A Rare Case of Synchronous Fumarate Hydratase-Deficient Renal Cell Carcinoma and Clear Cell Renal Cell Carcinoma With Fumarate Hydratase and von Hippel-Lindau Gene Mutations: A Clinicopathologic and Molecular Study

Introduction

Fumarate hydratase-deficient renal cell carcinoma (FH-deficient RCC) is characterized by either germline or somatic fumarate hydratase (FH) variants, newly named by the 2022 World Health Organization (WHO) classification of renal cell tumors. ¹ Hereditary leiomyomatosis and renal cell carcinoma (HLRCC)-associated renal cell carcinoma (RCC) is associated with germline FH mutations, which was first listed as a separate subtype in the 2016 WHO classification of renal cell tumors. ² The patients with HLRCC syndrome are characterized by FH-deficient cutaneous or uterine leiomyomas and FH-deficient RCCs. ² FH is located on chromosome 1q43 and encodes fumarate hydratase, which catalyzes the reversible hydration/dehydration of fumarate to malate in the tricarboxylic acid cycle. Studies have shown that accumulated fumarate stabilized hypoxia-inducible factor 1-alpha (HIF-1α) and spontaneously reacted with cysteine sulfhydryl groups to form 2-succinylcysteine (2SC), leading to dysregulation of cellular metabolism and promoting tumorigenesis. ³ It has been reported that negative FH staining combined with positive 2SC staining is sensitive and specific in diagnosing FH-deficient RCC. ⁴ FH-deficient RCC is a clinically aggressive tumor and frequently presents with locally advanced or metastatic disease.^5,6

Clear cell renal cell carcinoma (clear cell RCC) is the most common subtype of RCC. ⁷ The majority of clear cell RCCs result from somatic mutations, loss of heterozygosity (LOH), or promoter hypermethylation of the von Hippel-Lindau (VHL) gene, leading to abnormal expression of VHL and accumulation of HIFs, ultimately promoting angiogenesis and development of clear cell RCC. ⁸ CA9 is a downstream molecule in HIF-pathway. Accumulation of HIF results in the up-regulation of the expression level of CA9. ⁹ Multiple studies have documented that CA9 is overexpressed in most clear cell RCCs.^9,10

Bilateral synchronous RCC is rare and occurs in less than 5% of all RCCs patients. ¹¹ Most are associated with VHL syndrome. VHL syndrome is an inherited condition caused by VHL germline mutations. Patients with VHL syndrome are at an increased risk of developing clear cell RCCs. ¹² In patients with bilaterally heterogeneous RCC, clear cell RCC with contralateral papillary RCC or chromophobe RCC has been documented. ¹³ Two examples of bilateral renal tumors with clear cell RCC and FH-deficient RCC morphologies have been reported previously; however, neither of them showed mutations related to clear cell RCC in the clear cell RCC-like tumors.^14,15 In this study, we report a rare case of synchronous clear cell RCC (right) and FH-deficient RCC (left) likely driven by somatic VHL mutation and germline FH mutation, respectively, and discuss the difference between clear cell RCC and FH-deficient RCC.

Case Presentation

Clinical Data

A 60-year-old man was admitted to the West China Hospital (China) with synchronous bilateral renal tumors detected using color Doppler ultrasonography. Computed tomography (CT) revealed a 3.6 cm × 3.3 cm slightly low-density mass with unclear boundary in the right kidney and a 2.8 cm × 2.3 cm mixed density nodule with a poorly defined boundary in the left kidney (Figure 1A). During the enhancement phase, the right tumor (clear cell RCC) showed rapid heterogeneous cortical enhancement, with subsequent rapid washout during the medullary and delayed phases, whereas the left tumor (FH-deficient RCC) mainly manifested gradual enhancement (Figure 1B). Right partial nephrectomy was first performed, and the patient underwent the left partial nephrectomy 4 months later. Extensive FH-deficient RCC metastasis in the pelvic abdomen, peritoneum, and greater omentum was found on CT at 14 months after the first surgery. Sunitinib was used as the first-line treatment for 15 months. The patient was then started on second-line therapy with axitinib plus sintilimab for 4 months. The patient died in the 33rd month after the initial surgery. In the present case, the patient did not have cutaneous leiomyomas. None of the patient's first-degree relatives had RCCs or leiomyomas.

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Figure 1.

(A) CT revealed a slightly low-density mass in the right kidney (red box) and a mixed density nodule in the left kidney (blue box). (B) In the enhancement phase, the right tumor (clear cell RCC) showed rapid cortical phase heterogeneous enhancement (red box), whereas the left tumor (FH-deficient RCC) mainly manifested gradual enhancement (blue box). (C) The right tumor exhibits a nested growth pattern of large cells with clear cytoplasm. (D) The left tumor with a papillary architecture and eosinophilic cells showing prominent nucleoli. (E) Strong positive FH staining seen in the right tumor. (F) Most left tumor cells are negative for FH staining and retained FH staining is observed in a small area of the left tumor cells (G). (H) In the right tumor, 2SC immunostaining is negative. (I) The left tumor exhibits strong cytoplasmic and nuclear positivity for 2SC. (J) Diffusely positive CA9 staining seen in the right tumor. (K) CA9 is focally positive for the left tumor cells. CT, computed tomography.

Immunohistochemical Characteristics

Immunohistochemistry (IHC) was performed using the Roche BenchMark ULTRA automated staining system (Roche, Basel, Switzerland). Positive and negative controls for all markers were performed to follow the antibody instructions. In the right tumor (clear cell RCC), the tumor cells were positive for FH (Figure 1E), CA9 (Figure 1J), and CD10 and negative for 2SC (Figure 1H), KRT7, AMACR, and KSP-cadherin. In the left tumor (FH-deficient RCC), most tumor cells showed negative for FH (Figure 1F) and focally FH positive (Figure 1G). 2SC staining showed strong cytoplasmic and nuclear positivity (Figure 1I). The tumor cells were also focally positive for CA9 (Figure 1K), AMACR, CD10, and KSP-cadherin. Tumor cells were negative for KRT7.

In addition, the patient's peritoneal metastases (Figure 2A) showed positive for PAX8 (Figure 2B), negative for FH (Figure 2C), and positive for 2SC (Figure 2D), confirming that the peritoneal metastases were FH-deficient RCC.

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Figure 2.

(A) Peritoneal metastases showed a papillary pattern; (B) PAX8 was positive for the metastatic tumor cells; (C) FH was negative for the metastatic tumor cells; (D) 2SC was positive for the metastatic tumors.

Whole-Exome Sequencing (WES)

For WES, genomic DNA was extracted using the GeneRead DNA FFPE Kit (180134, QIAGEN). Exome capture was performed using the Agilent SureSelect Human All ExonV5 kit (Technologies, Santa Clara, CA, USA), followed by paired-end sequencing using an Illumina Novaseq6000 sequencer (Illumina Inc., San Diego, CA, USA) at GloriousMed Technology (Beijing, China). The right kidney tumor (clear cell RCC) showed a somatic VHL frameshift insertion (c.479_480insA, p.R161fs*13) and an FH germline mutation (c.563A > T, p.N188I). An extra mutation in PBRM1 (c.1148C > G, p.S383*) was found in the right tumor. The left tumor (FH-deficient RCC) harbored a pathogenic FH germline mutation (c.563A > T, p.N188I). Other gene mutations are summarized in Figure 3A.

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Figure 3.

(A) Molecular analysis of the right and left tumors. (B) Histologic features and FH and VHL alterations in the right tumor (clear cell RCC), left tumor (FH-deficient RCC), and normal tissues. An FH mutation (c.563A > T, p.N188I) was detected in the right tumor, the left tumor, and normal tissues, indicating a germline mutation. A somatic VHL frameshift insertion (c.479_480insA, p.R161fs*13) mutation was identified only in the right tumor.

Discussion

Bilateral synchronous RCC is uncommon. ¹¹ In this report, we describe a rare case of synchronous clear cell RCC (right) and FH-deficient RCC (left) in a patient's right and left kidneys. The right tumor (clear cell RCC) exhibited a nested growth pattern of large cells with clear cytoplasm and showed FH positive and 2SC negative. Germline FH c.563A > T and somatic VHL c.479_480insA mutations were detected in the right tumor. In contrast, the left tumor (FH-deficient RCC) demonstrated a high-grade papillary RCC morphology with FH negative and 2SC positive, and showed a germline FH c.563A > T mutation. Based on the different histological features, IHC results, and confirmed by molecular changes, the final diagnoses for the right and left tumors were clear cell RCC (right) and FH-deficient RCC (left), respectively.

Overexpression of CA9 is present in most clear cell RCCs. ¹⁰ Focal or diffuse positivity for CA9 also has been reported in FH-deficient RCCs.^16,17 One study hypothesized that the lack of FH caused the overexpression of fumarate, which lead to inhibiting degradation of HIF-1, ultimately resulting in the up-regulation of the expression level of CA9. ¹⁰ In this study, clear cell RCC showed diffuse positive staining for CA9 and the FH-deficient RCC was focally positive for CA9. So CA9 played little role in differentiating between clear cell RCC and FH-deficient RCC.

Synchronous bilateral RCCs are unusual and are mostly bilateral clear cell RCCs. ¹³ It is even rarer for FH-deficient RCC and clear cell RCC to simultaneously occur. To date, only 2 examples of bilateral renal tumors with clear cell RCC and FH-deficient RCC morphology have been reported; however, neither of them showed VHL mutation in the clear cell RCC-like tumor. In one case, only one FH germline mutation (c.989A > G, p.N330S) was identified using PCR-based sequencing in both tumors. Analysis of VHL mutation and LOH of chromosome 3p revealed no mutation or copy number change of VHL in the clear-cell tumors. The diagnosis of clear cell RCC was based on histopathological features. ¹⁴ In another report by Bai et al, the left FH-deficient RCC resulted from an FH somatic mutation (c.1256C > T, p.S419L), detected by next-generation sequencing, and the right tumor was diagnosed as clear cell RCC based on histological morphology. ¹⁵ In addition, a report documented a case of multifocal synchronous ipsilateral RCC of 3 different histologic subtypes, including unilateral clear cell RCC and bilateral multiple papillary RCCs. Tumor cells of the papillary subtype showed a large number of eosinophilic, atypical nuclei and prominent nucleoli, whose morphology was not excluded FH-deficient RCC, but no FH protein or molecular associated detection was performed to confirm it. ¹⁸

Here, we report a patient with detailed molecular analysis. In our case, clear cell RCC (right) was more likely caused by the VHL somatic frameshift mutation, whereas FH-deficient RCC (left) was caused by the FH germline mutation. A comparative analysis and additional details of above patients were summarized in Table 1. Moreover, clear cell RCC (right) had a PBRM1 mutation, whereas FH-deficient RCC (left) did not have a PBRM1 mutation in our case, further confirming the important role of PBRM1 in the development of clear cell RCC and highlighting the difference between the two RCCs. ¹⁹

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Table 1.

Comparison of 3 Cases With Synchronous of Clear Cell RCC and FH-deficient RCC.

	Patient 114	Patient 2 15	Patient 3 (the present patient)
Age (years)	23	54	60
Gender	Female	Male	Male
Family history
Skin leiomyoma	−	NA	−
Uterine leiomyoma	+ (her mother)	NA	−
RCC	−	NA	−
Patient history
Skin leiomyoma	−	−	−
Uterine leiomyoma	+	/	/
FH-deficient RCC
Size (cm)	2.3	1.5	2.8
Stage	NA	T1aN0M0	T1aN0M0
Histopathologic patterns	Cystic, papillary	Tubular, nested	Papillary, solid
ISUP /WHO grade	2	2	3
FH mutation analysis	c.989A > G, p.N330S	c.1256C > T, p.S419L	c.563A > T, p.N188I
VHL mutation analysis	Negative	NA	Negative
Molecular testing method	Sanger sequencing	NGS	WES and Sanger sequencing
Treatment	Partial nephrectomy	Partial nephrectomy	Partial nephrectomy
Follow-up time (months)	NA	12	29
Clear cell RCC
Size (cm)	13	2	3.6
Stage	NA	T1aN0M0	T1aN0M0
Histopathologic patterns	Nested	Nested	Nested
ISUP/WHO grade	2	2	2
FH mutation analysis	c.989A > G, p.N330S	-	c.563A > T, p.N188I
VHL mutation analysis	Negative	NA	c.479_480insA, p.R161fs*13
Molecular testing method	Sanger sequencing	NGS	WES and Sanger sequencing
Treatment	Radical nephrectomy	Partial nephrectomy	Partial nephrectomy
Follow-up time (months)	NA	7	33
Follow-up	NA	ANED	Dead

+, present; −, absent; NA, not available/not applicable; NGS, next-generation sequencing; ANED, alive and no evidence of disease.

Most patients with FH-deficient RCC have a poor prognosis. In one study, 50% of patients died of the disease, and 31% of patients were alive with active disease after a mean follow-up period of 16 months. ⁶ In contrast, the prognosis of clear cell RCC was better than that of FH-deficient RCC, with 5-year cancer specific survival rate of 76%. ²⁰ Patients with synchronous bilateral RCCs had significantly decreased 5-year recurrence-free survival compared to those with unilateral RCC. ²¹ In our patient, the patient experienced tumor recurrence and extensive FH-deficient RCC metastasis at 14 months, and died 33 months after the first surgery.

In conclusion, the present report describes an unusual case of synchronous bilateral RCCs, where the right clear cell RCC was more likely driven by a VHL somatic mutation and the left tumor was an FH-deficient RCC caused by an FH germline mutation. This report further emphasizes the rarity of subtypes of unconcordant bilateral synchronous RCCs and discussed the complexity of tumor driver gene pathogenesis.