Prospective Detection of Germline Mutation of Fumarate Hydratase in Women With Uterine Smooth Muscle Tumors Using Pathology-based Screening to Trigger Genetic Counseling for Hereditary Leiomyomatosis Renal Cell Carcinoma Syndrome

Abstract

Pathology-based screening of uterine smooth muscle tumors (uSMT) for morphology suggestive of fumarate hydratase deficiency (FH-d morphology) has been proposed as a method to identify women at increased risk for hereditary leiomyomatosis renal cell carcinoma (HLRCC) syndrome. For 5 years our clinical diagnostic practice has evaluated all women with any type of uSMT for FH-d morphology (defined, at low magnification, as staghorn shaped blood vessels and alveolar pattern edema and, at high magnification, as tumor macronucleoli surrounded by a halo and cytoplasmic eosinophilic globules) and, when present, used the pathology report to advise genetic counseling to further evaluate for HLRCC syndrome. We now report the results of this prospective screening strategy, with emphasis on the incidence and clinicopathologic features of FH-d morphology in uSMT, the rate of patient uptake of referral to genetic counseling, and the results of genetic testing for FH germline mutation. Among 2060 women with a uSMT, FH-d morphology was reported in 1.4% (30 women). Ten women elected to undergo FH genetic testing and 6 of 10 (60%) had a FH germline mutation: 5 were pathogenic mutations and 1 was a mutation variant of unknown significance. Therefore, the screening program led to a confirmed genetic diagnosis of HLRCC syndrome in 0.24% of all women with any type of uSMT. The women with a pathogenic mutation were ages 24 to 40 years. Although the majority of leiomyoma with bizarre nuclei exhibited FH-d morphology, the uSMT were conventional leiomyomas with FH-d morphology in 2 of 5 women found to have a pathogenic FH germline mutation. Relying on an abnormal FH immunostain result to trigger genetic counseling referral would have resulted in 2 of 5 (40%) cases with pathogenic FH germline mutation but normal FH immunoexpression going undetected, both of which were missense type mutations. There was no difference in the incidence of pathogenic FH germline mutation between FH-d morphology uSMT with an abnormal versus a normal FH immunostain result. Overall, this study demonstrates that prospective morphology-based screening, integrated with referral for genetic counseling, can result in the diagnosis of HLRCC syndrome in otherwise unselected women with uSMT. We conclude that this strategy should be incorporated in the routine pathologic examination of all uterine smooth muscle tumors.

Targeted sequencing of FH-deficient uterine leiomyomas reveals biallelic inactivating somatic fumarase variants and allows characterization of missense variants

“…This successful approach allowed us to identify a cluster of missense variants associated with immunohistochemical protein reduction, proving that missense variants contribute to FH deficient ULs. We agree with previous concerns (45,46) that some pathogenic missense variants in individuals with HLRCC might be missed using only FH-IHC as screening method. While screening of certain morphologic features in tumors has been shown to enrich for patients with HLRCC (46), the statistical performance of this approach can currently not be assessed reliably.…”

“…We agree with previous concerns (45,46) that some pathogenic missense variants in individuals with HLRCC might be missed using only FH-IHC as screening method. While screening of certain morphologic features in tumors has been shown to enrich for patients with HLRCC (46), the statistical performance of this approach can currently not be assessed reliably. Thus, a prospective tumor/normal sequencing study, which represent the current gold-standard (45), in a representative risk group is needed.…”

“…While biallelic CN-losses or the combination of a CN-loss of one allele and a truncating variant on the other allele (S03: splice-acceptor variant, S06: frameshifting variant) are expected to result in the lack of protein product, missense variants usually do not cause reduced protein dosage. Rabban and colleagues indeed reported normal FH immunoexpression in tumors of two females with pathogenic germline FH missense variants (46). However, we observed no difference in IHC and morphology for ULs with biallelic deletions, likely gene disrupting and missense variants in FH (Figure 3).…”

“…have been recently proposed and tested (45,46). Those recent studies highlighted the value of routine morphological assessment as strong screening tool assisted by adjunct IHC in the initial recognition of FH-related ULs.…”

“…The detection/identification of 5/13 ULs with biallelic CN-loss and 8/13 monoallelic CN-loss in this study, suggests CN-loss as a predominant mutational mechanism which has been underestimated, but can be reliably detected using panel sequencing. The combination of CN-and VAF-analyses not only allowed us to detect both FH mutations in all 13 tumors, but also raised the suspicion of intratumoral heterogeneity in one case (S05), a mechanism only recently proposed to further complicate detection of the cause of FH-associated ULs (46).…”