPP score was 6. Gapp grade of this type was 25% in low, 75% in intermediate, and 0% in high grade [Table 3]. Here, we present additional figures from metastatic PPGLs. Figure 1 is ACTH-producing PCC with liver metastasis. The catecholamine type was epinephrine plus norepinephrine plus dopamine type and belongs to Cluster 2. The patient survives for 13 years at present. This case was previously reported [ 37 ]. [Figure 1 ] Figure 2 is from aggressively proliferative type of retroperitoneal PGL who expired two years after the surgery. Histologically, small tumor cells with high nuclear cytoplasmic ratio diffusely proliferated and formed large and small irregular zellballen pattern. Catecholamine levels were low and there was no expression of tyrosine hydroxylase and SDHB immunohistochemistry. Ki67 index was 13% in the primary tumor and 23% in the metastatic lesion. GAPP score was 8. This case belongs to Cluster 1 [Figure 2 ]. Figure 3 is from a long survival group of retroperitoneal two PGLs. Tumor size was 7.0 cm, and GAPP score was 6. The patient had multiple bone metastases 19 years after initial operation for PGL. No metastasis in other organs was observed after operation for multiple pulmonary metastatic foci. The patient had germline SDHB-mutation (SDHB c201-2A > C). Histology is typical pseudo rosette type associated with loss of SDHB immunohistochemistry. This case also belongs to Cluster 1 [Figure 3 ]. Discussion PPGL are difficult tumors for predicting metastasis. Tumor tissues, especially metastatic PPGL, are usually immature in both morphology and biological function than those of the original organs of normal adrenal gland or paraganglia. What is immaturity in PPGL in morphology? PPGL derived from adrenal medulla and extra-adrenal paraganglia which have unique structure composed of nests of catecholamine-producing chief cells surrounded by sustentacular cells. Chief cells have catecholamine synthesizing enzymes such as TH and DBH, or chromogranins such as Chromogranin A and Chromogranin B, and sustentacular cells are positive for S100 protein or SOX10. All these cells have homeostatic balance in normal tissue as forming zellballen structure such as cell nests of chief cells surrounded by sustentacular cells and arrange in regular zellballen pattern associated with well- developed small vessels. However, PPGL shows irregular zellballen pattern due to irregular proliferation of chief cells and decrease of sustentacular cells. Furthermore, tumor tissues occasionally demonstrate peculiar structure similar to that of embryological structure named pseudorosette or psudopapillary pattern with lack or few sustentacular cells. This morphological structure is occasionally observed entirely or in part of tumor tissue. The immature tumor tissue reflects immature function of CA synthesis, such as TH-deficiency or nonfunctional PPGL, however, immaturity itself does not necessarily coincide with malignancy or metastatic potential. Immaturity is reflected by proliferation of smaller cells having scanty cytoplasmic organelle and smaller CA granules in the cytoplasm; however, immaturity itself is not necessarily coincidence with increase of proliferative activity [ 38 , 39 ]. If the immature tumor cells obtain proliferative ability such as high Ki67 and absence of S100 positive sustentacular cells, they rapidly grow and metastasize without any regulation as seen in Fig. 2 . Histologically, such tumor cells completely or incompletely lack tyrosine hydroxylase and produce low levels of catecholamine and may be resulting in dopamine-producing PGL or non-functional PGL. The role of sustentacular cells in PPGL is not clearly elucidated if sustentacular cells may control homeostasis of CA-producing cells. Irregular distribution of sustentacular cells is often observed in many PPGL. Complete loss of sustentacular cells is occasionally observed in increased sized PPGL and has been used as one of the indicators for malignant PPGL [ 40 ]. Ki67 is a marker for proliferating cells such as crypt cells in the intestinal mucosa, but they are usually not observed in normal adrenal medulla. Expression of Ki67 is usually low in PPGL in comparison to the epithelial malignant tumors. Ki67 Labeling Index (LI) is used for evaluating malignancy of epithelial neuroendocrine tumors (eNETs) of the pancreas, gastrointestinal tract or lung [ 41 ]. In PPGL, Ki67 LI has been considered 3% of more as border line levels between benign and malignant PPGL [ 11 , 14 , 15 ]. However, it was decided about 20 years ago, and now there is no official recommendation for what suitable Ki67 level is [ 42 ]. The cut-off value of Ki67 LI may be increasing recently in this author’s experience. The possible reason is due to increased sensitivity of Ki67 along with progress of IHC technique and automated image analysis. In this study of 35 metastatic PPGL, mean Ki67 LI was 7% of total cases including 11% of synchronous metastatic group and 7% of metachronous metastatic group, and 14% of expired group, and 5.4% of long survival metastatic group. Higher Ki67 LI more than 7% may be suitable cut off level for metastatic PPGL, and 14% for rapid tumor growth and poor prognosis from this limited amount of study. Further studies are necessary to decide Ki67 LI for prognostic marker. Another indicator of malignant grading for PPGL is GAPP which differentiates PPGL into well differentiated type, moderate differentiated type, and poorly differentiated type depending on the total score and classified them into low grade, intermediate grade, and high-grade malignancy [ 17 ]. There are some problems in the parameter of GAPP, 1) counting number of tumor cells may be sometimes difficult in some cases such as VHL-related PPGL etc. due to mixed highly proliferative vascular endothelial cells among tumor cells. In such cases, counting chromogranin A positive cells which is positive only for tumor cells, and differentiate them from non-tumor cells, and it is easy for counting number of tumor cells. 2) Another misunderstanding in GAPP parameter is large and small irregular zellballen pattern in Histological pattern. Any PPGL has irregular zellballen pattern as a character of neoplasm, however, it means 10 times difference between large and small zellballen size as described in the original article of GAPP [ 15 ]. In this study, 43% of the metastatic PPGL were compatible with SDHB -related tumors. This data is surprisingly identical with that of European study in which 50% of 169 patients with metastatic disease had cluster 1 tumors (42% SDHB -related tumors), only 4% had cluster 2 tumors, and 46% had apparently sporadic disease [ 24 ]. Although SDHB mutations in PPGL were highly detected in metastatic PPGL, it was not agreed that SDHB mutations as a major prognostic parameter in metastatic PPGL [ 7 , 17 , 24 ]. From histopathological experience, this author suspects that SDHB -deficient tumor cells monotonously proliferate without any regulation by sustentacular cells, and form norepinephrine or dopamine-producing PGL, and occasionally pseudorosette-forming PGL. There may be another molecule for tumor cell proliferation such as accelerator and breaks for abnormal proliferation in metastatic PPGL besides SDHB mutations. In addition, we had 23% of epinephrine type PPGL in this study, which is compatible with non- SDHB mutations. However, gene analysis has not been carried out for them. Further investigation is necessary. Although there have been two types of metastatic PPGL, such as benign-looking group and highly malignant group in the previous studies [ 6 , 7 ], however, the ratio of benign-looking group was smaller in our study. Larger scaled investigations are necessary in future to reveal the reasons for differentiating them. Declarations Informed Consent Statement : Not applicable. Declarations Competing interests: The authors declare no competing interests. Funding: This research received no external funding. Author Contribution Conception, design and figures: N.K., T.K, T.T, M.N., Data collection and analysis: all authors; Manuscript preparation and editing: NK, Approval of final manuscript: all authors. Acknowledgement The authors appreciate Hashimoto D, Mizugai Y, and Kotsuji H in Department of Diagnostic Pathology, National Hospital Organization Hakodate Medical Center for their technical assistance for pathological investigation. Data Availability: As this study is retrospective in nature, approval was deemed unnecessary according to national regulations. All individuals consented to participate in the study. References Neumann, H. P. H., Young, W. F. Jr & Eng, C. Pheochromocytoma and paraganglioma. N Engl. J. Med. 381 , 552–565. 10.1056/NEJMra1806651 (2019). Berends, A. M. A. et al. Incidence of pheochromocytoma and sympathetic paraganglioma in the Netherlands: a nationwide study and systematic review. Eur. J. Intern. Med. 51 , 68–73. 10.1016/j.ejim.2018.01.015 (2018). Ebbehoj, A. et al. Incidence and Clinical Presentation of Pheochromocytoma and Sympathetic Paraganglioma: A Population-based Study. J. Clin. Endocrinol. Metab. 106 , e2251–e2261. 10.1210/clinem/dgaa965 (2021). 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Endocri Pathol 34:79–97. doi.10.1007/s12022-023-09755-3 (2023). Thompson, L. D. R. et al. Data set for the reporting of pheochromocytoma and paraganglioma: explanations and recommendations of the guidelines from the International Collaboration on Cancer Reporting. Hum. Pathol. 110 , 83–97. 10.1016/j.humpath.2020.04.012 (2021). Tables Tables 1 to 3 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1Summaryof35patientswithmetastaticpheochromocytomaandparaganglioma.pdf Table2Summaryofexpired10patientswithmetastaticPPGL.pdf Table3SummaryoflongsurvivalfourpatientswithmetastaticPPGL.pdf 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. 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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-9585500","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":636661056,"identity":"d3367f44-4d19-42ac-b1d3-ae92af57f077","order_by":0,"name":"Noriko Kimura","email":"data:image/png;base64,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","orcid":"","institution":"National Hospital Organization Hakodate Medical Center","correspondingAuthor":true,"prefix":"","firstName":"Noriko","middleName":"","lastName":"Kimura","suffix":""},{"id":636661057,"identity":"673ab18f-0830-45e0-8dfa-dc0bde198659","order_by":1,"name":"TAKUYUKI KATABAMI","email":"","orcid":"","institution":"St. Marianna University Yokohama Seibu Hospital","correspondingAuthor":false,"prefix":"","firstName":"TAKUYUKI","middleName":"","lastName":"KATABAMI","suffix":""},{"id":636661058,"identity":"fd41c9f6-7e66-4d4f-abc9-eb9d7fdcd57a","order_by":2,"name":"Tomoaki Tanaka","email":"","orcid":"","institution":"Chiba University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Tomoaki","middleName":"","lastName":"Tanaka","suffix":""},{"id":636661059,"identity":"c26857cf-c43f-4037-b7d2-33d1541d21ca","order_by":3,"name":"Kazuhiro Takekoshi","email":"","orcid":"","institution":"University of Tsukuba","correspondingAuthor":false,"prefix":"","firstName":"Kazuhiro","middleName":"","lastName":"Takekoshi","suffix":""},{"id":636661060,"identity":"a65398d6-a7da-460a-9286-6474e1c981c5","order_by":4,"name":"Tatsuya Kondo","email":"","orcid":"","institution":"Faculty of Life Sciences, Kumamoto University","correspondingAuthor":false,"prefix":"","firstName":"Tatsuya","middleName":"","lastName":"Kondo","suffix":""},{"id":636661061,"identity":"1f6af518-1210-4e95-be86-ea74b95bbe70","order_by":5,"name":"Masato Ono","email":"","orcid":"","institution":"National Hospital Organization, Yokohama Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Masato","middleName":"","lastName":"Ono","suffix":""},{"id":636661062,"identity":"839c49eb-c622-46bb-a0c7-f60bd6a890c6","order_by":6,"name":"Ken Terui","email":"","orcid":"","institution":"Hirosaki University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Ken","middleName":"","lastName":"Terui","suffix":""},{"id":636661063,"identity":"ecc9fffd-3f53-4c28-8d83-6e78652a205f","order_by":7,"name":"Shinobu Takayasu","email":"","orcid":"","institution":"Hirosaki University Graduate School of Medicine and Hospital","correspondingAuthor":false,"prefix":"","firstName":"Shinobu","middleName":"","lastName":"Takayasu","suffix":""},{"id":636661064,"identity":"2ceeee12-c7f9-42b3-9238-2b35d956d941","order_by":8,"name":"Kenichi Inagaki","email":"","orcid":"","institution":"Okayama University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Kenichi","middleName":"","lastName":"Inagaki","suffix":""},{"id":636661066,"identity":"26a34524-fc14-44e0-a154-1a13db5354cb","order_by":9,"name":"Masato Yonamine","email":"","orcid":"","institution":"University of Tsukuba","correspondingAuthor":false,"prefix":"","firstName":"Masato","middleName":"","lastName":"Yonamine","suffix":""},{"id":636661068,"identity":"29d1ca1d-7a07-4dad-a8a9-8414c71eec46","order_by":10,"name":"Mitsuhide Naruse","email":"","orcid":"","institution":"Ijinkai Takeda General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Mitsuhide","middleName":"","lastName":"Naruse","suffix":""}],"badges":[],"createdAt":"2026-05-01 11:23:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9585500/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9585500/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108978334,"identity":"f8f9ab02-881e-4e3e-aea8-f32b801cda22","added_by":"auto","created_at":"2026-05-11 11:36:31","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":463724,"visible":true,"origin":"","legend":"\u003cp\u003ePheochromocytoma with multiple metastasis, tumor size:10 cm. GAPP: 4, Recurrence of the PCC: 10 years after the operation\u003c/p\u003e\n\u003cp\u003ePrimary tumor of the adrenal gland. The tumor is composed of diffuse proliferation of cells with basophilic cytoplasm (a). Tumor cells are positive for chromogranin (b), tyrosine hydroxylase (c), dopamine-beta-hydroxylase (d). Ki67 labeling index is 5% (e), and SDHB (f).\u003c/p\u003e\n\u003cp\u003eMetastatic tumor in the liver. Tumor cells are similar to those of the primary site with diffuse proliferation of basophilic tumor cells (g), Ki67 labeling index is 8% (h), and positive for ACTH which is ectopic producing as well as in the primary site of PCC (i).\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9585500/v1/f4bf19572ff0512f7fc79d47.jpg"},{"id":108978331,"identity":"4f201a59-ef32-4286-ae22-5c5a4da2ad83","added_by":"auto","created_at":"2026-05-11 11:36:28","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":469936,"visible":true,"origin":"","legend":"\u003cp\u003eParaganglioma, retroperitoneum with multiple metastasis, tumor size:19cm. GAPP: 8, The patient expired 2 years after the operation.\u003c/p\u003e\n\u003cp\u003ePrimary tumor of the peritoneum. The tumor shows large and small irregular tumor cell clusters (a) composed of diffuse proliferation of small monotonous cells of high nuclear cytoplasmic ratio (b). Tumor cells are negative for tyrosine hydroxylase (c), and positive for DBH (d). Ki67 labeling index is 13% in the primary site, and 23% in the metastatic site (e). SDHB immunohistochemistry is negative (f).\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9585500/v1/d261e90e3cc04c4f76e6a1df.jpg"},{"id":108978197,"identity":"ed8ca7c6-49bb-4d27-9540-c2fac5ae207f","added_by":"auto","created_at":"2026-05-11 11:34:53","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":495612,"visible":true,"origin":"","legend":"\u003cp\u003eParaganglioma, retroperitoneum, two, Tumor size:7.0 cm, GAPP 6, with multiple bone metastases 19 years after initial operation for PGL.\u003c/p\u003e\n\u003cp\u003eThe tumor shows pseudorosette (pseudopapillary) pattern which means tumor cells show perivascular arrangement with neuropile-like delicate cytoplasmic process (a). Tumor cells are positive for chromogranin A (b), tyrosine hydroxylase (c), S100 protein is sporadically positive in sustentacular cells (d), Ki67 index is 6% (e). SDHB is negative, only vascular endothelial cells are positive for SDHB (f).\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9585500/v1/2e56dfe0d4160d149c0cf807.jpg"},{"id":108980066,"identity":"467523ba-1d2b-4cf4-ba3c-3f7e10c91630","added_by":"auto","created_at":"2026-05-11 12:03:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1686159,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9585500/v1/464f5843-3a64-4961-a5d9-6970abebf0bb.pdf"},{"id":108977916,"identity":"fccea4fe-ca90-4ad0-bc19-10f255f6d910","added_by":"auto","created_at":"2026-05-11 11:33:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":130010,"visible":true,"origin":"","legend":"","description":"","filename":"Table1Summaryof35patientswithmetastaticpheochromocytomaandparaganglioma.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9585500/v1/6d13027e957f09f5bddf144e.pdf"},{"id":108978150,"identity":"779f71ed-f93f-4bc6-8728-104e8af6e0ce","added_by":"auto","created_at":"2026-05-11 11:34:22","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":96659,"visible":true,"origin":"","legend":"","description":"","filename":"Table2Summaryofexpired10patientswithmetastaticPPGL.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9585500/v1/088ef0dbe6d2375a3438d559.pdf"},{"id":108978196,"identity":"09e9fcc0-5136-4c84-8b37-8616b74d0175","added_by":"auto","created_at":"2026-05-11 11:34:52","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":127042,"visible":true,"origin":"","legend":"","description":"","filename":"Table3SummaryoflongsurvivalfourpatientswithmetastaticPPGL.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9585500/v1/986d2eb1f293939376aa0f92.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Metastatic Pheochromocytoma and Paraganglioma: Clinicopathological Analysis of 35 Cases","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePheochromocytoma (PCC) and sympathetic paraganglioma (PGL), together with PPGL, are adrenal and extra-adrenal paraganglion derived rare tumors. The incidence of PPGL is approximately 4.6\u0026ndash;6.6 cases per one million person-years [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Approximately 10 to 15% of PCC and up to 50% of PGL will exhibit metastatic behavior [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The 5-year survival among patients with metastatic PPGL is approximately 60% [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Mornex et al. reported that patients with metastatic PPGL are divided into two different groups; the one group seemed benign with complete recovery after the operation, but patients recurred on average 7.8 year later (range 1\u0026ndash;22 years). The other group had simultaneous metastasis and aggressively progressive type [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Hamidi and Young, Jr et al. reported large series of patients with metastatic PPGLs and divided them into two groups as rapidly progressive (n\u0026thinsp;=\u0026thinsp;29) and indolent disease (n\u0026thinsp;=\u0026thinsp;188), and resulted in shorter survival correlated with male sex, older age at the time of primary tumor, synchronous metastases, larger primary tumor size, elevated dopamine, and not undergoing primary tumor resection. There was no difference in the type of primary tumor or presence of SDHB mutation in their report [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Despite the rather high accuracy of biochemical and imaging analyses in pinpointing PPGLs in the clinical setting, pathology remains the gold standard for diagnostic purposes [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. A postoperative histological analysis of the resected specimen is standard practice [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The utility of tumor proliferative index (MIB-1/Ki-67) was 3% [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] and 5% [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] for differentiating malignant and benign PPGL. van der Harst et al. compared large series composed of 110 patients operated on for a total of 99 benign and 37 malignant PPGLs and emphasized the utility of tumor proliferative index (MIB-1/Ki-67) as 3.0% in malignant PPGL vs 0.2% in benign PPGLs (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0005), and Ki67 index can predict the occurrence of metastases [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The other factors such as depletion of S100-positive sustentacular cells (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0005), tumor size, extra-adrenal location and intratumor necrosis were also confirmed to be useful for differentiating between benign and malignant PPGLs [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Although there were many investigations for differentiating malignant and benign PCC, such as telomerase reverse transcriptase, heat-shock protein 90, vascular endothelial growth factor (VEGF), hypoxia-inducible transcription factor 1a (HIF1a), cyclooxygenase-2, N-cadherin, tenascin C, and specific cleavage products of chromogranin and secretogranin proteins, absence of labeling for cell adhesion molecule CD44-S, however, it was still uncertain whether these were useful tool to predict metastatic PPGLs [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Despite lots of efforts, it was still difficult to decide a single and direct marker for indicating malignant PPGL, then scoring system such as Pheochromocytoma of the Adrenal gland Scaled Score (PASS) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and Pathological grading for predicting metastasis in phaeochromocytoma and paraganglioma [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and Grading of Adrenal Pheochromocytoma and Paraganglioma (GAPP) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] were made. Then, WHO Endocrine Tumour Classification 4th (2017) declared that all PCC have some metastatic potential. Therefore, the previous categories of benign and malignant PCC have been eliminated in favor of an approach based on risk stratification [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. After that, the term malignant PPGL was changed with metastatic PPGL. The GAPP has good predictive performance; a higher GAPP score was significantly associated with higher risk of both recurrent disease overall and distant metastasis and was inversely associated with risk of new primary tumors, and GAPP was validated by the groups composed of endocrinologists and pathologists with some comments [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOn the other hand, PPGL have the highest rate of heritability among all tumors with around 30% to 35% of Caucasian patients showing germline mutations in susceptibility genes and further 35% to 40% of patients are affected by somatic driver mutations. Thus, around 70% of all patients with PPGL can be assigned to 1 of 3 main molecular clusters with different phenotypes and clinical behavior [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Based on driving genes, PPGLs can be categorized into 3 distinct clusters. Tumors in cluster 1, (pseudohypoxia pathway) are typically less differentiated, often extra-adrenal, and lack the complete catecholamine synthesis pathway, resulting in elevated dopamine and norepinephrine, along with their metabolites 3-methoxytyramine and normetanephrine. Cluster 2 (kinase cluster) tumors are generally more differentiated, adrenal in origin, express PNMT, secrete epinephrine, and have a lower risk of metastasis [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Cluster 3 tumors are currently poorly described, but aggressive behavior seems likely. In a subsequent study, 60.5% of all metastatic cases were in the cluster 1 group and only 2.3% in the cluster 2 group [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In a retrospective study investigating 169 patients, 50% of all patients with metastatic disease had cluster 1 tumors (42% SDHB-related tumors), only 4% had cluster 2 tumors, and 46% had apparently sporadic disease [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Thus, histopathological diagnosis should be related with cluster classification as possible. Here, we analyzed 35 metastatic PPGL using clinicopathological data such as catecholamine, mutation of SDHB, VHL, MAX, and GAPP score with Ki67 labeling index, and compared them with patients\u0026rsquo; clinical course and outcome.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eThis is a retrospective study of 35 metastatic PPGL of 20 male and 15 female from 17 to 81-year-old age from the list of about 300 cases of PPGL for histopathology examination during 2011\u0026ndash;2025, in Japan. The number and location of the tumors were 20 in the adrenal gland (PCC) and 14 in the retroperitoneum and 1 urinary bladder, total 15 PGL. Surgically operated tissues were fixed in 10% buffered formalin and embedded in paraffin, and three-micron thick sections were sent to Department of Diagnostic Pathology, National Hospital Organization, Hakodate Medical Center for pathological consultation. The sections were stained with hematoxylin eosin (H\u0026amp;E), and elastica-Masson trichrome stain (EM). Immunohistochemistry (IHC) was performed using a VENTANA Bench Mark ULTRA Slide Staining System (Roche Diagnostics, Indianapolis, IN, USA) with primary antibodies against chromogranin A (CgA) (DAKO; DAK-A3, 1:100), synaptophysin (Nichirei, Tokyo, Japan; clone 27G12, prediluted), CD56 (Nichirei clone MRQ42, prediluted), tyrosine hydroxylase (TH) (Chemicon, Temecula, CA, USA; rabbit polyclonal, 1:500), dopamine beta- hydroxylase (DBH) (Abcam, clone EPR209487, 1:3000), Ki67 (MIB1) (Ventana; clone 30\u0026thinsp;\u0026minus;\u0026thinsp;9, prediluted), S100 (Nichirei, rabbit polyclonal, prediluted), 1:3000), Ki67 (MIB1) (Ventana; clone 30\u0026thinsp;\u0026minus;\u0026thinsp;9, prediluted), carboxyl anhydrase 9 (CA9) (Abcam,clone15086, 1:100), succinate dehydrogenase B (SDHB) (Sigma, clone HPA002868, 1:200), MYC-associated factor X (MAX ) (Santacruz, SC-197, 1:600), and somatostatin receptor type 2 (SSTR2) (Nichirei, clone EP149, prediluted). Appropriate positive controls, such as human adrenal medulla for CgA, synaptophysin, CD56, S100, TH, and DBH [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] pancreatic tissue for SSTR2 [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], and duodenal mucosa for CA9 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] were added. IHC-positive locations were on the cell membrane for CA9 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] and SSTR2 [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], in the cytoplasm for SDHB [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] and in the nucleus for MAX [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. PBS was used as a negative control. Tissue localizations for identifying VHL, SDHB, and MAX mutations using IHC were as follows: CA9-IHC positivity localized on the cell membrane, SDHB-IHC negativity in the cytoplasm and MAX-IHC negativity in the nucleus provided supportive evidence for mutations in VHL, SDHB, and MAX, respectively. Genomic types were defined by the presence of gene mutations and/or immunohistochemistry. Gene analysis was performed in each institute retrospectively. Statistical analyses were performed using StatMate VI Software (Takahashi Y, ATMS, Tokyo, Japan).\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eEthics Approval:\u003c/strong\u003e \u003cp\u003eThe study was carried out in accordance with the Declaration of Helsinki and with the approval of the Ethics committee of the National Hospital Organization Hakodate Medical Center R7-1210001.\u003c/p\u003e \u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThirty-five patients with metastatic PPGL are summarized in Table\u0026nbsp;1. Tumor types were 20 PCC and 15 PGL of 20 male and 15 female, and patients mean age was 34 with range from 17 to 81-year-old. The mean tumor size was 8.6 cm varied from 2.5\u0026ndash;19 cm. The frequent metastatic organs were bones (50%) followed by lymph node (26%), liver (26%) and lung (23%). Metastatic types were 18 synchronous and 17 metachronous. Mean duration for postoperative metastasis was 5 years with range from 4 month to 19 years. The patient with extraordinarily long survival of 37 years after 1st operation was excluded from mean duration in Table\u0026nbsp;1 and included in Table\u0026nbsp;3 of long survival patients. Catecholamine types examined at pretreated state were 46% of norepinephrine (NE), 23% of epinephrine (E), 17% of NE plus dopamine (DA), and 14% of nonfunctional. There was no case of DA only in this study of metastatic PPGL.\u003c/p\u003e \u003cp\u003eGene analysis was performed in 5 cases in this series including 4 SDHB; SDHB (c424-delA), SDHB (c.G268\u0026thinsp;\u0026gt;\u0026thinsp;C/T), SDHB (c.137G\u0026thinsp;\u0026gt;\u0026thinsp;A (p.R46), SDHB (c201-2A\u0026thinsp;\u0026gt;\u0026thinsp;C), and one case with no gene mutations of SDHB, VHL, and MAX. There were no suspicious cases of VHL by CA9-immunoreactivity and MAX by MAX-immunonegativity in the examined cases. Immunohistochemical study of SDHB revealed that 43% of the 32 PPGL was negative, which was compatible with SDH-deficient PPGL [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], and four of them examined had SDHB gene mutations too.\u003c/p\u003e \u003cp\u003eKi67 labeling index was 7% (mean) in all patients and 11% in synchronous and 7% in metachronous types (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The mean GAPP score was 6 in entire cases. The ratio of patients in GAPP grade was 12% in low grade, 41% in moderate grade, and 47% in high grade [Table\u0026nbsp;1].\u003c/p\u003e \u003cp\u003eIn this series, we had 10 expired patients with metastatic PPGL. Mean tumor size was 10.6cm. Metastatic type was all metachronous type (100%). Mean survival duration after 1st operation was very short, as 21 months. Catecholamine types were 40% of NE, 30% of NE plus DA, 20% of nonfunctional, and 10% of E type. SDHB mutation was observed in 50%. Mean Ki67 labeling index was 14% and mean GAPP score was 8.5. GAPP grade of this type was 0% in low grade, 27% in intermediate grade, and 73% in high grade [Table\u0026nbsp;2].\u003c/p\u003e \u003cp\u003eAlthough the number of patients with long survival, more than 10 years, is small as 4 cases in our study. Mean tumor size of this type was 7 cm in diameter. Metastatic organs were limited in bone and lungs. Metastatic types were one synchronous, and three metachronous types. Two patients had multiple tumor metastases in only bone and survived 37 years in one patient and mor than 19 years in another patient. Both patients had a histologically typical pseudorosette pattern which was negative for SDHB-immunohistochemistry and SDHB mutations (SDHB c201-2A\u0026thinsp;\u0026gt;\u0026thinsp;C) in one patient. One of them was previously reported to have been 26 years long survival after operation for PCC [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The other one patient of long survival group had multiple small metastatic foci in the lung five year after operation of PCC, the patient underwent operation for all pulmonary metastatic foci, and then he is still healthy without recurrence of tumors. The rest patient had paraaortic PGL with metastases in the lymph node and lung at 9 years and 11 years after the first operation, however, the patient became complete remission by CVD therapy after that mean Ki67 labeling index was 5.4%, and mean GAPP score was 6. Gapp grade of this type was 25% in low, 75% in intermediate, and 0% in high grade [Table\u0026nbsp;3].\u003c/p\u003e \u003cp\u003eHere, we present additional figures from metastatic PPGLs. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e is ACTH-producing PCC with liver metastasis. The catecholamine type was epinephrine plus norepinephrine plus dopamine type and belongs to Cluster 2. The patient survives for 13 years at present. This case was previously reported [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. [Figure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e]\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e is from aggressively proliferative type of retroperitoneal PGL who expired two years after the surgery. Histologically, small tumor cells with high nuclear cytoplasmic ratio diffusely proliferated and formed large and small irregular zellballen pattern. Catecholamine levels were low and there was no expression of tyrosine hydroxylase and SDHB immunohistochemistry. Ki67 index was 13% in the primary tumor and 23% in the metastatic lesion. GAPP score was 8. This case belongs to Cluster 1 [Figure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e is from a long survival group of retroperitoneal two PGLs. Tumor size was 7.0 cm, and GAPP score was 6. The patient had multiple bone metastases 19 years after initial operation for PGL. No metastasis in other organs was observed after operation for multiple pulmonary metastatic foci. The patient had germline SDHB-mutation (SDHB c201-2A\u0026thinsp;\u0026gt;\u0026thinsp;C). Histology is typical pseudo rosette type associated with loss of SDHB immunohistochemistry. This case also belongs to Cluster 1 [Figure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePPGL are difficult tumors for predicting metastasis. Tumor tissues, especially metastatic PPGL, are usually immature in both morphology and biological function than those of the original organs of normal adrenal gland or paraganglia. What is immaturity in PPGL in morphology? PPGL derived from adrenal medulla and extra-adrenal paraganglia which have unique structure composed of nests of catecholamine-producing chief cells surrounded by sustentacular cells. Chief cells have catecholamine synthesizing enzymes such as TH and DBH, or chromogranins such as Chromogranin A and Chromogranin B, and sustentacular cells are positive for S100 protein or SOX10. All these cells have homeostatic balance in normal tissue as forming zellballen structure such as cell nests of chief cells surrounded by sustentacular cells and arrange in regular zellballen pattern associated with well- developed small vessels. However, PPGL shows irregular zellballen pattern due to irregular proliferation of chief cells and decrease of sustentacular cells. Furthermore, tumor tissues occasionally demonstrate peculiar structure similar to that of embryological structure named pseudorosette or psudopapillary pattern with lack or few sustentacular cells. This morphological structure is occasionally observed entirely or in part of tumor tissue. The immature tumor tissue reflects immature function of CA synthesis, such as TH-deficiency or nonfunctional PPGL, however, immaturity itself does not necessarily coincide with malignancy or metastatic potential. Immaturity is reflected by proliferation of smaller cells having scanty cytoplasmic organelle and smaller CA granules in the cytoplasm; however, immaturity itself is not necessarily coincidence with increase of proliferative activity [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. If the immature tumor cells obtain proliferative ability such as high Ki67 and absence of S100 positive sustentacular cells, they rapidly grow and metastasize without any regulation as seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Histologically, such tumor cells completely or incompletely lack tyrosine hydroxylase and produce low levels of catecholamine and may be resulting in dopamine-producing PGL or non-functional PGL.\u003c/p\u003e \u003cp\u003eThe role of sustentacular cells in PPGL is not clearly elucidated if sustentacular cells may control homeostasis of CA-producing cells. Irregular distribution of sustentacular cells is often observed in many PPGL. Complete loss of sustentacular cells is occasionally observed in increased sized PPGL and has been used as one of the indicators for malignant PPGL [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eKi67 is a marker for proliferating cells such as crypt cells in the intestinal mucosa, but they are usually not observed in normal adrenal medulla. Expression of Ki67 is usually low in PPGL in comparison to the epithelial malignant tumors. Ki67 Labeling Index (LI) is used for evaluating malignancy of epithelial neuroendocrine tumors (eNETs) of the pancreas, gastrointestinal tract or lung [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In PPGL, Ki67 LI has been considered 3% of more as border line levels between benign and malignant PPGL [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. However, it was decided about 20 years ago, and now there is no official recommendation for what suitable Ki67 level is [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The cut-off value of Ki67 LI may be increasing recently in this author\u0026rsquo;s experience. The possible reason is due to increased sensitivity of Ki67 along with progress of IHC technique and automated image analysis. In this study of 35 metastatic PPGL, mean Ki67 LI was 7% of total cases including 11% of synchronous metastatic group and 7% of metachronous metastatic group, and 14% of expired group, and 5.4% of long survival metastatic group. Higher Ki67 LI more than 7% may be suitable cut off level for metastatic PPGL, and 14% for rapid tumor growth and poor prognosis from this limited amount of study. Further studies are necessary to decide Ki67 LI for prognostic marker.\u003c/p\u003e \u003cp\u003eAnother indicator of malignant grading for PPGL is GAPP which differentiates PPGL into well differentiated type, moderate differentiated type, and poorly differentiated type depending on the total score and classified them into low grade, intermediate grade, and high-grade malignancy [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. There are some problems in the parameter of GAPP, 1) counting number of tumor cells may be sometimes difficult in some cases such as VHL-related PPGL etc. due to mixed highly proliferative vascular endothelial cells among tumor cells. In such cases, counting chromogranin A positive cells which is positive only for tumor cells, and differentiate them from non-tumor cells, and it is easy for counting number of tumor cells. 2) Another misunderstanding in GAPP parameter is large and small irregular zellballen pattern in Histological pattern. Any PPGL has irregular zellballen pattern as a character of neoplasm, however, it means 10 times difference between large and small zellballen size as described in the original article of GAPP [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, 43% of the metastatic PPGL were compatible with \u003cem\u003eSDHB\u003c/em\u003e-related tumors. This data is surprisingly identical with that of European study in which 50% of 169 patients with metastatic disease had cluster 1 tumors (42% \u003cem\u003eSDHB\u003c/em\u003e-related tumors), only 4% had cluster 2 tumors, and 46% had apparently sporadic disease [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Although \u003cem\u003eSDHB\u003c/em\u003e mutations in PPGL were highly detected in metastatic PPGL, it was not agreed that \u003cem\u003eSDHB\u003c/em\u003e mutations as a major prognostic parameter in metastatic PPGL [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. From histopathological experience, this author suspects that \u003cem\u003eSDHB\u003c/em\u003e-deficient tumor cells monotonously proliferate without any regulation by sustentacular cells, and form norepinephrine or dopamine-producing PGL, and occasionally pseudorosette-forming PGL. There may be another molecule for tumor cell proliferation such as accelerator and breaks for abnormal proliferation in metastatic PPGL besides \u003cem\u003eSDHB\u003c/em\u003e mutations.\u003c/p\u003e \u003cp\u003eIn addition, we had 23% of epinephrine type PPGL in this study, which is compatible with non-\u003cem\u003eSDHB\u003c/em\u003e mutations. However, gene analysis has not been carried out for them. Further investigation is necessary.\u003c/p\u003e \u003cp\u003eAlthough there have been two types of metastatic PPGL, such as benign-looking group and highly malignant group in the previous studies [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], however, the ratio of benign-looking group was smaller in our study. Larger scaled investigations are necessary in future to reveal the reasons for differentiating them.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eInformed Consent\u003c/h2\u003e \u003cp\u003e \u003cb\u003eStatement\u003c/b\u003e: Not applicable.\u003c/p\u003e \u003c/p\u003e \u003cdiv class=\"Heading\"\u003eDeclarations\u003c/div\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests:\u003c/strong\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis research received no external funding.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConception, design and figures: N.K., T.K, T.T, M.N., Data collection and analysis: all authors; Manuscript preparation and editing: NK, Approval of final manuscript: all authors.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors appreciate Hashimoto D, Mizugai Y, and Kotsuji H in Department of Diagnostic Pathology, National Hospital Organization Hakodate Medical Center for their technical assistance for pathological investigation.\u003c/p\u003e\u003ch2\u003eData Availability:\u003c/h2\u003e \u003cp\u003eAs this study is retrospective in nature, approval was deemed unnecessary according to national regulations.\u003c/p\u003e\u003cp\u003eAll individuals consented to participate in the study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNeumann, H. 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Pathol.\u003c/em\u003e \u003cb\u003e110\u003c/b\u003e, 83\u0026ndash;97. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.humpath.2020.04.012\u003c/span\u003e\u003cspan address=\"10.1016/j.humpath.2020.04.012\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 3 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"pheochromocytoma and paraganglioma, Ki67, GAPP, SDHB, prognosis, mortality","lastPublishedDoi":"10.21203/rs.3.rs-9585500/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9585500/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePheochromocytoma (PCC) and sympathetic paraganglioma (PGL), together PPGL are rare tumors of the adrenal medulla and extra-adrenal paraganglion, and 10\u0026ndash;15% of PCC and 30\u0026ndash;50% of PGL metastases. Histopathological analysis of metastatic PPGL has not been well defined. We analyzed 35 metastatic PPGLs based on clinicopathological data and the grading of adrenal pheochromocytoma and paraganglioma (GAPP) which is one of the tools for evaluating malignancy. We added succinate dehydrogenase type B (SDHB) immunohistochemistry with partly \u003cem\u003eSDHB\u003c/em\u003e gene analysis and compared them to the patient prognosis. The mean age of patients was 34-year-old (range: 17\u0026ndash;81). Mean tumor size was 8.6 cm. Synchronous metastasis was 18 (51.4%) cases, and metachronous metastasis occurred in 17 cases from 1 to 19 years (mean duration: 5 years) after first operation. Catecholamine types were 8 epinephrine, 16 norepinephrine, 6 norepinephrine plus dopamine, and 5 nonfunctional. Loss of SDHB-immunohistochemistry was observed in 43%. Ki67 labeling index was 11% in synchronous and 7% in metachronous metastasis (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Mean Ki67 was 6.0 in all and 14% in expired patients. GAPP score was 6 in all and 8.5 in expired patients. GAPP risk stratification was high in 47%, intermediate in 41%, and low in 12% in all patients, and high in 73%, intermediate in 27%, and low in 0% in expired patients. In conclusion, metastasis occurred in any type of PPGL. \u003cem\u003eSDHB\u003c/em\u003e mutation was not confirmed as a major prognostic parameter in metastatic PPGL. Both higher Ki67 labeling index and higher GAPP score were histological indicators for poor prognosis.\u003c/p\u003e","manuscriptTitle":"Metastatic Pheochromocytoma and Paraganglioma: Clinicopathological Analysis of 35 Cases","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-11 10:55:20","doi":"10.21203/rs.3.rs-9585500/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":"35e7fb64-586e-4d29-95cd-6d502b275c1f","owner":[],"postedDate":"May 11th, 2026","published":true,"recentEditorialEvents":[{"type":"checksComplete","content":"","date":"2026-05-08T07:07:47+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-05-08T06:33:14+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":67762760,"name":"Biological sciences/Cancer"},{"id":67762761,"name":"Health sciences/Diseases"},{"id":67762762,"name":"Health sciences/Oncology"}],"tags":[],"updatedAt":"2026-05-11T10:55:21+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-11 10:55:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9585500","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9585500","identity":"rs-9585500","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}