Keratin 20 positive SDH-deficient renal cell carcinoma: a case report and literature review

Abstract

This study aims to broaden the morphological scope of SDH-deficient renal cell carcinoma and to assist clinicians and pathologists in better understanding this entity to prevent misdiagnosis. This study used immunohistochemistry staining and the first-generation sequencing Sanger method for gene detection. It retrospectively analysed the clinical pathology, molecular characteristics, biological behaviour, and treatment information of one case of SDH-deficient renal cell carcinoma. The patient was a 57-year-old female with right back pain for more than 20 days and had no personal or family history of kidney tumours. In addition, the tumour cells had clear boundaries in morphology, and residual normal renal tubules could be seen around them. There were also ossification and adipose tissue around the tumour centre. The tumour cells were arranged in a glandular tubular and cord-like manner. Vacuolar and eosinophilic inclusion bodies could be observed in the cytoplasm. The nucleus was regular, the chromatin distribution was fine, and there were no obvious nucleoli. They were low-grade nuclei. In addition, no atypical mitosis or necrosis could been found. Furthermore, immunohistochemistry staining showed SDHB-negative and keratin 20 -positive tumour. Meanwhile, the first-generation sequencing also pointed out the presence of SDHB gene mutations in the tumour. After 12 months of follow-up, there was no evidence of disease recurrence in the patient. SDH-deficient renal cell carcinoma is a rare tumour associated with SDH gene germline mutations, and suspected cases should undergo SDHB immunohistochemistry staining. Most SDH-deficient renal cell carcinomas have a good prognosis, but undifferentiated cases require long-term follow-up.

Introduction

Succinate dehydrogenase (SDH) is an enzyme complex composed of four subunits (SDHA, SDHB, SDHC, and SDHD). In addition, SDH plays an important role in converting of succinate to succinate and in the mitochondrial electron transport chain. SDH is also essential for cellular energy metabolism. Whereas SDH-deficient renal cell carcinoma (SDH-deficient RCC) was first discovered in 2004 [1]. Then, it was included in the Vancouver classification of the International Society of Urological Pathology (ISUP) in 2013 [2] and was accepted by the World Health Organization as a morphologically unique subtype of RCC in 2016 [3, 4]. This type of renal cell carcinoma is rare, accounting for approximately 0.05% to 0.20% of all renal cell cancers [5]. At present, there are few reports on its clinical pathology and molecular characteristics. To improve our understanding of this type of renal cell carcinoma, we introduced a keratin 20-positive SDH-deficient RCC case and reviewed relevant literature.

Case presentation

Clinical information

A 57-year-old woman presented the right back pain 20 days ago without obvious cause, which aggravated after activity. For further treatment, she went to the outpatient department of our hospital few days later. The specialist examination revealed percussion pain in the right renal area. The patient was recently diagnosed with hypertension for 3 months. She denied other chronic medical histories and infectious diseases, underwent laparoscopic cholecystectomy was performed 15 years prior to presentation due to gallstones. She also denied other major trauma and surgery histories as well as any family history of hereditary diseases.

Imaging features

Enhanced computed tomography (CT) examination of the upper abdomen showed that a round-like abnormal enhancement was seen in the right kidney with a clear boundary, of about 3.5 cm. The uneven enhancement was also presented in the arterial phase, with patchy hypo-dense shadows and small flake fat density shadows (Fig. 1A). While the enhancement degree decreased in the venous phase and delayed phase (Fig. 1B-C). No enlarged lymph nodes were found in the periphery. To further confirm the diagnosis, the upper abdominal magnetic resonance imaging (MRI) plain scan was also performed and found a circular long T1 and T2 signal in the right kidney. The DWI signal was high, with a diameter of about 3.5 cm. The internal signal was uneven, with a slight fat signal (Fig. 1D-E). According to the preoperative imaging examination results, the patient was considered to have a tumour in the right kidney. Then, a right laparoscopic partial nephrectomy was operated. The postoperative CT plain scan of the upper abdominal showed the postoperative changes in the right kidney after mass resection, with a local absence of the right kidney. A dense strip-like shadow was visible in the surgical area, with mild exudation around the right kidney (Fig. 1F). There was no tumour recurrence after 12 months of tumour resection.

Fig. 1

CT and MRI scans showed a tumour in the right kidney. A Enhanced CT examination of the upper abdomen showed that a round-like anomaly enhancement may be seen in the right kidney, the boundaries were still clear, about 3.5 cm, and the arterial phase of the enhanced scanning was unevenly enhanced, with patchy hypo dense shadows and small flake fat density shadows in it. B-C The degree of enhancement in the venous phase and delayed phase was reduced. D-E Upper abdominal MRI plain scan found a circular long T1 and T2 signal on the right kidney, and a diameter of about 3.5 cm. The internal signal was uneven and there was a slight fat signal. F Postoperative upper abdominal CT plain scan showed postoperative changes in the right kidney after mass resection, with a local absence of the right kidney. A dense strip-like shadow was observed in the surgical area, with slight exudation around the right kidney

Diagnostic histological features

Resected tumour specimen was fixed in 10% neutral buffer formalin and immunohistochemistry staining was performed according to standard protocols. Paraffin-Embedded blocks were cut into 5 μm thick sections, and stained with hematoxylin and eosin, as well as various antibodies. The clones, working dilutions and commercial sources of those antibodies were listed in Table 1.

Table 1 Antibodies used in this case

In gross examination, a piece of tissue was incised with a size of 4.5 × 3.0 × 2.8 cm. A mass of 4.5 × 3.0 × 2.3 cm was seen at 0.4 cm from the cut margin. The cut surface was greyish yellow, and the lesion area was dark red with an external capsule. A grey-white area could be seen in the centre of the mass, with calcification in the lesion area, ranging from approximately 0.5 × 0.4 cm.

Histologically, low magnification showed that the tumour cells had clear boundaries, with fibrous capsule-like tissue on the outside and residual normal renal tubules around (Fig. 2A). Ossification and adipose tissue were presented in the centre of the tumour, with haemorrhage in some areas (Fig. 2B). The tumour cells were arranged in a glandular tubular and cord-like pattern, with vitreous stroma, benign tubules invasion, and scattered mast cells (Fig. 2C-D). Under the high-power microscope, tumour cells were cubic or elliptical, flocculent cytoplasm, vacuolar or eosinophilic cytoplasm, eosinophilic inclusion bodies, regular nuclei, fine chromatin, and indistinct nucleoli (Fig. 2E-F).

Fig. 2

Microphotographs showing the histopathological features of the tumour. A The tumour cells had clear boundaries, with fibrous capsule-like tissue on the outside, and residual normal renal tubules could be seen around them (H&E; × 40). B There was ossification and adipose tissue in the centre of the tumour, and some areas were bleeding (H&E; × 40). C The tumour cells were arranged in a glandular tubular and cord-like pattern, with glassy stroma and benign tubule invading (H&E; × 100). D The tumour cells were arranged in a glandular tubular and cord-like pattern, with many scattered mast cells visible within the tumour (H&E; × 100). E Tumour cells appeared cubic or oval in shape, with flocculent cytoplasm and vacuolar or eosinophilic cytoplasm (H&E; × 200). F Eosinophilic inclusion bodies could be seen, with regular nuclei, delicate chromatin, and indistinct nucleoli (H&E; × 400)

Immunohistochemistry results

IHC showed diffuse cytoplasmic positivity of SDHA (Fig. 3A). Tumour cells showed negative expression of SDHB (Fig. 3B), keratin 7 (Fig. 3C), CD10 (Fig. 3D), and carbonic anhydrase 9 (CA9). Mast cells scattered throughout the tumour expressed CD117 (Fig. 3E), while tumour cells diffusely expressed keratin 20 (Fig. 3F). The Ki-67 positive marker index for tumour cell proliferation was 3% (Table 1).

Fig. 3

Immunohistochemically analysis of the tumour. A IHC showed diffuse cytoplasmic positivity of tumour cells towards SDHA (original magnification × 40). B Tumour cells showed negative expression for SDHB in the IHC image (original magnification × 40). C IHC image showing cells were negative for keratin 7 (original magnification × 40). D IHC image showing cells were negative for CD10 (original magnification × 40). E Mast cells scattered throughout the tumour expressed CD117 in the IHC image (original magnification × 100). F IHC of keratin 20 showing diffuse cytoplasm positivity (original magnification × 100)

Molecular detection results

The first-generation sequencing Sanger method was used for gene testing, and the results showed two mutations. Heterozygous mutation in exon 3 of SDHB: C.201-36G > T. Heterozygous mutation occurred in exon 8 of SDHB: C.725G > A (Fig. 4). Based on these morphological features, as well as immunohistochemistry and genetic testing results, SDHB-deficient renal cell carcinoma was diagnosed pathologically.

Fig. 4

Sanger sequencing test results. A Heterozygous mutation in exon 3 of SDHB: C.201-36G > T. B Heterozygous mutation in exon 8 of SDHB: C.725G > A

Discussion

Succinate dehydrogenase (SDH), also known as mitochondrial complex 2, is a key enzyme that oxidizes succinate to fumaric acid in the tricarboxylic acid cycle. It consists of SDHA, SDHB, SDHC, and SDHD protein subunits. It also participates in the tricarboxylic acid cycle, mitochondrial electron transfer chain, as well as electron transfer in the respiratory chain [6]. The absence of any subunit of SDH may lead to the instability of these complexes, resulting in complete loss of enzyme function, accumulation of succinic acid in cells and induction of tumour development. In addition, mutations or abnormal expression of SDH-related genes are associated with a variety of diseases, including pheochromocytoma/ paraganglioma, gastrointestinal stromal tumours, renal cell carcinoma, and pituitary adenoma.

SDH-deficient renal cell carcinoma (RCC) is a rare subtype of RCC, accounting for approximately 0.05%-0.20% of all RCCs [5], and was recently recognized as a unique subtype in the 2016 World Health Organization classification [3, 4]. The RCC with SDH deficiency is mainly seen in young people, with an average age of 37 (patients 14 to 76 years old), with males predominating, with a male-to-female ratio of 1.7:1 [6, 7]. Tumours are highly correlated with genetics. Patients often have germline mutations in SDH-related genes, mainly SDHB mutations, followed by SDHC, SDHA, and SDHD, which are rare and cause mitochondrial complex 2 dysfunctions, leading to tumour development. It is estimated that the lifetime risk of developing kidney tumours in patients with SDHB gene mutations is 14% [7]. Clinically, most SDH-deficient renal cell carcinoma presents as small organ-localized tumours occasionally found on imaging, or as lower back pain or accidental findings. In rare cases, tumours may manifest as metastatic diseases. At long-term follow-up, approximately 30% of patients presented with multifocal or bilateral renal tumours, 15% had a personal history of gastrointestinal stromal tumours (GIST), 15% had a personal history of paragangliomas (PGL), 22% had a family history of RCC, 26% had a family history of PGL positivity, and 4% had a family history of GIST positivity [6, 7]. Our patient was a 57-year-old woman with a right renal mass due to back pain. No personal or family history of renal cell carcinoma, GIST, pheochromocytoma, or PGL exists. The clinical presentation of this patient differs from previous reports, which may expand the clinical spectrum of the disease.

Pathologically, the tumour is 0.7 to 20.0 cm in size, with an average of 5.1 cm [8]. The tumour is well-defined and can be surrounded by a pseudo capsule. Its cross-section is brownish or reddish brown. Haemorrhage or partial cystic changes can also be seen, usually without necrosis. Microscopically, SDH-deficient RCC has clear boundaries, but it is often seen that glomeruli and renal tubules are located at the "edge" (i.e. outside) of the tumour, but interestingly, they are sometimes located within the tumour. Tumours are arranged in solid patchy or glandular tubular shapes, with tumour cells appearing round or oval in shape, round nuclei, evenly distributed chromatin, and blurry nucleoli. The most typical features are eosinophilic, vacuolar, or flocculent inclusion bodies in the cytoplasm of tumours. In most cases, SDH-deficient RCC is a low-grade tumour, but high-grade nucleoli, sarcomatous changes, or coagulation necrosis may also be present. In addition, mast cell infiltration might be seen in the tumour stroma. The morphologic appearance of our patients essentially maintained the described typical appearance caused by SDH-deficient RCC.

When any component of mitochondrial complex 2 undergoes genetic inactivation, the entire complex becomes unstable, leading to the degradation of SDHB subunits. Therefore, the absence of SDHB immunohistochemistry staining is necessary for diagnosing SDH-deficient RCC. In RCC with SDHB, SDHC, and SDHD deficiency, tumour cells show SDHB-negative while SDHA-positive. In contrast, tumour cells in SDHA-deficient RCC are negative for both SDHA and SDHB [9], and the immunohistochemical interpretation of SDHB expression requires comparison with surrounding normal renal tissue. SDH-deficient RCC typically exhibits immune reactivity to PAX-8, EMA, and Ksp-cadherin, but is negative for keratin 7, keratin 20, AE1/AE3, CD117, RCC antigens, P63, CA9 and vimentin. Neuroendocrine and epithelial markers are also generally negative, but CD117 may highlight mast cells in tumours. Under electron microscopy, in SDH-deficient RCC, the cytoplasm of tumour cells contains abundant mitochondria, and the cytoplasmic inclusion bodies correspond to mitochondria with degenerated or compressed cristae and matrix abnormalities [10] Molecular genetics indicates that SDH-deficient RCC has strong heritability and a germline mutation in one of the SDH-related genes. Among them, SDHB mutations are the most common, followed by SDHC and SDHD. Ricketts et al. [11] studied 14 cases of SDHB-deficient renal cell carcinoma and found that 8 exons of the SDHB gene could be mutated. Our patient had heterozygous mutations in exons 3 and 8 of SDHB, C.201–36 G > T; C.725 G > A. All of the above mutations can affect the expression of proteins, which is consistent with the SDHB-negative result detected by immunohistochemistry. There had been many reports of mutations in the third exon of SDHB, while in the cases reported by Zhu Q et al. [6], there were gene mutations at the same site as our cases: C.725 G > A (guanine > adenine), resulting in changes in amino acids R242H (arginine > histidine), which had been reported as a pathogenic mutation [12]. In addition, our patient showed a positive expression for keratin 20. We reviewed previous cases (Table 2) [6, 13,14,15,16,17,18,19,20,21,22,23,24,25] and literature [7] and found that 5 patients underwent keratin 20 staining [7, 14, 15, 23], and we were the first reported case of SDHB-deficient RCC expressing keratin 20.

Table 2 The clinical and pathological characteristics of SDH-deficient RCC were reported in the past five years

The differential diagnosis of SDH-deficient RCC is mainly other renal tumours with eosinophilic morphology, such as oncocytoma, chromophobe renal cell carcinoma, clear cell renal cell carcinoma, eosinophilic solid cystic renal cell carcinoma, etc. Oncocytoma exhibits characteristic island-like structures, central scar, round or oval nucleus, and relatively mild nuclear atypia. Immunohistochemistry mostly expresses CD117, while keratin 7 is scattered or focal stained. Most SDH-deficient RCCs usually show negative results for CD117 and keratin 7. In addition, one of the diagnostic features of SDH-deficient RCC is the presence of eosinophilic, vacuolar, or flocculent inclusion bodies in the cytoplasm. Whereas, eosinophilic cells are usually absent in oncocytoma. Chromophobe cells of the eosinophilic subtype RCC are also a factor to consider in differential diagnosis. The tumour cell membrane is clear, the nucleus is wrinkled like a raisin, and paranuclear halos are often visible. Immunohistochemistry expressions of keratin 7, CD117, and SDHB are associated with the absence of intracytoplasmic inclusion bodies, which helps to distinguish this entity from SDH-deficient RCC. When eosinophilic cytoplasm appears in clear cell RCC, it is mostly a high-grade nucleus, and the classic clear cell region may exist elsewhere. Immunohistochemistry shows positive results for SDHB, CA9, vimentin, and CD10, all of which are different from SDH-deficient RCC. Eosinophilic solid cystic renal cell carcinoma (ESC RCC) is a tumour with or without nodular sclerosis. The tumour is mainly composed of eosinophils, forming solid and cystic structures. Tumours also have abundant eosinophilic cytoplasm, and in some cases, eosinophilic glomeruli and vacuoles can be seen in the cytoplasm. The characteristic immunohistochemistry in ESC RCC is keratin 20 positivity (patchy or diffuse), keratin 7 deficiency or weak positivity, and CD117 is usually negative [26]. And the RCC with TFEB amplification can also be positive for keratin 20 [27]. However, in this case, SDHB-deficient RCC also expressed keratin 20. Therefore, keratin 20 positivity cannot distinguish these three types of tumours, and gene abnormalities need to be detected through immunohistochemically expression and sequencing of SDHB and SDHA. Attention should also be paid to distinguishing SDH-deficient RCC from RCC/FH-deficient RCC associated with hereditary leiomyomatosis and RCC syndrome. A few FH-deficient RCCs have also been reported to exhibit low-grade eosinophilic morphology [28]. Tumours have flocculent and vacuolated eosinophilic cytoplasm, which is very similar to SDH-deficient RCC. However, immunohistochemistry shows FH deficiency, of 2-succinylcysteine (2-SC) overexpression and SDHB retention, all different from SDH-deficient RCC.

There are no evidence-based medical guidelines for the treatment of SDH-deficient renal cell carcinoma. Surgical resection is still the preferred treatment method. If the tumour is early or small, partial nephrectomy or tumour ablation is usually chosen to preserve the kidney. For advanced renal cell carcinoma, FDG-PET examination is recommended to determine the current growth status of the tumour. For metastatic diseases, molecular targeted therapy with vascular endothelial growth factor (VEGF), mammalian target of rapamycin (mTOR), and tyrosine kinase (TK) has been previously administered [8, 29]. It is suggested that molecular analysis of SDHB may not be necessary if the patient is over 40–45 years old and has no family history of renal tumours. However, for patients under the age of 45, even if there is no family history, the possible diagnosis of SDH-deficient RCC should be considered [30]. Through this case, we find that for patients with kidney tumours, it is necessary to inquire about the disease condition and family history of the tumour in detail. Regardless of the patient's age, SDHB immunohistochemically staining and genetic testing are necessary for diagnosis and can guide clinical treatment decisions.

Most SDH-deficient RCCs are low-grade tumours with good prognosis. Long-term follow-up shows that the metastasis rate is about 11% [31], which can be cured by complete resection alone. However, coagulation necrosis, high-grade nuclei, and sarcomatous changes have been reported in some tumours, and they have more positive progression and higher metastasis rates (possibly up to 70%) [5], requiring curative treatment. Metastasis in the lungs, liver, bones, brain, and lymph nodes has been reported [30]. Therefore, it is necessary to conduct long-term follow-up and monitoring of SDH-deficient tumours.

In conclusion, SDH-deficient RCC is a rare tumour associated with SDH gene germline mutations, with unique clinical and pathological features. For renal eosinophilic tumours with inclusion bodies in the cytoplasm, pathologists should consider the possibility of SDH-deficient RCC. Even if there is no inclusion body or inclusion body is not obvious, SDHB immunohistochemistry staining should be performed on suspected cases. After diagnosis, patients and immediate family members should monitor other SDH-deficient tumours, including pheochromocytoma, paraganglioma, and GIST. Most SDH-deficient RCCs have a good prognosis, but for undifferentiated cases, the prognosis is poor and requires long-term follow-up. For tumours without adverse pathological features, separate resection may be a reasonable choice. With the improvement of physician awareness, patients can receive correct diagnosis and timely treatment. Therefore, pathologists and clinicians should hold a highly sceptical attitude towards this eosinophilic renal tumour.