The clinicopathologic and molecular features, and treatment outcome of fumarate hydratase-deficient renal cell carcinoma: a retrospective comparison with type 2 papillary renal cell carcinoma

Background: To compare clinicopathologic, molecular features, and treatment outcome between fumarate hydratase-deficient renal cell carcinoma (FH-dRCC) and type 2 papillary renal cell carcinoma (T2 pRCC). Methods: Data of T2 pRCC patients and FH-dRCC patients with additional next-generation sequencing information were retrospectively analyzed. The cancer-specific survival (CSS) and disease-free survival (DFS) were primary endpoint. Results: A combination of FH and 2-succino-cysteine (2-SC) increased the rate of negative predictive value of FH-dRCC. Compared with T2 pRCC cases, FH-dRCC cases displayed a greater prevalence in young patients, a higher frequency of radical nephrectomy. Seven FH-dRCC and two T2 pRCC cases received systemic therapy. The VEGF treatment was prescribed most frequently, with an objective response rate (ORR) of 22.2% and a disease control rate (DCR) of 30%. A combined therapy with VEGF and checkpoint inhibitor reported an ORR of 40% and a DCR of 100%. FH-dRCC cases showed a shortened CSS (P = 0.042) and DFS (P < 0.001). The genomic sequencing revealed 9 novel mutations. Conclusions: Coupled with genetic detection, immunohistochemical biomarkers (FH and 2-SC) can distinguish the aggressive FH-dRCC from T2 pRCC. Future research is awaited to illuminate the association between the novel mutations and the clinical phenotypes of FH-dRCC in the disease progression.


INTRODUCTION
As a rare subtype of renal cell carcinoma (RCC), fumarate hydratase-deficient renal cell carcinoma (FH-dRCC) is characteristic of pathologic germline/ somatic mutations in the FH gene [1][2][3] while lesions with germline mutations are associated with hereditary leiomyomatosis and RCC (HLRCC) syndrome.The AGING latter, an autosomal dominant disorder, usually predisposes individuals to cutaneous leiomyomas (CL), multiple uterine leiomyomas (MUL), and RCC [4][5][6].Currently, FH-dRCC is recognized as a separate category in the World Health Organization (WHO) classification of RCC [7].FH-dRCC is the preferred term for RCC with compatible morphology, negative FH immunohistochemical (IHC) staining, positive 2-SC IHC staining, and/or identification of a mutation in the FH gene in the tumor, when the clinical and family history of CL and MUL is uncertain and the genetic status is unknown [8].Clinically, FH-dRCC is an aggressive tumor that commonly presents itself with locally advanced or metastatic disease, even in the setting of a small primary tumor, and with a high rate of malignancy progression and mortality [3,9].So far, National Comprehensive Cancer Network (NCCN) guidelines recommend radical nephrectomy (RN) for such patients and erlotinib plus bevacizumab for advanced patients [10].Therefore, it is of particular significance to explore the strategies for a timely diagnosis and effective surgical intervention.
Clinically, the high aggressiveness of FH-dRCC poses a particular challenge to effective management, FH-dRCC is often misdiagnosed as type 2 papillary RCC (T2 pRCC) when only by histopathology [11][12][13].After the release of 2016 WHO classification criteria, studies have documented FH deficiency in many T2 pRCC cases [14,15].Recent investigations into the images of papillary type II HLRCC-associated RCC and FH-dRCC have identified some distinct features that may help clinical practitioners in differentiating FH-dRCC from T2 pRCC and pathologists in IHC or even gene sequencing [16][17][18].However, few studies have systematically compared the clinical features and prognostic outcomes of patients with FH-dRCC and T2 pRCC.
Therefore, the current study tackled this very issue by retrospectively analyzing the clinical data of patients with FH-dRCC and T2 pRCC in a single-center database.We found that when compared with their T2 pRCC counterparts, FH-dRCC patients reported distinct features in age, disease severity, and prognostic outcomes.Moreover, we discovered 9 novel gene mutations in the FH-dRCC cohort.These findings may be of great importance to the clinical management of FH-dRCC patients.

Comparison clinical and histologic characteristics between FH-dRCC and T2 pRCC
The clinical and pathological features of FH-dRCC and T2 pRCC are summarized in

Treatment response and survival outcomes
A total of 9 patients received systemic therapy and were evaluable for response by RECIST v1.1, including 2 metastatic T2 pRCC patients and 7 metastatic FH-dRCC patients.
One patient with T2 pRCC had metastases detected 24 months after surgery and died of the disease 59 months later after receiving chemotherapy and multiple lines of TKIs drugs.Another patient, who had bone metastatic at the time of surgery, developed lung metastases and distant LN metastases 10 months after receiving Sunitinib and died 2 months later.

DISCUSSION
FH-dRCC is a rare and recently described entity tumor with variable clinical and pathologic features and closely related to the RCC found in HLRCC syndrome [19][20][21].At present, observations suggest that the clinical behavior of FH-dRCC and HLRCC-associated RCC is similar.[2,11,14].All of FH-dRCC cases in our cohort had no known family history of HLRCC and due to the lack of definitive genetic or clinical information at their initial presentations, we use the term "FH-dRCC" to include both HLRCC-associated RCC and disseminated cases of FH-dRCC.
This study analyzed the baseline characteristics, immunohistochemical features, gene mutation and prognosis of 16 FH-dRCC patients and compared with those of 44 patients with T2 pRCC.Many of our findings support the conclusions described in previous studies [14,18], including that FH-dRCC tend to present at a younger age than T2 pRCC (median age of 43.5 years), behave aggressively (50% of cases presented at tumor stage pT3a or higher) and more common in males.Lymph node and distant metastases were more frequent in FH-dRCC, as shown in the present study, metastases are commonly observed in the LNs, liver, lungs, bone and peritoneal seeding, and portend a poor prognosis.To date, a total of 14 FH-dRCC younger than 20 years old have been reported, but the disease is not limited in younger patients [22].In this study, 9 of 16 patients (56.3%) were older than 40 years, and in a previous study [3], 62.5% patients were older than 40 years old.In addition, MUL were frequently observed in FH-dRCC, whereas CL were not observed.Similarly, CL are rare in patients with HLRCC in Japan and China.The phenotype of HLRCC may differ between Asians and Caucasians [1,23,24].HLRCC only presented as single CL is usually disregarded and misdiagnosed.The participants in this study were RCC patients.Patients with personal and family history of MUL all had germline mutations, while patients with somatic mutations had no history of MUL, CL and RCC.
Although pathological features play a pivotal role in the diagnosis of FH-dRCC, it may be confused with T2 pRCC when only through microscopic examination, FH and 2-SC IHC is a useful tool.Studies have shown that FH-/2-SC+ has optimal sensitivity and specificity for FH-dRCC, the sensitivity even reaching 100% [14,25].
In this study, we performed FH and 2-SC IHC in all patients and sensitivity and specificity were also 93.75% and 100% when FH and 2-SC were used together.Most of FH-dRCC (68.8%) showed FH staining deficiency and the vast majority (93.8%, 15/16) showed 2-SC staining positivity, of which 73.3% showed cytoplasmic and nuclear staining to varying degrees and 26.7% showed only cytoplasmic positive staining, one of the patients confirmed by gene test showed FH+/2-SC-with a meaningful mutation (c.1189G>A:p.G397R), similar cases have never been reported in previous articles.While in 56.8% of T2 pRCC patients was cytoplasmic staining only in 2-SC immunoreactivity but no FH mutation was detected.While Chen's study showed that all confirmed cases showed diffuse and strong nuclear and cytoplasmic staining, a small subset of unclassified and T2 pRCC exhibited immunoreactivity to 2-SC, but did not harbor FH germline or somatic alterations.That is, FH gene mutation may also be detected in other RCC when 2-SC IHC was cytoplasmic staining only.Therefore, the diagnosis of 2-SC IHC staining pattern in FH-dRCC needs to be more cautious.Other IHC stains also are useful in conjunction with FH/2-SC IHC in distinguishing FH-deficient RCC from its morphologic mimics, such as CK7, p63, and GATA3 in differentiating FH-deficient RCC from CDC [3,26].Zhang et al. found that AKR1B10 is a new sensitive and specific marker for FH-dRCC, which in conjunction with 2-SC and FH can help in the diagnosis of the disease and reduce the rate of leakage and misdiagnosis [27].
As of November 2023, the ClinVar database includes 268 pathogenic and 147 likely pathogenic single gene variants within the FH gene [28].This data still being updated.In our study, 21 different FH gene mutations were detected.Two are frame shift mutations, 16 are missense mutations, 3 are splicing positions, 9 are recent discoveries.Of the 9 newly discovered mutations, 3 are damaging, 3 are benign, and 3 are still uncertain.Germline mutations were found in both patients with a personal and family history, as well as in other three patients without any history.Immunohistochemistry of tumor specimens from 1 patient suggested FH-/2-SC-, but no mutation was found, and we suspected large fragment deletion.
As found in our study, the prognosis of FH-dRCC is worse than T2 pRCC.The proportion of patients with distant metastasis was higher in our study (81.3% vs. 13.6%)than in the study by Yang, while that of LNs metastasis was lower (37.5% vs. 6.8%)[18].Since FH-dRCC is significantly more invasive than T2 pRCC, RN and lymph node dissection are recommended.
A study showed the median survival for metastatic disease was 18 months [9].In another report [3], a small number of (19%, 5/26) patients with FH-dRCC showed no evidence of disease, a portion of (31%, 8/26) patients were alive with disease, and most patients (50%, 13/26) were died of disease after a median follow-up of 16 months (range, 1 to 118 mo) in 26 patients.However, in this study, 18.8% of patients had no postoperative recurrence or metastasis, which may be related to low DNA methylation, most patients survived.This may be related to a relatively low genome-wide DNA methylation [29].Xu's study [30] found that ICI/TKI combination therapy was associated with more favorable outcomes compared to other first-line therapies, including Bevacizumab/Erlotinib combination therapy and TKI monotherapy, suggesting a AGING combination of immunotherapy and TKI could be promising in advanced FH-dRCC.Lucia's study [31] found that ORR for treatments were 50% for cabozantinib, 43% for sunitinib, 63% for other antiangiogenics, and 30% for Bevacizumab/Erlotinib, whereas ORR was 0% for mTOR inhibitors and 18% for ICBs.In our study, only a subset (43.75%, 7/16) received systemic treatment and all developed resistance, disease progression, even death.But ICI/TKI combination therapy was associated with more favorable outcomes compared to other first-line therapies, suggesting a combination of immunotherapy and targeted therapy could be promising in advanced FH-dRCC.We also tried Axitinib/Toripalimab, Lenvatinib/Tislelizumab, Cabozantinib/Toripalimab, Lenvatinib/Everolimu and other treatment protocols, failed but no death.Interestingly, 18.8% patients are alive without disease and 1 patient with lung metastases discovered shortly after resection of the metastases survived without receiving systemic therapy and did not show disease progression, which may be related to the relatively good prognosis morphology.Compared with targeted therapy alone, immune-based combination medication can improve the ORR and DCR of patients.Dong's [32] study also found that in patients with germline mutations, the ORR and DCR of immunebased treatment were higher.This is because there is abundant CD8+T cell infiltration in fumarate hydratasedeficient renal cell carcinoma, and the depletion of T cells is related to poor immune efficacy in FH-dRCC patients.Current anti-PD-1 /PD-L1 antibodies cannot reverse the depletion of CD8+T cells in such patients.In addition, Liang [33] et al. observed abnormalities in several negative immunomodulators, which may be related to immune escape.However, there have been no studies on the correlation between FH-dRCC mutation sites and treatment programs.In our study, case 5 (c.562A>G:p.N188D) and case 9 (c.439A>G:p.T147A) showed no response to systemic treatment such as TKIs, PD-1 or PD-1/TKIs and developed resistance in a short time.Patients with mutation (c.1189G>A:p.G397R) was the most common (31.3%).Although it was reported to be damaging in previous studies, 60% of patients didn't show strong aggressiveness, however patients with mutation (c.385G>A:p.E129K and c.1189G>A:p.G397R) died after receiving chemotherapy.Patients with mutation (c.1276_1277insAGATG:p A426fs and c.1189G> A:p.G397R) experienced PR soon after receiving PD-1/CTLA-4 and TKIs, with more than 50% reduction in focal size.Therefore, we hypothesized that patients with c.1189G>A:p.G397R and c.1276_1277insAGATG:p A426fs mutations would benefit more.The newly discovered mutation in case 13 (c.454A>T:p.P304fs, c.911delC: p.N152Y), despite multiple metastases, was PR after treatment with TKIs and is now stable.Given the limited number of cases, it is not possible to draw definitive conclusions regarding the relationship between gene variations and drug sensitivity or prognostic assessment in patients.However, the diverse range of gene variations identified in this study will serve as a foundation for building a comprehensive understanding of FH-dRCC through future molecular typing studies.These findings contribute to the initial stages of research in this field, facilitating further exploration and characterization of FH-dRCC.
Our study also has some limitations.First, the limited sample size and short follow-up period may lead to inaccurate statistical results and prognostic judgments.However, this is still the first time that the largest sample of FH-dRCC has been considered for reproductive/physiological mutations and compared with T2 pRCC in terms of prognosis.Second, in some cases, we could not obtain confirmation of the presence of FH mutations in uterine smooth muscle tumors, nor could we obtain a complete family history regarding specific HLRCC features.

Patients
Ethical approval for this study (Ethical Committee NO.2019KJCX037) was provided by the Ethical Review Committee of Fujian Medical University Union Hospital on 28 November 2019 and all eligible participants provided written informed consent.It recruited from June 2014 to September 2022, 16 FH-dRCC patients with pathological IHC FH-confirmed RCC (defined as FH-and/or 2-SC+) or FH mutation and 44 pathologically-diagnosed T2 pRCC patients.Clinical data were collected, including patients' age, sex, tumor size, surgical data, pathological results, staging, treatment, follow-up time, outcome, and family history of CL or MUL or RCC within secondary consanguinity.The flowchart of patient selection was shown in Figure 3.

Histologic features
Each tumor was diagnosed by two experienced expert pathologists.The tumor specimens were respectively stained and analyzed using FH antibody reagents and 2-SC antibody reagents, which were ready-to-use kits purchased from Fuzhou Maixin Biotechnology Development Co., Ltd.In the non-tumor renal parenchyma, when an internal positive control in inflammatory cells or interstitial cells, or a FH-positive staining in cytoplasm was present, tumor cells with FH negative staining were considered negative.When adjacent non-tumor cells showed a 2-SC-negative staining (internal negative control), tumor cells with 2-SC positive staining in cytoplasm/nucleus were considered positive.Histological types of tumors were categorized according to the 2016 classification of the WHO, and tumors were graded according to the WHO/International Society of Urological Pathology (ISUP) grading system.

Mutational analyses
ANNOVAR (http://annovar.openbioinformatics.org/en/latest/) was referred to for all the released mutations with the latest group database, function, database, and known disease information such as comparative analyses.The following SNV/InDel sites were consulted for

Follow up
Patients were followed up with regular postoperative visits with radiological surveillance imaging of the chest, abdomen and pelvis was performed at various time intervals.The last information on file was considered as the final follow-up time.The primary endpoint (PE) of the study was cancer-specific survival (CSS), which was defined as the length from the date of surgery to death from renal cell carcinoma, and the secondary endpoint (SE) was the disease-free survival (DFS), which was defined as the interval from the date of nephrectomy until the diagnosis of tumor recurrence, metastasis or last follow-up.The main therapeutic effects of systemic treatment were ORR and DCR.

Statistical analysis
All statistical analyses were performed using IBM SPSS version 26.0 (IBM Corporation, Armonk, NY, USA).Continuous and categorical variables were expressed as mean ± SD and percentage, respectively, and compared between groups by unpaired Student's t-test or chisquare test.PE and SE were assessed by Kaplan-Meier estimation and log-rank test.Statistical significance was defined as a two-tailed P-value < 0.05.

Table 4
metastasis, and 6 had distant metastases.Both LN and distant metastases were more frequent in the FH-dRCC (P = 0.011 and P < 0.001).