Original Research

Succinate dehydrogenase-deficient renal cell carcinoma: A retrospective study, 1999–2018

Ella Morrison, Jacqueline Goedhals, Shireen Pretorius, Gina Joubert, Anthony Gill, Gerhard van der Westhuizen

Received: 17 Oct. 2022; Accepted: 16 Feb. 2023; Published: 08 June 2023

Copyright: © 2023. The Author(s). Licensee: AOSIS.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background: Succinate dehydrogenase-deficient (SDH-deficient) renal cell carcinoma (RCC) is a rare subtype of RCC. Approximately 0.05% – 0.2% of RCCs are SDH-deficient. It is usually associated with a pathogenic germline variant of the enzyme. Although the tumour is often indolent, it has a strong association with hereditary disease, most commonly paraganglioma and/or pheochromocytoma, and genetic testing is advised. Succinate dehydrogenase-deficient RCC is diagnosed by immunohistochemical (IHC) staining for SDH subunit B (SDHB), with loss of staining confirming SDHB deficiency. The incidence of SDH-deficient RCC in South Africa is unknown.

Aim: To determine the incidence of SDH-deficient RCC in the Free State Province and its correlation with the worldwide incidence of 0.05% – 0.2%.

Setting: Department of Anatomical Pathology, Universitas Academic Hospital and National Health Laboratory Service (NHLS) in Bloemfontein, South Africa.

Methods: A retrospective descriptive study was performed. All primary RCCs diagnosed over a 20-year period (1999–2018) were included. An SDHB IHC stain was performed on all cases. A diagnosis of SDH-deficient RCC required loss of staining in all tumour cells in the presence of internal positive controls.

Results: The study included 187 RCC cases. Two cases of SDH-deficient RCC were identified, representing an incidence of 1.1%. Both patients were male and were 22 and 44 years of age, respectively.

Conclusion: The incidence of SDH-deficient RCC in central South Africa is slightly higher than global findings.

Contribution: This study provides new data on SDH-deficient RCC in the South African context.

Keywords: renal cell carcinoma; RCC; succinate dehydrogenase; SDH; SDH-deficient RCC; incidence; diagnosis.

Introduction

Succinate dehydrogenase- (SDH)-deficient renal cell carcinoma (RCC) is a rare subtype of RCC, estimated to comprise 0.05% – 0.2% of all cases of RCC globally.^1,2 It was recently added to the expanding list of subtypes of RCC.³ The mean age at presentation is 38 years, with a wide age range (14–76 years) and a male predominance of 1.7:1.¹

Succinate dehydrogenase is a respiratory enzyme that forms part of both the respiratory chain and the tricarboxylic acid pathway, or Krebs cycle.^2,4 It is the only enzyme involved in both these cycles.^2,4,5 Succinate dehydrogenase consists of four subunits, namely subunits A-D (SDHA, SDHB, SDHC and SDHD), all encoded by autosomal deoxyribonucleic acid (DNA). Succinate dehydrogenase assembly factor 2 (SDHAF2), also encoded by autosomal DNA, combines the subunits at the inner mitochondrial membrane where it forms mitochondrial complex 2 (SDH/succinate-ubiquinone oxireductase). Succinate dehydrogenase assembly factor 2 is needed for the flavination and functioning of SDHA.²

When SDH is inactivated, oxidative phosphorylation is impaired, which requires the cell to employ glycolysis to produce energy (‘aerobic glycolysis’) that, according to the Warburg hypothesis, is one of the hallmarks of cancer.^6,7,8 Inactivation of SDH occurs due to the loss of one of the subunits or the assembly factor. Loss of a subunit is caused by bi-allelic inactivation of the specific gene encoding for the subunit, leading to either failure or instability in SDH complex formation. This results in the release of SDHB into the cytoplasm where it is degraded.^2,4 The degradation of SDHB leads to the loss of immunohistochemical (IHC) staining with SDHB antibodies, which occurs in SDH-deficient neoplasms, including SDH-deficient RCC.²

Currently, it is accepted that bi-allelic inactivation almost exclusively occurs when a germline variant is present, in addition to a somatic second hit.⁴ Isolated cases of sporadic SDH-deficient RCC due to purely somatic events have been reported, although they are very rare.^9,10 Similarly, SDH deficiency not associated with syndromic disease has been reported in paraganglioma, but is exceptional.¹¹ Therefore, if SDH deficiency, that is loss of SDHB IHC staining, is detected in a tumour, then genetic counselling and testing for germline mutation in the SDH genes are recommended.²

Succinate dehydrogenase deficiency was first described in pheochromocytomas and paragangliomas followed by gastrointestinal stromal tumours (GIST) and RCCs.¹² Succinate dehydrogenase-deficient RCC was first reported by Vanharanta et al.¹² in 2004 as an entity related to ‘SDHB-associated heritable paraganglioma’. Since its initial description in 2004, the understanding of SDH-deficient RCC has evolved to a great extent. Succinate dehydrogenase-deficient RCC was included as a provisional entity in the classification of renal neoplasia by the classification working group of the International Society of Urological Pathology (ISUP) in 2013 before its addition to the World Health Organization (WHO) classification as a recognised entity.^1,2,13

The majority of SDH-deficient RCCs are indolent neoplasms. A small proportion shows tumour necrosis or high-grade transformation and has a higher risk of metastatic disease.^2,5 Patients with SDH deficiency are prone to develop other tumours, including paraganglioma, GIST and recurrent or bilateral RCCs as part of hereditary paraganglioma/pheochromocytoma syndrome.^1,2,5

Identification of patients with germline SDH deficiency is crucial as they need both genetic counselling and/or testing and regular surveillance for other SDH-deficient tumours. Syndromic disease includes SDH-deficient pheochromocytoma/paraganglioma, GIST and RCC.⁵ Pituitary adenomas are also associated with SDH deficiency but are relatively rare.² Because of the association of a germline variant, genetic testing must be performed to identify the affected gene. Family members can then be offered cascade genetic testing allowing regular screening and early intervention.^14,15,16 The risk of developing RCC in patients with an SDHB variant is not yet clear, but is estimated to be up to 10% – 15%.¹⁵

Suspicion of the presence of heritable RCC should be raised in the presence of bilateral, multicentric RCC or recurrent RCC. Associated tumours, such as GIST or pheochromocytoma/paraganglioma, are red flags for SDH deficiency.¹⁵

Some hereditary RCC syndromes have set treatment guidelines. For example, tumours of Von-Hippel Lindau and Birt-Hogg Dube syndromes are followed up until a size of 3 cm is reached and then excised with nephron-sparing surgery.¹⁵ Fumarate hydratase- (FH)-deficient RCC is considerably more aggressive and should be excised as soon as detected.¹⁵ Specific guidelines for treatment of SDH-deficient RCC detected at an early stage have not yet been compiled; however, nephron-sparing surgery may be appropriate.¹

To the authors’ knowledge, no similar studies have been conducted in Africa or South Africa to determine the incidence of SDH-deficient RCC in an African setting. A single case report of a 13-year-old female patient with bilateral SDH-deficient RCCs, treated in Gaborone, Botswana, could be located in the literature.¹⁷ In light of this lack of information, this study set out to evaluate the number of patients diagnosed with SDH-deficient RCCs in the public sector of the Free State province during a 20-year period. In addition, the morphology of the tumours in this study’s population was compared with the typical morphology described in the literature.

Methods

Study design and sample acquisition

A retrospective descriptive study was performed. A Systemized Nomenclature of Medicine (SNOMED) search of the National Health Laboratory Service (NHLS) electronic databases was performed to identify all primary RCCs and RCC subtypes diagnosed by the Department of Anatomical Pathology, NHLS Universitas Academic Laboratories and University of the Free State (UFS).

Data collection

Cases of metastatic RCC were excluded from the study if no biopsy of the primary renal tumour was available. Specimens obtained by means of biopsy, enucleation, partial and total nephrectomy from 01 January 1999 to 31 December 2018 were included. Information for the period 2005 – August 2014 was obtained from the Disa*Lab Laboratory Information System (LIS) (DISA system), while the LabTrak system was searched for the period September 2014–2018. A manual search of archived paper records was performed for the period 1999–2004. Once all the cases had been identified, the patient’s age, gender, relevant history, clinical presentation and results of other IHC stains were obtained from the pathology report and recorded in a Microsoft Excel spreadsheet designed for data collection. Only the pathology reports of the RCC cases were available for study and it was therefore not possible to obtain additional clinical data suggestive of syndromic disease or any family histories, unless it was stated on the pathology request form.

Laboratory methods

The slides from the cases identified were retrieved from the departmental archives and reviewed by a registrar (resident in training) and a pathologist. During the review to select a representative slide of each case, the tumour was classified according to the most recent WHO classification published in 2016,³ as many of the new entities had not yet been described during the earlier years of the study period. The cases from 1999 to 2004 and from 2015 to 2018 were classified by the registrar and the pathologist. The data reported in an unpublished Master’s dissertation (ethics reference number HSREC UFS-HSD2017/1432), ‘Renal cell carcinoma diagnosed by the Department of Anatomical Pathology at the University of the Free State, South Africa: a 10-year histopathological review’,¹⁸ were used for 2005–2014.

The wax block of the selected representative slide was retrieved from the departmental archives. Sections of 4 µm were cut and stained with anti-SDHB mouse monoclonal antibody (AB14714, Biocom Diagnostics; Centurion, South Africa). A dilution of 10:1000 was used. The stains were performed with both positive and negative controls, using a Benchmark XT automated slide preparation system (Ventana Medical Systems Inc., Tucson, Arizona, United States). The slides were then counterstained with Mayer’s haematoxylin, dehydrated and cover-slipped.

The IHC stains were evaluated separately by a registrar and a pathologist to determine whether the tumour retained SDHB staining or demonstrated loss of staining. Any discrepancies in their findings and all cases with negative IHC staining were referred to a second pathologist for confirmation. The opinion of an internationally renowned expert in the field of SDH-deficient neoplasia^1,2,4 was also obtained on cases showing loss of SDHB staining.

Retained SDHB staining was recorded with granular cytoplasmic staining in the tumour cells. Loss of SDHB staining was recorded with complete absence of staining in the tumour cells in the presence of retained staining of internal and external controls. Internal controls included normal renal parenchyma and endothelial cells. The cases showing loss of SDHB IHC staining were then stained with anti-SDHA mouse monoclonal antibody (AB14715, Biocom Diagnostics; Centurion, South Africa) using a dilution of 8:1000. The same criteria applied for SDHB staining were used for the interpretation of the SDHA stains.

Statistical analysis

Statistical analysis was performed by the Department of Biostatistics, UFS. Results were expressed in frequencies and percentages (categorical variables) and medians and interquartile ranges (IQR) (numerical variables due to skew distributions). A 95% confidence interval (CI) was calculated for the main outcome. The statistical analysis was performed using SAS version 9.4 (SAS Institute Inc., Cary, North Carolina, United States) software.

Ethical considerations

Approval to perform the study was obtained from the Health Sciences Research Ethics Committee (HSREC) of the UFS (ethics reference number UFS-HSD2018/0428/3107). Due to the retrospective nature of the study and using archived specimens, patient informed consent was not required.

Results

During the 20-year study period (1999–2018), a total of 215 RCC diagnoses were identified. Twenty-eight cases did not meet the inclusion criteria and the final study sample comprised 187 cases.

Loss of SDHB IHC staining in all the tumour cells was detected in two (1.1%, 95% CI: 0.3% to 3.8%) of the 187 cases in the presence of adequate positive internal and external controls. Retained granular cytoplasmic staining was therefore present in 185 (98.9%) cases. Both cases with loss of SDHB staining showed retained SDHA staining in the tumour cells. The two SDH-deficient RCC cases are briefly described below.

Case 1

The first patient with SDH-deficient RCC was a 41-year-old male patient who initially presented with metastatic RCC of the iliac wing. On further examination, a left-sided renal mass was detected on which a needle biopsy was performed. The size of the tumour was not known. The initial diagnosis was RCC unclassified.

Histologic examination (see Figure 1) showed the presence of RCC with a nested growth pattern. The individual cells had indistinct cell borders and the cytoplasm was eosinophilic with multiple vacuoles and cytoplasmic inclusions. The nuclei were large with prominent nucleoli, consistent with an ISUP nuclear grade 3. No necrosis was present in the tissue submitted to the laboratory.

FIGURE 1: Case 1. Succinate dehydrogenase (SDH)-deficient renal cell carcinoma. (a) Haematoxylin and eosin (H&E) of case 1 (100× magnification). (b) H&E of case 1 (200× magnification). (c, d) SDHB immunohistochemical stain (both 400× magnification). (e) SDHA immunohistochemical stain (400× magnification). (f) SDHA immunohistochemical stain (200× magnification).

The tumour cells all showed loss of SDHB IHC staining. No non-neoplastic renal tissue was present in the biopsy but scattered inflammatory cells and endothelial cells were present, which provided an internal control. The internal controls showed distinct granular cytoplasmic staining. The SDHA IHC stain showed retained SDHA staining (Figure 1).

Images of the haematoxylin and eosin (H&E) slides and both the SDHB and SDHA stains were sent to an expert in the field for his professional opinion. His comment was that the morphology is not typical but is not inconsistent either, and that the loss of staining was sufficient to be considered diagnostic. The tumour displayed a higher nuclear grade (ISUP 3), which occurs in 20% of SDH-deficient RCCs.¹⁴

This case is a clear illustration of the relationship between higher nuclear grade (ISUP/WHO grade 3) and increased risk of metastasis.^1,5 As noted, the patient initially presented with metastatic RCC to the iliac wing.

Case 2

The second patient with SDH-deficient RCC was a 22-year-old male patient with a left-sided renal tumour for which a radical nephrectomy was performed. The tumour measured 110 mm in maximum diameter, which correlated with the American Joint Committee on Cancer (AJCC) tumour stage of pT2b.¹⁹ No mention was made of metastases.

Histological examination (see Figure 2) showed a well-circumscribed but unencapsulated primary RCC. Benign renal tubules were entrapped at the periphery. The tumour cells were polygonal with indistinct cell borders and abundant eosinophilic cytoplasm. Cytoplasmic vacuoles were clearly evident. Some of the tumour cells contained wispy eosinophilic material, with flocculent eosinophilic cytoplasmic inclusions in others. The nuclei were round with dispersed chromatin and inconspicuous nucleoli, consistent with ISUP nuclear grade 2.³ The growth patterns observed were solid, nested and tubular.

FIGURE 2: Case 2. Succinate dehydrogenase (SDH)-deficient renal cell carcinoma. (a) Haematoxylin and eosin (H&E) of case 2 (200× magnification). (b) H&E of case 2 (400× magnification). (c) SDHB immunohistochemical stain showing loss of staining in tumour cells. Entrapped normal tubules showing retained staining (200× magnification). (d) SDHB immunohistochemical stain with loss of staining (400× magnification). (e) Retained SDHA immunohistochemical stain (200× magnification). (f) Retained SDHA immunohistochemical stain (200× magnification).

Succinate dehydrogenase subunit B (SDHB) IHC staining was negative in all the tumour cells, with obvious granular cytoplasmic staining of the surrounding renal parenchyma and endothelial cells. Both the internal and external controls were positive with granular cytoplasmic staining. The SDHA IHC stain showed retained staining. The staining pattern confirmed the diagnosis of SDH-deficient RCC.

The morphological features of this case have been reported as part of an international series of SDH deficient RCC.²⁰ Briefly, compared to Case 1, this case demonstrated more classical morphological features of SDH-deficient RCC, which included eosinophilic cells in a nested growth pattern,^1,2,3,5 the presence of cytoplasmic vacuoles and inclusion-like spaces and entrapped normal tubules at the periphery.^1,21

Discussion

Two cases of SDH-deficient RCC were identified in a study population of 187 cases, comprising an incidence of 1.1% with a 95% CI of 0.3% to 3.8%, which is slightly higher than the worldwide incidence of 0.05% – 0.2%.¹

Succinate dehydrogenase-deficient RCC usually demonstrates characteristic morphological features. Macroscopically, the tumours usually consist of well circumscribed, mostly solid, sometimes cystic lesions, which reveal a brown or red interior surface on sectioning.^1,3,5 Succinate dehydrogenase-deficient RCC most commonly presents as a single tumour ranging between 7 mm and 90 mm, with an average size of 51 mm.⁵ In 30% of cases, tumours occur bilaterally or are multifocal.^1,5

The specific microscopic features exhibited by the tumours include eosinophilic cells in diffuse sheet-like or nested growth patterns and cytoplasmic vacuoles.^2,14 The presence of cytoplasmic vacuoles and inclusion-like spaces is described as being the most typical finding and were found in both cases in this study.^1,2,21 However, these inclusions may be inconspicuous. The cell borders are indistinct. The cytoplasm is described as flocculent, or as having wispy pale material in the cytoplasm. It is important to note that the cytoplasm is not granular, as seen in oncocytes. This is an important distinction, as SDH-deficient RCC is often mistaken for oncocytoma.² Benign tubules are frequently found entrapped within the tumour.^1,2,5,14

More than half (55%) of SDH-deficient RCCs have a low ISUP nucleolar grade of 1 or 2. International Society of Urological Pathology nucleolar grade 3 and grade 4 features are found in 20% and 25% of tumours, respectively. The grade 4 component is usually only focal.¹⁴

Coagulative necrosis is mostly absent,¹ but when present, its occurrence is only focal.¹⁴ Succinate dehydrogenase-deficient RCC is usually associated with a low risk (11%) of metastasis and generally has a good prognosis. However, high-grade transformation has been described.^1,2 The occurrence of sarcomatoid change, higher nucleolar grade and coagulative necrosis predicts a worse prognosis, with an increased potential metastatic rate of 70%.^2,5

To interpret both SDHA and SDHB IHC stains, certain criteria should be fulfilled. It is specifically required that an internal positive control is present in the form of non-neoplastic cells.^2,4 Only when the non-neoplastic tissue stains positive, the IHC for SDHB can be reported as negative, therefore showing loss of staining. When the positive control is not present, the stain cannot be interpreted. Positive staining is characterised by a granular cytoplasmic pattern of staining, indicating a mitochondrial pattern.^2,4 Negative staining refers to the complete absence of the granular cytoplasmic staining pattern in all the malignant cells. Gill et al.⁴ stated that ‘only cases which are completely negative yet maintain a positive internal control in non-neoplastic cells, are considered informative’.⁴

Care should be taken when faced with a tumour in which the cells have abundant clear cytoplasm, as they erroneously can be interpreted as showing loss of staining. An attempt should be made to locate the area of the tumour with the most eosinophilic cytoplasm and the IHC stain should be interpreted in that region.² An example of clear cell RCC (CCRCC) with scanty cytoplasmic staining observed during the evaluation of cases for this study, is depicted in Figure 3. In the same field, cells with granular cytoplasmic staining are noted, confirming retained SDHB staining.

FIGURE 3: (a) Clear cell renal cell carcinoma (RCC) with focal apparent loss of staining (200× magnification). (b) Clear cell RCC with focal apparent loss of staining (400× magnification). Retained granular staining can be seen in the upper left corner of the image. (c) Clear cell RCC with sarcomatoid change (100× magnification). Retained granular staining is only focally present. (d) Clear cell RCC with sarcomatoid change (200× magnification).

Another approach when interpreting IHC staining in a tumour with clear cytoplasm is to use another mitochondrial IHC stain, such as FH. When both SDHB and FH display negative staining, the likelihood of true SDH deficiency decreases markedly.²

An observation made during the review of the SDHB slides in this study was that some tumours with clear cytoplasm gave the impression that the cytoplasm was displaced to the periphery of the cell, creating an imitation of ‘membranous’ staining, with obvious retained staining as depicted in Figure 4. This pattern has not previously been reported.

FIGURE 4: Clear cell renal cell carcinoma with retained SDHB staining giving an impression of a membranous staining pattern. Tumour cells on the lower right show the expected granular cytoplasmic staining (200× magnification).

Both of the patients with SDH-deficient tumours in this study were young males. The cases, however, were too few to make a reliable comment on gender ratio. The mean age of the two cases was 31.5 years, which corresponded to findings published in the literature, as the average age at presentation with SDH-deficient RCC is wide.¹ Gill et al. reported that the mean age of presentation is 38 years,¹ while Maclean et al.¹⁴ found that the mean age is 43.1 years, although both of these studies indicated a wide range in age at presentation.^1,14 The findings of the 41-year-old male in this study (Case 1) were in keeping with the observation of increased risk of metastasis in tumours with higher nuclear grade (ISUP/WHO grade 3).^1,5 As noted, the patient initially presented with metastatic RCC to the iliac wing.

The typically described morphology does not occur in all cases of SDH-deficient RCC, and recently variant morphologies have been reported more frequently.²⁰ One should therefore consider the patient profile and history along with the histological features.⁴ For example, in an effort to reclassify 33 eosinophilic tumours in young patients (< 35 years) as FH-deficient, SDH-deficient or eosinophilic solid and cystic carcinoma, Li et al.²² detected eight SDH-deficient RCCs that had previously been labelled as unclassified. Only four of the eight tumours displayed the morphology described as typical. Two cases mimicked oncocytoma and two tumours had a biphasic appearance.²²

Although SDH-deficient RCC is a rare subtype of RCC, a high index of suspicion should be maintained when faced with a tumour with cells containing eosinophilic cytoplasm, especially in children and young adults, or patients with unclassified or oncocytic renal tumours. Therefore, a low threshold for SDHB immunohistochemistry is recommended in the assessment of any renal tumour that is difficult to classify, particularly if there is eosinophilic cytoplasm.^20,22,23,24 The morphological features such as intra-cytoplasmic vacuoles and flocculent cytoplasm can be subtle and should be searched for carefully.^4,25 Although SDH-deficient RCCs are frequently low-grade tumours, variant morphologies, sarcomatoid transformation and coagulative necrosis are associated with a high risk of metastatic disease.^1,20,24,25 Also, variant morphologies are increasingly being recognised.^20,22

Conclusion

Succinate dehydrogenase-deficient RCC is a rare subtype of RCC, generally displaying indolent behaviour. This study identified two (1.1%) out of 187 cases of RCC over a 20-year period. This finding is slightly higher than the worldwide incidence of 0.05% – 0.2%.¹

Although rare, it is important to identify SDH-deficient RCC because of the strong association with syndromic disease – most commonly paraganglioma/pheochromocytomas. This has important implications for the patient and the family, as they require counselling and testing with regular follow-up for syndromic disease.¹⁶

According to the authors’ knowledge, no other studies exploring the incidence of SDH-deficient RCC have been performed in Africa or South Africa. Further studies to ascertain the incidence in the other provinces of South Africa, and also in the private healthcare sector, can be beneficial. The inclusion of oncocytomas should also be strongly considered, as it forms part of the differential diagnosis of RCC.

Acknowledgements

The authors would like to thank Dr. Nicholas Pearce for guidance on the data analysis, the NHLS Universitas Academic Unit for the use of the facilities, and Dr. Daleen Struwig, medical writer/editor, Faculty of Health Sciences, UFS, for technical and editorial preparation of the article.

The authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article.

E.M. was the principal investigator, applied for funding, searched the databases for relevant cases, did the data collection and analysis of the data, retrieved slides and wax blocks from the archives, analysed and interpreted the IHC stains, identified the SDH-deficient RCC cases and wrote the first draft of the article. J.G. conceptualised the study, assisted with application for funding, was available in an advisory position, was consulted in identifying and confirming SDH-deficient RCC cases. S.P. assisted with retrieval of archived slides and wax blocks and prepared and performed the IHC stains. G.J. performed the statistical analysis of the data and assisted with interpretation of the results. A.J.G. reviewed cases and provided expert consultation opinions. G.v.d.W. was the primary supervisor, assisted in identifying the cases included in the study and identification of the SDH-deficient RCC cases. All the authors reviewed and approved the final version of the article.

This research was funded by the National Health Laboratory Service Research Trust.

All data generated or analysed during the study are included in this published article. Any further enquiries can be directed to the corresponding author, E.M.

The views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors.

References