In this study we demonstrated that one fifth of RCC, diagnosed either as “unclassified RCC, high grade” or “unclassified RCC with papillary pattern,” are FH deficient by IHC and were almost invariably accompanied by FH mutations. Only 0.5% of all cases diagnosed previously as papillary RCC, and 3% of those considered type 2 papillary RCC, showed FH deficiency by IHC and FH mutations, whereas all other evaluated renal tumors showed retained FH expression. The FH-deficient RCCs shared remarkable clinico-pathologic similarities with HLRCC-associated RCC, including younger age at presentation, aggressive clinical behavior, and adverse morphologic features, and were characterized predominantly by papillary architecture, typically admixed with other growth patterns, with invariable presence of at least focal macronucleoli. In fact, we were able to document an association with HLRCC syndrome in 8/23 (35%) patients. By IHC, these tumors typically demonstrated an FH-deficient profile (FH−) with aberrant succination, resulting in diffuse and strong 2SC reactivity (2+), which has previously been shown to be strongly associated with HLRCC-related RCC.13,14 The IHC profile, along with the morphology, aggressive clinical behavior, and the presence of FH mutations, provides strong justification to consider these tumors as part of the spectrum of the HLRCC-associated RCC.
We believe that both antibodies, FH and 2SC, should be used simultaneously to enhance the IHC potential in detecting FH-deficient RCC. Combined negative staining for FH and strong positive staining for 2SC demonstrated very good sensitivity for FH-deficient RCC profile and excellent specificity. That is, a normal pattern of staining for FH and 2SC can be used to rule out FH deficiency in the great majority of renal carcinomas encountered. However, we identified 2 tumors (patient #19 and #21, second tumor) with variably retained FH expression by IHC; both tumors showed FH point mutations (2SC 2+ in both cases). As previously shown, possible missense or other in-frame FH mutations may be associated with retained FH expression, resulting from a synthesis of a stable, but inactive enzyme.13–15,21 In addition, all cases considered “indeterminate” on IHC due to 2SC 1+ (21 cases) or 2SC 2+ (3 cases), but showing retained FH expression (FH+), exhibited wild-type FH. Therefore, restricting the evaluation to only 1 of the antibodies would be limited by the relatively lower sensitivity of FH and the lower specificity of 2SC. We also found it difficult to reliably confirm whether 2SC reactivity was nuclear, when diffuse and strong cytoplasmic reactivity was present. This is in contrast to Chen et al14 who found both cytoplasmic and nuclear 2SC to be present in HLRCC-associated RCC, allowing them to distinguish it from the “cytoplasmic only” pattern observed in a proportion of papillary RCC, type 2, and some unclassified, high-grade RCC cases. The clinical utility of the 2SC antibody is also currently limited, because it is not yet commercially available and cannot be routinely used in surgical pathology laboratories. Given the lower specificity and the difficulty in interpretation of 2SC, negative FH appears to be a more specific and comparably sensitive test at the present time.
Recent studies have also shown that IHC reactivity for 2SC22 or 2SC in combination with FH21,23 aids in identifying FH-deficient leiomyomas in younger patients, associated with HLRCC syndrome. We have previously shown that, although the great majority of patients with HLRCC syndrome will have FH-deficient leiomyomas, 1% of all sporadic uterine leiomyomas are FH deficient usually due to somatic inactivation.23 This is in contrast to the current study, in which germline FH mutations were identified in all patients with FH-deficient RCC with sufficient material for testing.
The RCC associated with HLRCC syndrome have been reported in about 30% of HLRCC families.4,12 HLRCC-associated RCC are particularly difficult to manage because they are highly aggressive and present with advanced-stage and metastatic disease, resulting in death of disease in 40% to 50% patients.9,10 Therefore, active surveillance is not recommended for the management of even small HLRCC-associated renal tumors in families with HLRCC syndrome, and wide surgical excision is recommended when any renal tumor is detected.10 RCCs associated with HLRCC syndrome are, however, quite rare and clinically challenging to diagnose in practice, because patients frequently do not exhibit the whole spectrum of the clinical presentations, and the family association is either unknown or not apparent; clinical manifestations can also differ within families.9,10 The initial report described renal tumors in 32% of the patients, all with metastatic disease at presentation,2 with a prevalence of 14% reported by the National Cancer Institute (NCI) group in a North American cohort.4 Kidney cancers have lower penetrance than the skin or uterine leiomyomas in the HLRCC-affected families, and they typically occur more than a decade later.4,9,12 Therefore, patients may initially present only with skin or uterine leiomyomas or less commonly with renal cell carcinoma, and renal tumors may demonstrate a delayed presentation, or the patients may lack the other HLRCC syndromic features. In the largest cohort of 38 patients with renal tumors, reported by Merino et al9 from the NCI, 39% had documented skin leiomyomas, and 55% had uterine leiomyomas. The morphology remains crucial in recognizing these tumors in routine practice.
In a recent study of comprehensive molecular characterization of papillary RCC, 3.1% (5/161) of all papillary RCCs and 8.3% (5/60) of those diagnosed as “papillary RCC type 2” demonstrated germline or somatic FH mutations, which were associated with the CpG methylator phenotype.24 Similar to our study, these patients were younger at presentation, and had a lower probability of overall survival than other patients with papillary RCC. A subset of these papillary RCC type 2 tumors, designated as CpG methylator phenotype associated, shared the FH-deficient profile observed in HLRCC-associated RCC, on the basis of their molecular features, allowing for more accurate characterization, which may lead to disease-specific targeted therapies.24 This also highlights the fact that the differential diagnosis of FH-deficient RCC will typically include “papillary RCC type 2,” which is a relatively commonly diagnosed renal tumor. However, it is becoming apparent that “type 2 papillary RCC” may not constitute a single entity, but instead represents a pattern that may be seen in a variety of neoplasms, including, for example, Xp11 translocation RCC and collecting duct carcinoma, as acknowledged in the 2016 WHO Renal Tumor Classification. The frequent papillary morphology in combination with additional architectural patterns, and at least focal presence of macronucleoli, should be regarded as morphologic clues to undertake additional IHC testing for FH and 2SC in this setting.
Currently, there is a lack of uniformly accepted definition of HLRCC-associated RCC, which is defined not just by mutational analysis, but also clinically. The NIH definition requires that the diagnosis of HLRCC be established with the identification of a heterozygous pathogenic variant in FH in combination with multiple cutaneous leiomyomas, with at least 1 histologically confirmed leiomyoma, a single leiomyoma in the presence of a positive family history of HLRCC, and/or 1 or more tubulopapillary, collecting duct, or papillary type 2 renal tumors with or without a family history of HLRCC. For the time being, “FH-deficient RCC” may be the most appropriate nomenclature for tumors that show IHC-negative staining for FH and strong 2SC reactivity, in the setting of uncertain clinical and family history and unknown genetic status.25,26 Taking a pragmatic approach, we would recommend that if FH-deficient RCC is diagnosed, the possibility of HLRCC should be first considered clinically. If there is a suggestive personal or family history, a presumptive diagnosis of HLRCC can be made pending confirmation with formal genetic counseling and germline mutation testing. When FH-deficient RCC is diagnosed in the absence of features suggesting syndromic disease, there are little data to indicate the risk of germline mutation. On the basis of our limited data (the finding of germline FH mutation in all 9 patients who had sufficient material available for testing), at this stage we believe the risk of germline mutation (that is HLRCC) is very high, and therefore performing genetic counseling and mutational analysis would be appropriate in all patients with FH-deficient renal carcinoma. This recommendation may be modified in the future if, similar to FH-deficient uterine leiomyoma, a low rate of germline mutation is found in follow-up studies.
The tumorigenic effect of mutated FH results in fumarate accumulation, which acts as a metabolic tumor suppressor, resulting in a metabolic shift toward aerobic glycolysis with decreased oxidative phosphorylation (so-called Warburg effect). It has been postulated that this alteration has possible downstream effects by inhibiting the hypoxia-inducible factor prolyl hydroxylase and increasing the hypoxia-inducible factor 1 alpha (HIF1alpha), which targets vascular endothelial growth factor, erythropoietin, and glucose transporter 1, and produces additional epigenetic alterations of genome-wide histone and DNA methylation, leading to increased cell proliferation and tumorigenesis.3,26–28 On a molecular level, these changes have also been characterized by increased oxidative stress and activation of the NRF2-antioxidant response elements pathway.24,29,30
Limitations of this study include its retrospective nature, which allowed us to confirm association with HLRCC syndrome only in about a third of patients with FH-deficient RCC. For example, in some cases we were not able to obtain a dermatological confirmation of skin leiomyomatosis, and a complete family history on specific HLRCC features was not available. Although mutational analysis was performed on FFPE neoplastic tissue in the majority of tested cases, a formal FH genetic testing was done only in a subset of cases, perhaps with a selection bias toward patients with a high likelihood of familial disease, limiting the ability to confirm germline FH mutations. In 2 FH-deficient cases by IHC, we could not confirm the presence of FH mutations; 1 of the 2 cases demonstrated low DNA quality, and additional studies to investigate for possible FH mutations in these 2 cases, for example, by MLPA to screen for large scale FH deletions, were not performed.
In summary, we found that a substantial number of cases considered either as “unclassified RCC, high grade” or “unclassified RCC with papillary pattern,” and a small percent of cases diagnosed as papillary RCC type 2, demonstrated an FH-deficient pattern (FH−, 2SC 2+) by IHC and were invariably accompanied by FH mutations at the molecular level. Although we could document an unequivocal association with HLRCC syndrome in only about a third of the patients, there is clearly a high likelihood of syndromic disease in patients presenting with FH-deficient RCC. Furthermore, even apparently sporadic FH-deficient RCCs show striking clinicopathologic similarities to unequivocally HLRCC syndrome–associated renal carcinomas, including a younger age and adverse features at presentation, aggressive clinical behavior, and frequent papillary architecture in combination with other growths patterns, with invariable presence of at least focal macronucleoli. In addition to the careful morphologic evaluation, IHC for FH and 2SC is a useful aid that allows recognition of the RCC with an FH-deficient profile with FH IHC being more specific and 2SC being more sensitive.
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Keywords:Copyright © 2016 Wolters Kluwer Health, Inc. All rights reserved.
renal cancer; HLRCC; fumarate hydratase; FH; fumarate hydratase–deficient RCC; succination; 2SC