Fumarate Hydratase–Deficient Renal Cell Carcinoma With Predominant Tubulocystic Features Mimics Tubulocystic Renal Cell Carcinoma

A total of 97 FH-deficient RCCs were retrieved from the electronic records of the 8 medical centers. The data of 6 patients with tubulocystic RCC were obtained from a single center. Architectural patterns included papillary, solid, tubulocystic, cribriform, sarcomatoid, and rhabdoid. Morphologic features were reevaluated by 2 expert pathologists. The average value was determined as the percentage of each architectural pattern. Tubulocystic FH-deficient RCC was defined as FH-deficient RCC exhibiting a tubulocystic component in 80% or more of the tumor area, with pure tubulocystic architecture observed in at least 1 H&E section. Papillary FH-deficient RCC was defined as FH-deficient RCC with a papillary architecture 80% or more of the tumor area. Tubulocystic RCC was diagnosed when only tubulocystic architecture was observed and other renal neoplasms were excluded. Verbal informed consent was obtained from patients or legally authorized representatives by telephone. This multi-institutional study was approved by the Department of Pathology of Ruijin Hospital (Shanghai, China).

The following IHC markers were used: cytokeratin 7 (CK7; OV-TL 12/30, prediluted, DAKO), CK20 (Ks20.8, prediluted, DAKO), TFE3 (OTI1H6, prediluted, ZSGB-BIO), TFEB (ab2636, 1:100; Abcam), ALK (1A4, prediluted, ZSGB-BIO), INI-1 (ZA-0696, prediluted, ZSGB-BIO), FH (ab233394, 1:100; Abcam), GATA3 (EP368, prediluted, ZSGB-BIO), 2SC (RAB-1015, prediluted, MXB), and Ki-67 (GA62661, prediluted, DAKO).

Alterations in chromosomes 7, 17, and Y have been reported in tubulocystic RCC and FH-deficient RCC.^4,9  In this study, we used probes for chromosomes 7, 17, and Y (GPMEDICAL) to explore whether tubulocystic RCC or FH-deficient RCC displayed chromosomal alterations. Three or more signals in more than 10% of tumor cells were determined as trisomy 7 or 17 chromosomes. One signal in more than 70% of tumor cells was viewed as loss of chromosome Y.

DNA was extracted from fresh-frozen tumor tissue or retrieved formalin-fixed paraffin-embedded tissue. Whole-exome sequencing or 2 gene panels, (1 panel included 688 cancer-associated genes, and the other panel included 19 RCC-related genes) were performed on 6 FH-deficient RCCs to explore their molecular features.

We identified a group of 10 tubulocystic FH-deficient RCCs and 6 tubulocystic RCCs. Clinicopathologic characteristics of the patients are summarized in Table 1. In the FH-deficient RCC group, there were 8 men and 2 women with a mean age of 51.8 years (range, 37–66 years). Skin leiomyomas were not observed during initial presentation. Two female patients had a history of uterine fibroids. There was no family history of RCC or other types of cancer. Patient 1 had metastasis to the pelvic cavity, and patient 4 had lung and bone metastases at the time of diagnosis. All the patients underwent radical or partial nephrectomy. The tumor sizes ranged from 4.5 to 13.0 cm. Perinephric or lymphovascular invasion was observed in 5 patients. The follow-up duration ranged from 3 to 89 months. Seven patients developed recurrence or metachronous metastases, and 3 patients died of the disease. The tubulocystic RCC group included 5 men and 1 woman with a mean age of 58.3 years (range, 50–77 years). The tumor size ranged from 1.8 to 3.0 cm. Adverse morphologic factors, such as perinephric or lymphovascular invasion, were not observed. The follow-up duration ranged from 3 to 67 months. None of the patients experienced any adverse clinical events at the time of presentation.

The morphologic features and immunohistochemical and molecular results are presented in Table 2. The integrated clinicopathologic features of tubulocystic FH-deficient RCCs and tubulocystic RCCs are presented in Table 3. In at least 1 H&E section, pure tubulocystic component can be observed in tubulocystic FH-deficient RCC (Figure 1, A and B). The diagnosis of FH deficiency was made based on loss of FH and diffuse 2SC expression (Figure 1, C and D). In these tumors, tumor cells exhibited eosinophilic cytoplasm and prominent nucleoli. Perinucleolar haloes and eosinophilic nucleoli can be observed in at least focal areas, which were not observed in tubulocystic RCC (Figure 2, A). In addition, in FH-deficient RCC, a variable component of other patterns (for example, single-cell invasion in Figure 2, B, poorly differentiated foci in Figure 2, C and D) can always be observed after reviewing all the H&E sections. No sarcomatoid or rhabdoid differentiation was observed in the tubulocystic FH-deficient RCC group. In contrast, tubulocystic RCC exhibited a pure tubulocystic architecture (Figure 3, A and B), and nontubulocystic patterns were not observed. Psammomatous calcification was observed in 1 tubulocystic RCC (Figure 3, C) but not in other renal neoplasms. CK7 expression was mostly negative in FH-deficient RCC. Only 2 FH-deficient RCCs exhibited focal expression. CK7 showed heterogeneity in tubulocystic RCC. Diffuse or focal positivity and negative expression can be observed. GATA3 was positive in 1 FH-deficient RCC and negative in tubulocystic RCC. In tubulocystic RCC, INI1 and FH were retained (Figure 3, D), and 2SC showed negative expression. Ki-67 index was relatively high in FH-deficient RCC and low in tubulocystic RCC. All 6 tubulocystic RCCs were negative for TFE3, TFEB, ALK, and CK20 expression. Chromosomal alterations were identified using fluorescence in situ hybridization (FISH) in 3 FH-deficient RCCs. Gene analysis was performed on 6 FH-deficient RCCs. Three patients had somatic mutations and 3 had germ-line mutations. FISH was performed on 6 tubulocystic RCCs. All 6 tumors were negative for chromosome 7. Four tumors had trisomy 17 and 3 tumors had loss of Y.

A group of 25 papillary FH-deficient RCCs was also identified, and related clinical data are shown in Supplemental Table 1 (see the supplemental digital content, containing 2 tables and 1 figure at https://meridian.allenpress.com/aplm in the December 2024 table of contents). Integrated clinicopathologic data of the clinicopathologic features of tubulocystic or papillary FH-deficient RCC are shown in Supplemental Table 2. Papillary FH-deficient RCC may occur at a younger age. Bilateral masses were observed in 6 patients. In addition, papillary FH-deficient RCC can exhibit a pure papillary architecture (Supplemental Figure 1, A and B), whereas a pure tubulocystic pattern was not observed in our study. Loss of FH and diffuse GATA3 expression can be observed in papillary FH-deficient RCC (Supplemental Figure 1, C and D). Except for morphologic patterns, there were no clear differences between FH-deficient RCCs with predominant papillary or tubulocystic architecture.

The concept of tubulocystic RCC was updated in the 5^th edition of the WHO.¹  Most importantly, renal tumors cannot be diagnosed as true tubulocystic RCC if a nontubulocystic component (papillary, sarcomatoid, etc) is identified. Previous studies have demonstrated that tubulocystic RCC and papillary RCC are closely related entities.¹⁰  However, the tubulocystic RCC included in previous studies morphologically exhibited partial or exclusive tubulocystic architecture mixed with variable papillary components. Such tumors cannot be diagnosed as tubulocystic RCC now. A previous study reported 12 pure tubulocystic RCCs,¹¹  in which all tumors were 50 mm or less in size, and all tumors were negative for trisomy 7 or 17. Moreover, they often had a high nuclear grade, but neither recurrence nor metastasis was observed. They demonstrated that pure tubulocystic RCC is an indolent renal tumor with a good prognosis and is immunohistochemically and genetically distinct from papillary RCC. However, another study explored the molecular alterations of 9 pure tubulocystic RCCs by targeted next-generation sequencing and found that all the tumors exhibited loss of chromosomes 9 and Y, and gain of chromosome 17.¹²  The average size of the 9 tumors was 5.1 cm, with the largest size at 13.0 cm. Similarly, none of the patients had metastasis, indicating a favorable prognosis. In the current study, we introduced 6 patients with tubulocystic RCC. Tubulocystic RCC was frequently observed in male patients, and there was no significant laterality predominance. Tubulocystic RCCs were usually small, with all 6 tumors less than 4.0 cm (pT1a). Morphologically, tumors exhibited pure tubulocystic architecture, with a relatively low Ki-67 index (<5%) and retained FH expression. Alterations of chromosomes 17 and Y can be identified by FISH. However, chromosome 7 was often intact. None of the 6 tubulocystic RCC cases exhibited adverse clinical outcomes, indicating a relatively favorable prognosis.

FH-deficient RCC is a highly aggressive cancer caused by somatic or germline mutations in FH. Morphologically, tumor cells also have eosinophilic cytoplasm with prominent nucleoli. The nuclei usually have prominent inclusion-like eosinophilic nucleoli surrounded by perinucleolar haloes. However, prominent perinucleolar haloes can also be observed in FH-wild papillary RCC,³  indicating limited specificity. FH-deficient RCC usually demonstrates 2 or more growth patterns, of which papillary is the most common and dominant pattern.^13,14  Other common morphologic patterns include solid, tubulocystic, cribriform, and cystic architectures. Therefore, it is not uncommon for FH-deficient RCC to exhibit papillary combined with solid or tubulocystic architecture. However, FH-deficient RCC exhibiting predominant tubulocystic architecture is rare. FH and 2SC have been validated as useful tools for diagnosing FH-deficient RCC.^3,13,14  In the current study, we introduced 10 tubulocystic FH-deficient RCCs. In such tumors, pure tubules and cysts can be observed in at least 1 H&E section, and nontubulocystic architecture can be observed in only a few areas (<20%). Although the morphology of a large number of FH-deficient RCCs was reevaluated in our study, we did not encounter an FH-deficient RCC with a pure tubulocystic architecture. A nontubulocystic architecture was always observed. Tubulocystic FH-deficient RCCs were relatively large in size, with a relatively high index (≥5%), loss of FH, and diffuse 2SC expression. Two female patients presented with a history of leiomyomas, and FH germline mutations were validated using genetic analysis. Recurrence, metastasis, or death was observed in more than half of tubulocystic FH-deficient RCCs, indicating its highly aggressive behavior. We noticed that different morphologies within 1 renal entity may lead to different prognoses. One study reported that papillary RCC with a microcystic architecture may indicate extrarenal invasion and metastatic disease.¹⁵  We compared the clinicopathologic features of tubulocystic and papillary FH-deficient RCC. However, 2 groups had similar clinicopathologic features. Normal FH expression was observed in 2 papillary FH-deficient RCCs and was not observed in tubulocystic FH-deficient RCC. We observed that FH-deficient RCC can exhibit a pure papillary architecture, whereas no pure tubulocystic architecture was observed. Therefore, it may be necessary to perform multiple sections to identify the presence of a nontubulocystic architecture to exclude the diagnosis of tubulocystic RCC. Previously, we observed GATA3 positivity in half of the patients with FH-deficient RCC.⁹  GATA3 IHC was performed in this study. However, only 1 tubulocystic FH-deficient RCC showed moderate positivity. Other tumors exhibited negative GATA3 expression. Alterations in chromosomes 17 and Y were both observed in FH-deficient RCC and tubulocystic RCC, indicating their limited value in differentiating 2 entities. However, trisomy 7 was only observed in FH-deficient RCC and may be a useful tool for excluding tubulocystic RCC.

Tubulocystic architecture has been described in many renal entities, including clear cell papillary renal cell tumor, chromophobe RCC, papillary RCC, FH-deficient RCC, TFE3-rearranged RCC, and ALK-rearranged RCC.^{2,10,13,16–19}  However, tubulocystic architecture is usually not the dominant morphologic feature, and tumors often exhibit multiple architectural patterns. As defined by the WHO and emphasized by various studies,^1,11,12  tubulocystic RCC should be diagnosed when it presents as pure tubulocystic architecture with a lack of other components, such as papillary, solid, or poorly-differentiated. Therefore, caution should be exercised when encountering RCC with a predominant tubulocystic architecture. A previous study reported 2 tubulocystic RCCs confirmed during fine-needle aspiration.²⁰  Here, we did not recommend the diagnosis of tubulocystic RCC when conducting renal mass biopsy, even if pure tubulocystic morphology was observed under the microscope. Considering that only a few tissues are obtained for morphologic evaluation during renal mass biopsy, and many other renal entities, including tubulocystic FH-deficient RCC, may exhibit tubulocystic architecture and affect the diagnosis, the diagnosis of tubulocystic RCC should be raised only when the tumors were sampled in toto with other renal entities excluded and the absence of nontubulocystic architecture.

An early study reported a case of tubulocystic RCC exhibiting a large size (15.1 cm in the largest size) and bone metastasis.²¹  Considering that real tubulocystic RCC usually has a small size and favorable prognosis, and FH-deficient RCC can also exhibit predominant tubulocystic architecture, we cannot exclude the diagnosis of FH-deficient RCC if FH/2SC or genetic analysis is not performed. A large number of “malignant tubulocystic RCCs” were reported in the past, with local invasion, recurrence, or metastases to liver, bone, lymph nodes, pelvic cavity, peritoneum, omentum, and abdominal wall.^21–29  It is noteworthy that in these studies, FH staining was not performed on the tumors and FH-deficient RCC cannot be excluded. In addition, they demonstrated a morphology of tubulocystic RCC coexisting with a papillary, poorly differentiated, or other component, which cannot be viewed as true tubulocystic RCC according to the 5^th edition of the WHO classification. Together with previous reports on true tubulocystic RCC,^11,12  our study validated that none of the patients with tubulocystic RCC experienced any adverse clinical events, indicating that true tubulocystic RCC may present with a favorable prognosis.

Our study had some limitations. First, tubulocystic RCC and FH-deficient RCC both accounted for a low proportion of renal cancers, and the number of FH-deficient RCCs exhibiting predominant tubulocystic architecture was even smaller. We demonstrated potential differences between tubulocystic RCC and FH-deficient RCC in terms of tumor size, architectural pattern, Ki-67 index, and FH/2SC staining. However, the small number of cases included in this study cannot support the statistical analysis. Further studies are required to validate these results. Second, the limited number of cases and follow-up duration resulted in a lack of survival curves.

In conclusion, we reported a series of tubulocystic FH-deficient RCCs. Such morphologic features may mislead to the diagnosis of tubulocystic RCC. This small cohort study observed that tubulocystic FH-deficient RCCs may have a larger tumor size and a higher Ki-67 index than tubulocystic RCCs. Moreover, the presence of a nontubulocystic pattern (such as papillary, cribriform, or solid architecture) and FH/2SC staining can differentiate between the 2 renal entities. Therefore, multiple sections are recommended when encountering a renal neoplasm with tubulocystic pattern. The diagnosis of tubulocystic RCC is not recommended even if encountering pure tubulocystic morphology in renal mass biopsy, because pure tubulocystic morphology can be confirmed only when tumors are sampled completely.