Colorectal paragangliomas with immunohistochemical deficiency of succinate dehydrogenase subunit B

Abstract

Recent progress in paraganglioma (PGL) revealed genotype-phenotype relationship, especially succinate dehydrogenase complex subunit B (SDHB) gene mutation-related to the extra-adrenal origin and metastasis. SDHB-immunohistochemistry can detect all types of SDH-subunit mutations, and is a useful tool to detect SDH-mutation tumors. PGLs usually occur along with sympathetic, and parasympathetic chains, however, colorectal paraganglioma is extremely rare. We have experienced one sigmoid colon PGL and one rectal PGL. These colorectal PGLs: a sigmoid colon PGL measuring 25 mm associated with a gastrointestinal stromal tumor (GIST) of the stomach, and a rectal PGL measuring 75 × 45 mm with elevated norepinephrine level were analyzed by immunohistochemistry for INSM1, chromogranin A, synaptophysin, tyrosine hydroxylase, dopamine-beta-hydroxylase, and SDHB and SDHA. The tumors were strongly positive for above markers, however, negative for SDHB. Both PGLs negative for SDHB immunohistochemistry were defined SDHB-deficient PGLs. Histologic grading of the PGLs by GAPP was well differentiated in sigmoid PGL versus poorly differentiated in rectal PGL. Although these PGLs were the same Stage II of TNM classification, the patient with sigmoid colon PGL had neither recurrence nor metastasis for 5 years after the operation, however, the patient with rectal PGL suffered the recurrent multiple metastases and expired 5 years after the operation. Herein, we compared these colorectal PGLs in regard to the patients’ prognostic factors. Patient prognosis with these colorectal PGLs was mostly related to the tumor size and histologic grade under the same situation of SDH-deficiency.

PARAGANGLIOMAS (PGLs) are rare neural crest-derived non-epithelial neuroendocrine tumors, which locate around the sympathetic and parasympathetic ganglia and nerves. PGLs in the colorectum have been very rarely reported [1-3]. Among more than 20 types of genetic and somatic mutations that are related to PGLs, succinate dehydrogenase (SDH) enzyme complex II, that is comprised of four subunit A, B, C, or D gene mutation-related paragangliomas account for approximately 10% of the total mutations and for more than 40% of the total metastatic PGLs [4]. Alterations occurring in any of the SDH genes lead to the loss of SDH enzymatic function [5] and to the absence of SDHB expression [6, 7]. SDHx mutations cause hereditary paraganglioma-pheochromocytoma syndrome (HPPS) including Carney-Stratakis dyad. Of these, the SDHB gene mutation was highly correlated with extra-adrenal manifestations and the early onset of PGL, as well as with recurrence and malignancy [8]. The Carney-Stratakis dyad is a syndrome which combined gastrointestinal stromal tumor (GIST) and paraganglioma. Loss of signaling of SDHB, as observed via immunohistochemistry, is characteristic for the pathologic diagnosis of PGL with SDHx mutations in HPPS, as well as in GIST and PGL in the Carney-Stratakis dyad [9]. Although there have been very few reports of PGL in unusual sites with a SDHB deficiency [10, 11], it is uncertain as to whether PGLs in unusual sites have a tendency toward exhibiting a SDHB deficiency. Herein, we report on two cases of colorectal PGL with loss of SDHB: one was Carney-Stratakis dyad, and the other was a sporadic, functioning PGL with multiple metastases. We compared histological differences in these PGLs, and patients’ prognosis.

Case Report

Case 1. A 40-year-old male underwent endoscopic examinations for lower abdominal pain, which revealed submucosal tumors in the sigmoid colon measuring 25 mm, as well as tumors in the anterior wall of the lesser curvature of the stomach measuring 18 mm. Both colonic tumors and gastric tumors were simultaneously operated. He had no remarkable in past history or familial history, nor clinical syndrome of hypertension. The patient had neither recurrence nor metastasis for 5 years after the operation and he was withdrawn from clinical observation.

Case 2. A 57-year-old female underwent operation for a noradrenaline-producing rectal paraganglioma measuring 75 × 45mm in size. Three years after the operation, the metastases of the PGLs in the lymph nodes, and invasion to the inferior vena cava were observed. Since that time, she suffered the recurrent tumor metastases and died of multiple metastases of paraganglioma 6 years after the initial operation. Patient 2 was previously reported with clinicopathological data and chemotherapy in detail by Araki et al. [3], however, SDHB-immunohistochemistry was first carried out this time. In the present paper, we added data of SDHB-immunohistochemistry and compared pathologic findings of Case 1 and Case 2. Genetic testing for SDH mutations was not investigated in both cases due to various reasons. The colorectal PGLs previously reported in English were summarized in Table 1.

Table 1 Cases of colorectal paragangliomas reported in English literature

Cases (# References)	Location	Age/Gender	Tumor size	Histological Grading	Plasma CA levels (pg/mL)	SDHB-IHC	Metastasis	Prognosis
1 (#1)	Sigmoid colon	58/F	6 mm	n/e	n/e	positive	no metastasis	unknown
2 (#2)	Rectum	37/M	40 × 40 mm	PASS: 6	n/e	n/e	lymph node	well, 9 mo. a/o
3 (#3)	Rectum	57/F	75 × 45 mm	GAPP: 7	E: 50, NE: 6016, DA: 349	n/e	lymph node	died, 6 yrs. a/o
4 (Present Case 2)	Rectum	57/F	75 × 45 mm	GAPP:7		negative	Lymph node	died, 6 yrs. a/o
5 (Present Case 1)	Sigmoid colon	40/M	25 mm	GAPP: 2	n/e	negative	no metastasis	well, 5 yrs. a/o

Case 3 and 4 is the same patient reported by Araki (Ref. 3), and the present Case 2.

E, epinephrine; NE, norepinephrine; DA, dopamine; SDHB-IHC, succinate dehydrogenase subunit B-immunohistochemistry; n/e, not examined; mo, month; a/o, after the operation.

Materials and Methods

Immunohistochemistry

The tumor tissues were fixed in 10% buffered formalin and embedded in paraffin. Three-micron thick sections were stained with hematoxylin eosin (H&E), and elastica-Masson trichrome stain (EM). Immunohistochemistry was performed on a BenchMark ULTRA Slide Staining System (Roche Diagnostics, Indianapolis, IN, USA) using the primary antibodies directed against INSM1 (Santa cruz, monoclonal A-8, dilution 1:500), chromogranin A (DAKO, monoclonal DAK-A3, dilution 1:100), synaptophysin (Nichirei, Tokyo, monoclonal 27G12, prediluted), S100 (Nichirei, polyclonal, prediluted), tyrosine hydroxylase (Chemicon, Temecula, CA, polyclonal, 1:500), dopamine-b-hydroxylase (Abcam, monoclonal EPR20385, 1:5,000), Ki67: (Ventana, MIB1, monoclonal, prediluted), SDHB (Sigma, polyclonal, 1:200), SDHA (Abcam, monoclonal ab14715, 1:500), c-Kit (CD117) (Nichirei, polyclonal, 1:800), CD34 (DAKO monoclonal QBEend10, prediluted), podoplanin (Cell Marque (D2-40), prediluted), cytokeratin (BD, monoclonal CAM5.2, prediluted), and DAKO, monoclonal AE1/AE3, prediluted). The adrenal medulla was simultaneously stained for each slide as a positive control. Phosphate buffered saline was used as a negative control.

Histological analyses

These PGLs were evaluated histological grade by the Grading of Adrenal Pheochromocytoma and Paraganglioma (GAPP) scoring system [12].

Case 1: The tumor of the sigmoid colon (Fig. 1A–1F) was irregularly shaped nodules invading from the mucosal layer to the subserosal layer with negative for surgical margins and diagnosed as T2, N0, M0, Stage II by AJCC Prognostic Stage Groups of Pheochromocytoma/Sympathetic paragangliomas [13]. The tumor cells had a clear cytoplasm and hyperchromatic nuclei, and they formed cell nests surrounded by abundant capillaries. Immunohistochemistry demonstrated that the tumor cells were positive for INSM1, chromogranin A, synaptophysin, tyrosine hydroxylase (TH), and dopamine-beta-hydroxylase (DBH), and these cell nests were surrounded by S100-positive sustentacular cells, which resulted in a zellballen pattern and led to the diagnosis of sympathetic paraganglioma. Furthermore, tumor cells were completely negative for SDHB but were strongly positive for SDHA, which ruled out SDHA mutations. The sigmoid colon tumor was diagnosed as a SDHx (SDHB, SDHC, and SDHD)-related paraganglioma. The Ki67 labeling index was 1.7%, and the GAPP score was 2 points, well differentiated type.

Fig. 1

Paraganglioma in the sigmoid colon, and GIST in the stomach of Case 1

Paraganglioma in the sigmoid colon (1A–1F).

Gross findings of cut surface of the colonic tumor. Although the tissue was not completely fixed in buffered formalin, blue arrows indicate tan-colored, irregularly shaped tumors invading mainly the submucosal layer to adjacent adipose tissue (1A). Histologically, the tumor mainly located in the submucosal layer to subserosal layer, and focally invade to the mucosal layer (1B). The tumor invades to the propria muscularis. Zellballen pattern which is composed of clear cell nests of chief cells surrounded by capillaries is evident. (1C). Immunohistochemistry of tyrosine hydroxylase (1D) and S100 protein (1E) demonstrated strongly positive in tumor cells and sustentacular cells, respectively, however, SDHB immunohistochemistry is completely negative except for capillary endothelial cells (1F).

Gastrointestinal stromal tumor of the stomach (1G–1L).

Gross findings of step sections of the resected stomach. A yellowish submucosal nodule with cavity-like degeneration is GIST (arrow) (1G). Tumor cells have spindle-shaped nuclei with indistinct cytoplasmic borders forming palisading pattern (1H). Immunohistochemistry demonstrated c-Kit (1I) and CD34 (1J), but negative for SDHB with positive internal control of capillary endothelial cells (IK). The GIST is strongly positive for podoplanin (1L).

The tumor of the stomach of Case 1 (Fig. 1G–1L) was mainly located in the muscular layer and had focally invaded into the subserosal layer. The tumor was composed of bundles of smaller tumor cells with spindle-shaped nuclei that were intermingled with each other. There were also foci of palisading patterns of the tumor cell nuclei that area was epithelioid component. There were nearly no mitotic figures. Immunohistochemically, the tumor cells were moderately positive for c-Kit (CD117) and strongly positive for CD34. The Ki67-labeling index was 2%. Therefore, this tumor was a gastrointestinal tumor (GIST) of a mixed type, representing a very low-risk group [14]. Furthermore, the tumor cells were immunohistochemically negative for SDHB but were strongly positive for SDHA in the cytoplasm. In addition, podoplanin (D2-40) was strongly positive on the cytoplasmic border. Genetic testing of the tumor revealed no mutations in KIT exon 9 and exon 11. Subsequently, this tumor was SDH-deficient GIST. Finally, Case 1 was Carney-Stratakis dyad that combined SDH-deficient colonic paraganglioma and SDH-deficient gastric GIST.

Case 2: The rectal tumor (Fig. 2) was a submucosal tumor with a cut surface of homogeneous and nodular lesions with a focal hemorrhage. The depth of invasion was subserosal layer, and negative for surgical margins. The tumor was T2, N0, M0, Stage II by AJCC staging. The tumor had a Zellballen pattern with well-developed vessels, and sometimes associated coagulation necrosis, and a pseudo-rosette pattern. Immunohistochemically, the tumor cells were positive for INSM1, chromogranin A, synaptophysin, TH, and DBH, and these tumor cells were surrounded by S100-positive sustentacular cells. The Ki67 labeling index was 41%. In addition, the tumor cells were completely negative for SDHB but were strongly positive for SDHA. The rectal tumor was diagnosed a SDH (SDHB, SDHC, and SDHD)-related sympathetic paraganglioma. Histological evaluation using the GAPP score was 7 points, poorly differentiated type.

Fig. 2

Rectal Paraganglioma of Case 2

The tumor located mainly in the submucosal layer showing zellballen patterns with well-developed vessels (2A), and coagulation necrosis in center of cell nest is sometimes observed (2B). Immunostaining of INSM 1 is positive in tumor nuclei (2C) in addition to chromogranin A, synaptophysin, tyrosine hydroxylase, and dopamine-beta-hydroxylase these are all markers for paraganglioma. SDHB immunostaining showed loss of reactivity except endothelial cells of capillaries (arrow) (2D), however SDHA was strongly positive in tumor cells (2E).

An appropriate institutional review board (IRB) approved the experiment (No. R3-730002).

Discussion

Colorectal paragangliomas

There have been only a few cases of colorectal PGLs reported in the English literature (Table 1), and it is difficult to review the general features of colorectal PGLs. Two colon PGLs, Case 1 and Case 5 (Case 1 in the present article) had no metastasis. Case 1 was incidentally found to have a small-sized PGL in a diverticulum of the sigmoid colon that might be innocent [1]. In Case 5 (present Case 2), a sigmoid colon PGL neither recurred nor metastasized after the operation. However, both rectal PGLs had metastasized. Case 2 had metastasis at the time of the initial operation, received no adjunctive therapy after surgery and was well at the 9-month follow-up [2]. Case 3 had lymph node metastasis three years after the operation, suffered multiple metastases in different organs during her life, and expired approximately 5 years after the operation [3]. The common characteristic of our cases, Case 4 and Case 5 was the lack of SDHB-positive immunohistochemistry (SDHB-deficiency).

Paragangliomas in Carney-Stratakis dyad

Mutations of the SDH gene, including SDHA, SDHB, SDHC, SDHD, and SDHAF2, are causes of various hereditary pheochromocytoma-paraganglioma syndromes, including Carney-Stratakis dyad [9]. Carney [11] reported that the locations of origin for 60 paragangliomas including tumors of the Carney triad and Carney-Stratakis dyad, from 34 patients include 20 head and neck tumors (8 carotid body, 7 middle ear, 2 vagus, 2 larynx, and 1 thyroid tumors), 14 chest tumors (3 superior mediastinum, 9 aorticopulmonary body and 2 sympathetic chain tumors), and 26 abdomen tumors (7 retroperitoneum, 6 adrenal, 9 sympathetic chain, 2 organ of Zuckerkandle, and 2 liver tumors). Of these tumors, combinations of GIST and extra-adrenal PGL (Carney-Stratakis dyad) were found in 19 tumors (24%) from 79 patients. The locations of most PGLs in HPPS were the same as those of ordinary PGLs, with a few exceptions in the liver. PGLs in the colorectum were not included in Carney’s series. The Carney-Stratakis dyad is caused by any of the SDH genes [15]. The expression of podoplanin in GIST is also positive evidence of the Carney-Stratakis dyad [16].

Factors predicting patient prognosis

The patient in Case 1 was indolent 5 years after the operation and was withdrawn from clinical observation. Additionally, the patient in Case 2 died of multiple metastases of paraganglioma 5 years after the initial operation. Tumor staging was based on the TNM staging system according to the 8^th edition of the AJCC, which added a new chapter for pheochromocytoma/paraganglioma in the Cancer Staging Manual in 2017 [13]. Both tumors from Case 1 and Case 2 were T2, N0, Stage II at the time of the initial operation. However, the prognosis of these patients was quite different for these SDH-deficient colorectal PGLs. The important differences between these two PGLs were tumor size and histological grade. Hamidi and Young et al. reported the prognostic factors of PPGLs in 272 patients for over 55 years in which a larger primary tumor size was associated with rapid disease progression and a higher mortality risk [17]. Similarly, a larger tumor size (generally >4.5 to 5 cm) has been reported to be a a strong predictor of persistent or recurrent disease and mortality in several other studies, In particular, a primary tumor size ≥6 cm was found to be an important independent risk factor for metastatic disease [18]. Kimura et al. reported that the mean tumor size was 5.1 ± 0.3 cm in a nonmetastatic group (range: 1.1–20.0 cm) and 8.7 ± 0.7 cm in a metastatic group (range: 3.0–16.5 cm) from a total of 163 PPGLs (p < 0.001) [12]. The tumor sizes of the present colorectal PGLs were 7.5 cm and 2.5 cm in Case 2 and Case 1, respectively.

In addition to tumor size, Kimura et al. proposed a histological grading system for the pathological diagnosis of adrenal phaeochromocytoma and paraganglioma called GAPP to predict the possibility of metastasis and patient prognosis [12]. GAPP scores range from 0 to 10 points and are graded as one of three types: well-differentiated (0–2 points), moderately differentiated (3–6 points) and poorly differentiated (7–10 points). These types were correlated with metastasis and patient prognosis. The GAPP grading of the PGL in Case 1 was 2 points, (well differentiated type), and in Case 2, it was 7 points, (poorly differentiated type). The AJCC commented on the difficulty of reproducing histological grading due to interobserver inconsistency [13, 19]. However, GAPP was improved to be more consistent through the use of objective parameters for diagnosis, and the utility was validated by pathologists [20, 21]. In addition to our cases, Yu and Wang [2] reported their rectal PGL with lymph node metastasis that was 40 mm in size, and the histological grading by PASS was 6. These findings are highly suggestive of potentially aggressive biological behavior [22].

Patients with SDH-deficient PGLs progress with various clinical courses that range from indolent to highly malignant, or they can have a long life with multiple bone metastases [17, 23]. Therefore, the higher prevalence of malignancy in sympathetic PGL cannot be explained in every case by an association between the genetic background and tumor site alone [13]. Although the data are limited to only four cases of colorectum PGLs, it is necessary to evaluate the tumor size and histological grade of tumors to determine treatments and predict patient prognoses, even if the PGLs are in unusual sites.

In conclusion, the present cases demonstrated that SDH-mutations themselves are not necessarily an independent marker for worse prognosis. The combination of tumor size and histological grading, in addition to TNM classification, is important for predicting patient prognosis. PGLs in unusual sites including the gastrointestinal tract, may increase with the advancements of endoscopic tools, imaging, laboratory examination and genetic analysis in the future, and extensive pathologic investigation is necessary to evaluate patient prognosis.

Disclosure

None of the authors have any potential conflicts of interest associated with this research.

Disclosure Statement

The authors have nothing to disclose.

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