FLCN-mutated eosinophilic renal tumors: clinicopathologic and molecular analysis of five cases highlighting morphologic heterogeneity.

FLCN -associated eosinophilic renal tumors mainly refer to hybrid oncocytic/chromophobe tumors (HOCT) and other oncocytic tumors related to Birt-Hogg-Dubé (BHD) syndrome, which can sometimes occur sporadically. Accurate diagnosis of FLCN -associated tumors is challenging due to their morphologic heterogeneity and the lack of reliable biomarkers. We evaluated the clinicopathologic and IHC profiles of 18 eosinophilic renal tumors with targeted DNA sequencing-confirmed FLCN mutations, including 10 typical HOCT and 8 unclassified tumors. Fourteen of these, plus 45 cases from the control group, were profiled transcriptionally by RNA-seq. Ten typical HOCT displayed consistent mosaic morphology and immunohistochemical patterns. Eight unclassified FLCN -mutated tumors exhibited diverse morphologies, including chromophobe renal cell carcinoma (ChRCC)-like, succinate dehydrogenase-deficient renal cell carcinoma (SDH-RCC)-like, and histiocyte-rich patterns, lacking obvious hybrid cellular components and typical immunohistochemical features. Despite this heterogeneity, glycoprotein non-metastatic melanoma protein B (GPNMB) was identified as a highly sensitive biomarker for FLCN -mutated tumors, showing strong and diffuse positivity in both typical HOCT, unclassified FLCN -mutated tumors, and in the oncocytosis surrounding the tumors. RNA sequencing revealed that typical HOCT formed a unique gene expression cluster, distinct from recognized renal tumor types. Some unclassified FLCN -mutated tumors were grouped with HOCT, while others remained unclassified among known kidney tumors, existing independently. This study expanded the morphologic spectrum of FLCN -mutated renal tumors and highlighted GPNMB as a valuable diagnostic marker for both typical and unclassified FLCN -mutated tumors. GPNMB should be utilized to screen eosinophilic renal tumors that cannot be classified, aiding in the precise diagnosis and management of BHD or sporadic FLCN mutation-related patients.

BackgroundThe Birt-Hogg-Dubé syndrome is a rare cancer susceptibility syndrome characterised by renal tumours, lung cysts and pneumothoraces, and fibrofolliculomas. It is caused by dominantly inherited mutations in FLCN. Our objective was to report renal tumour characteristics in a large series of patients with the Birt-Hogg-Dubé syndrome.MethodsWe studied French Birt-Hogg-Dubé patients with a history of renal tumour.ResultsWe included 33 patients with 21 distinct germline FLCN mutations. Median age at diagnosis of first renal tumour was 46, and age varied from 20 to 83. Twenty cases had one renal tumour, the remainder had two or more tumours. Most cases (23/33, 70%) had oncocytoma or renal cell carcinoma of the chromophobe or hybrid chromophobe-oncocytoma type, three had clear cell carcinoma (9%), and the other seven had carcinoma of papillary, undifferentiated or undetermined histology. Four cases had metastatic disease, although none died of it.ConclusionsAge at renal tumour diagnosis was highly variable, highlighting the need for regular surveillance from young adulthood to old age. Most cases had tumour types classically associated with Birt-Hogg-Dubé, i.e. oncocytoma or renal cell carcinoma of the chromophobe or hybrid type. Nevertheless, 9% had clear cell renal cell carcinoma. Geneticists, urologists and oncologists should therefore be alert to the possibility of Birt-Hogg-Dubé in patients with renal cell carcinoma of clear cell histology, especially if there are associated manifestations. Finally, the behaviour of metastatic carcinoma seemed more indolent than in sporadic renal cancers.

Birt–Hogg–Dubé Syndrome: An Update

Actualización en el síndrome Birt-Hogg-Dubé

A 62-year old female patient was initially diagnosed with a histologically confirmed trichodiscoma. Due to the association of trichodiscomas with Birt-Hogg-Dube Syndrome (BHD), the patient underwent screening for renal tumors, which revealed a heterogeneous right adrenal mass (Figure 1a). The patient did not have any clinical hormone excess. Adrenalectomy revealed a tumor measuring 6.2cm and weighing 104g with features diagnostic for an adrenal oncocytoma (Figure 1b). While the tumor did not fulfill the established criteria for malignancy in oncocytic adrenal neoplasms, the borderline Ki67 labeling of 5% suggested the diagnosis of at least uncertain malignant behavior rather than a benign lesion 1. The traditional Weiss scoring system to differentiate benign and malignant lesion fails in oncocytomas as they almost invariably are positive for at least two of the criteria, leading to overdiagnosis of malignant lesions 1.The patient remains disease free 24 months following surgery. Review of a chest CT imaging revealed two small cysts. The patient’s family history was suggestive of BHD, with a maternal cousin with a history of spontaneous pneumothorax (Figure 1c). The patient’s mother had a diagnosis of basal cell carcinoma and one maternal aunt died of lung cancer. The patient’s father died of pancreatic cancer. The patient underwent germline genetic testing, revealing a deleterious mutation in exon 11 of FLCN (c.1252delC; p.Leu418TrpfsX50) leading to a premature stop codon and confirming the diagnosis of BHD. Unfortunately, insufficient tumor tissue precluded loss of heterozygosity analysis. Figure 1 (a) heterogeneous adrenal mass on T1 weighted MRI. (b) oncocytic adrenal neoplasm with diffuse growth pattern. (c) Pedigree. Proband (III.1) indicated with the arrow. A maternal cousin fulfilled clinical criteria for BHD (III.3), making the patient’s ... Adrenocortical tumors, benign and malignant, are well known to occur as part of hereditary cancer susceptibility syndromes2. Adrenocortical carcinoma (ACC) is a core malignancy of Li Fraumeni syndrome. ACC and adrenocortical adenomas are observed in Beckwith Wiedemann syndrome, Multiple Endocrine Neoplasia type 1 and Familial Adenomatous Polyposis. Oncocytic adrenal tumors are rare, have biological behavior which can be difficult to predict and have not been reported in association with hereditary syndromes1. BHD was first described in 1977 with the classical manifestations of benign hair follicle associated tumors. In the decades to follow, BHD has been defined as an autosomal dominant hereditary syndrome, characterized by the cutaneous triad of fibrofolliculomas, trichodiscomas and acrochordons, lung cysts and renal tumors. BHD is caused by mutations in FLCN (Online Mendelian Inheritance in Man #13510, http://omim.org/entry/135150, for photograph of typical skin lesions see Ref. 3). Renal oncocytomas, as well as chromophobe and mixed chromophobe oncocytic adrenal tumors, are typical for BHD 3. In order to explore an adrenal phenotype in BHD patients, we identified 14 patients from 11 unique families with genetically confirmed FLCN mutations in the University of Michigan Cancer Genetics Registry. A stable 1.2cm adrenal nodule was observed in a 22 year old male patient. Next, we retrospectively reviewed 359 patients diagnosed with ACC using the Michigan Endocrine Oncology Repository. None of the 359 patients had reported history of trichodiscoma or fibrofolliculoma. One patient reportedly had 2 lung cysts sized 1.2cm. Three patients had histories of renal tumors, including one oncocytoma. Five patients reported first-degree relatives with kidney cancer. No patient fulfilled clinical criteria for BHD, had undergone germline genetic testing for FLCN mutations, or had a reported family history of BHD. In summary, adrenal tumors may be more common in BHD patients, but BHD is not common amongst ACC patients. Review of the literature yielded three reports of adrenal tumors in BHD patients. First, a study of 23 BHD patients with confirmed FLCN mutations presenting with lung cysts reported one patient with an adrenal nodule4. This adrenal nodule was small (1.5 x 2.0cm) and non-hormone-secreting (personal communication Dr. Seyama). Second, Juszczak et al. reported a large oncocytic adrenal tumor in a 36 year old female patient with BHD and confirmed FLCN mutation 5. The third report of an adrenal tumor describes a presumed ectopic renal tumor in the adrenal gland of a BHD patient 6. Our case, together with the case described by Juszczak et al,. suggest that adrenal oncocytomas might be part of the BHD tumor spectrum. With the exception of trichodiscoma, fibrofolliculoma and typical kidney tumors, no specific association of other tumors with BHD has been proven, despite several reports of benign and malignant tumors in BHD patients3. As is often the case with rare cancer susceptibility syndromes, predisposition to tumors other than the core malignancies is not well-defined. Furthermore, the phenotype of BHD can be subtle, leading to underdiagnosis. Suspicion of BHD should be raised in patients with adrenal oncocytomas. Dermatologic exam and review of chest imaging can reveal other hallmark clinical features of BHD.

BackgroundBirt-Hogg-Dubé syndrome is an autosomal dominant hereditary condition caused by mutations in the folliculin-encoding gene FLCN (NM_144997). It is associated with skin lesions such as fibrofolliculoma, acrochordon and trichodiscoma; pulmonary lesions including spontaneous pneumothorax and pulmonary cysts and renal cancer.MethodsGenomic DNA was extracted from peripheral venous blood samples of the propositi and their family members. Genetic analysis was performed by whole exome sequencing and Sanger sequencing aiming at corresponding exons in FLCN gene to explore the genetic mutations of these two families.ResultsIn this study, we performed genetic analysis by whole exome sequencing and Sanger sequencing aiming at corresponding exons in FLCN gene to explore the genetic mutations in two Chinese families. Patients from family 1 mostly suffered from pneumothorax and pulmonary cysts, several of whom also mentioned skin lesions or kidney lesions. While in family 2, only thoracic lesions were found in the patients, without any other clinical manifestations. Two FLCN mutations have been identified: One is an insertion mutation (c.1579_1580insA/p.R527Xfs on exon 14) previously reported in three Asian families (one mainland family and two Taiwanese families); while the other is a firstly reviewed mutation in Asian population (c.649C > T / p.Gln217X on exon 7) that ever been detected in a French family.ConclusionsOverall, The detection of these two mutations expands the spectrum of FLCN mutations and will provide insight into genetic diagnosis and counseling of Birt-Hogg-Dubé syndrome.

Birt-Hogg-Dubé syndrome in an overall view: Focus on the clinicopathological prospects in renal tumors

Birt-Hogg-Dube (BHD) is a rare genetic disorder characterized by multiple lung cysts, typical skin manifestations, and renal tumors. We prospectively enrolled thirty-one subjects from four South Korean institutions with typical lung cysts, and next-generation sequencing was conducted. We prospectively enrolled thirty-one subjects from four Korean institutions with typical lung cysts. Next-generation sequencing was performed to investigate mutations in the following genes: FLCN, TSC1, TSC2, CFTR, EFEMP2, ELN, FBLN5, LTBP4, and SERPINA1. BHD was diagnosed in 11 of the 31 enrolled subjects (35.5%; FLCN mutations). Notably, we identified three novel mutations (c.1098G>A, c.139G>T, and c.1335del) that have not been previously reported. In addition to FLCN mutations, we also observed mutations in CFTR (16.1%), LTBP4 (9.7%), TSC2 (9.7%), TSC1 (3.2%), ELN (3.2%), and SERPINA1 (3.2%). According to a systematic review of 45 South Korean patients with BHD, the prevalence of pneumothorax (72.7%) was greater in South Korea than in the rest of the world (50.9%; p = 0.003). The prevalence of skin manifestations (13.6%) and renal tumors (9.1%) was lower in Korea than in the rest of the world, at 47.9% [p < 0.001] and 22.5% [p = 0.027], respectively). This study confirmed a significant prediction model for BHD based on age, number of lung cysts (>40), and maximal diameter of lung cysts (>2 cm) regardless of skin manifestations and renal tumors. Importantly, three novel mutations (c.1098G>A, c.139G>T, and c.1335del) were identified. In conclusion, South Korean patients with BHD display characteristics that are different from those observed in patients of other nationalities. Detailed characterization of lung cysts is needed to define BHD, especially in South Korea, even if patients do not present with skin or renal lesions.

The Birt–Hogg–Dubé (BHD) syndrome is a rare, autosomal dominant disorder caused by germline mutations in the FLCN gene, which encodes a tumor suppressor protein. The syndrome is characterized by cutaneous fibrofolliculomas, pulmonary cysts, spontaneous pneumothoraces, and renal tumors. We report a rare familial case of BHD complicated by primary lung adenocarcinoma in a 69-year-old man. The patient presented with a persistent cough, bilateral pulmonary cysts, and a 4 × 3 cm tumor in the left lung lobe. Genetic testing revealed a novel pathogenic FLCN mutation (c.295_311del p.Asp99Ter) in the patient and several family members, including his son and brother, both of whom exhibited pulmonary cysts and histories of pneumothoraces. The patient was diagnosed with invasive lung adenocarcinoma. Although tumor progression was stabilized with treatment, the clinical course was complicated by severe hyperthyroidism, liver injury, and myelosuppression. This case highlights the complexities of managing lung cancer in BHD patients and suggests a potential role for FLCN mutations in tumorigenesis. Our findings underscore the importance of early diagnosis, genetic testing, and a multidisciplinary approach to the management of BHD in order to improve patient outcomes and prevent complications. In addition, we conducted a literature review of previously reported FLCN mutations in BHD syndrome.

Maladies kystiques pulmonaires de l’adulte d’origine génétique

Approximately 10% of patients who have a spontaneous pneumothorax have a positive family history. We sought to identify DNA sequence variations that confer susceptibility to pneumothoraces. We collected 12 families that had at least 2 first-degree relatives with a spontaneous pneumothorax. All affected family members had no obvious stigmata of known genetic disorders associated with pneumothoraces. We used haplotype analysis, DNA sequencing, and restriction fragment analysis of mutations to evaluate the individuals in these families. In 2 of the 12 families the disorder cosegregated with markers flanking a candidate locus, FLCN. Sequencing the linked alleles revealed 2 mutations predicted to introduce premature stop codons in 2 of the 12 families. Most mutations in FLCN cause a rare disease, Birt-Hogg-Dubé syndrome, characterized by autosomal dominant inheritance of multiple benign skin lesions, renal tumors, pulmonary blebs, and pneumothoraces. None of the family members with the nonsense mutations had the skin manifestations of Birt-Hogg-Dubé syndrome or renal cancer. Pathologic examination of lung tissue from three affected nonsmokers revealed blebs and underlying emphysema. Isolated familial spontaneous pneumothorax can be caused by mutations of the FLCN gene. Because development of a pneumothorax and/or pulmonary blebs may be the earliest or the only clinical manifestation of FLCN mutations, pulmonologists should be alert to the contribution of this gene toward this familial form of emphysema.

Parathyroid carcinoma (PC) is rare, poses challenges in definitive diagnosis and treatment, and carries a high rate of recurrence and mortality. Atypical parathyroid tumors (APT) have histopathologic features of malignancy but lack unequivocal evidence of invasion and are of uncertain malignant potential. Analysis of genetic alterations that may play a role in PC development, particularly those that might serve as markers of malignant potential, is important for further understanding this disease. FLCN is a gene found on chromosome 17 that encodes folliculin, a ubiquitously expressed protein with roles in multiple cellular processes such as apoptosis and cell signaling. Inactivating FLCN mutations cause Birt-Hogg-Dubé syndrome (BHD), an autosomal dominant condition characterized by benign fibrofolliculomas, pulmonary cysts and spontaneous pneumothorax, and increased risk of renal cancer. Benign parathyroid tumors had previously been reported in a few BHD patients and recently one study has reported a link between FLCN mutations and parathyroid carcinoma. Germline frameshift mutations in FLCN, accompanied by somatic loss of the normal allele, were reported in two unrelated PC patients, both with clinical features suggestive of BHD, but no established diagnosis of BHD. An identical somatic frameshift FLCN mutation was reported in one PC and one APT. Interestingly, the authors noted low coverage of FLCN on whole exome sequencing, resulting in the failure to detect variants identified by Sanger sequencing, thus raising the possibility that prior studies may have missed FLCN variants. To better understand the frequency of germline or somatic FLCN mutations in PC and APT, we performed Sanger sequence analysis of the entire coding region of the FLCN gene on available tumor DNA from 11 PCs and 15 APTs. We identified no inactivating FLCN mutations in any of the PC or APT samples examined. A germline missense variant, resulting in a p.Gly325Val change, predicted to be benign/tolerated by in silico analyses, was seen in one PC and a synonymous variant (c.1233G&amp;gt;A) was seen in one APT. The absence of pathogenic mutations in our series parathyroid carcinoma and atypical parathyroid tumors suggests that FLCN mutations are rare in these parathyroid tumors. Nevertheless, germline FLCN testing and/or additional screening for BHD-related lesions in parathyroid carcinoma patients merits further consideration and study. Citation Format: Callie Burke, Justin Bellizzi, Jessica Costa-Guda, Andrew Arnold. FLCN variants and their role in parathyroid cancer and atypical parathyroid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6265.

Objective: To investigate the expression of glycoprotein non metastatic melanoma protein B (GPNMB) in renal eosinophilic tumors and to compare the value of GPNMB with CK20, CK7 and CD117 in the differential diagnosis of renal eosinophilic tumors. Methods: Traditional renal tumor eosinophil subtypes, including 22 cases of renal clear cell carcinoma eosinophil subtype (e-ccRCC), 19 cases of renal papillary cell carcinoma eosinophil subtype (e-papRCC), 17 cases of renal chromophobe cell carcinoma eosinophil subtype (e-chRCC), 12 cases of renal oncocytoma (RO) and emerging renal tumor types with eosinophil characteristics [3 cases of eosinophilic solid cystic renal cell carcinoma (ESC RCC), 3 cases of renal low-grade eosinophil tumor (LOT), 4 cases of fumarate hydratase-deficient renal cell carcinoma (FH-dRCC) and 5 cases of renal epithelioid angiomyolipoma (E-AML)], were collected at the Affiliated Drum Tower Hospital of Nanjing University Medical School from January 2017 to March 2022. The expression of GPNMB, CK20, CK7 and CD117 was detected by immunohistochemistry and statistically analyzed. Results: GPNMB was expressed in all emerging renal tumor types with eosinophil characteristics (ESC RCC, LOT, FH-dRCC) and E-AML, while the expression rates in traditional renal eosinophil subtypes e-papRCC, e-chRCC, e-ccRCC and RO were very low or zero (1/19, 1/17, 0/22 and 0/12, respectively); the expression rate of CK7 in LOT (3/3), e-chRCC (15/17), e-ccRCC (4/22), e-papRCC (2/19), ESC RCC (0/3), RO (4/12), E-AML(1/5), and FH-dRCC (2/4) variedly; the expression of CK20 was different in ESC RCC (3/3), LOT(3/3), e-chRCC(1/17), RO(9/12), e-papRCC(4/19), FH-dRCC(1/4), e-ccRCC(0/22) and E-AML(0/5), and so did that of CD117 in e-ccRCC(2/22), e-papRCC(1/19), e-chRCC(16/17), RO(10/12), ESC RCC(0/3), LOT(1/3), E-AML(2/5) and FH-dRCC(1/4). GPNMB had 100% sensitivity and 97.1% specificity in distinguishing E-AML and emerging renal tumor types (such as ESC RCC, LOT, FH-dRCC) from traditional renal tumor types (such as e-ccRCC, e-papRCC, e-chRCC, RO),respectively. Compared with CK7, CK20 and CD117 antibodies, GPNMB was more effective in the differential diagnosis (P<0.05). Conclusion: As a new renal tumor marker, GPNMB can effectively distinguish E-AML and emerging renal tumor types with eosinophil characteristics such as ESC RCC, LOT, FH-dRCC from traditional renal tumor eosinophil subtypes such as e-ccRCC, e-papRCC, e-chRCC and RO, which is helpful for the differential diagnosis of renal eosinophilic tumors.

Birt-Hogg-Dube syndrome (BHD, OMIM#135150) is a rare disease in clinic; it is characterized by skin fibrofolliculomas, pulmonary cysts with an increased risk of recurrent pneumothorax, renal cysts, and renal neoplasms. Previous studies have demonstrated that variants in folliculin (FLCN, NM_144997) are mainly responsible for this disease. In this research, we enrolled two BHD families and applied direct sequencing of FLCN to explore the genetic lesions in them. Two FLCN mutations were identified: one is a novel deletion variant (c.668delA/p.N223TfsX19), while the other is a previously reported insertion mutation (c.1579_1580insA/p.R527QfsX75). And the pathogenicity of both variants was confirmed by cosegregation assay. Bioinformatics analysis showed that c.668delA may lead to functional haploinsufficiency of FLCN because mRNA carrying this mutation exhibits a faster degradation rate comparing to the wild type. Real-time qPCR also confirmed that the mRNA level of FLCN expression in the proband was decreased significantly compared with the controls, which may disrupt the mTOR pathway and lead to BHD. The insertion mutation (c.1579_1580insA) was predicted to cause a prolonged amino acid sequence of FLCN. The present identification of two mutations not only further supports the important role of tumor suppressor FLCN in BHD and primary spontaneous pneumothorax, but also expands the spectrum of FLCN mutations and will provide insight into genetic diagnosis and counseling of families with BHD.

Background Birt-Hogg-Dubé (BHD) syndrome is a hereditary kidney cancer syndrome, which predisposes patients to develop cutaneous fibrofolliculomas, pulmonary cysts and renal tumors with various types of histology including chromophobe renal cell carcinoma (chRCC), hybrid oncocytic/chromophobe tumor (HOCT), clear cell renal cell carcinoma (ccRCC), papillary renal cell carcinoma (pRCC) and oncocytoma. The responsible gene FLCN is a tumor suppressor for kidney cancer which plays an important role in energy homeostasis through the regulation of mitochondrial oxidative metabolism. In previous studies, we demonstrated that kidney-targeted Flcn knockout mice develops hyperproliferative polycystic kidney; however, this mouse model dies at three weeks of age before developing kidney cancer, suggesting that mutations in addition to FLCN are necessary for the development of renal neoplasms. To understand the molecular mechanisms by which alterations in metabolism under FLCN-deficiency lead to kidney cancer development, we conducted whole-exome sequencing analysis as well as metabolite analysis of BHD-associated kidney cancer. Results Whole-exome sequencing analysis of BHD-associated kidney cancer revealed that copy number variations (CNV) of BHD-associated kidney cancer are considerably different from those already reported in sporadic cases. We observed less CNV in chRCC, HOCT and oncocytoma compared to ccRCC and pRCC, which might reflect the indolent nature of chRCC, HOCT and oncocytoma. Somatic mutation analysis revealed that very few genes are commonly mutated in BHD-associated kidney cancer; however, chromatin remodeling genes were frequently mutated in BHD-associated kidney cancer (17/29 tumors, 59%), suggesting that alterations in epigenome regulation might drive kidney cancer development in cooperation with FLCN mutations. Metabolite analysis of BHD-associated kidney cancer revealed the metabolic reprogramming towards mitochondrial oxidative metabolism and upregulated lipolysis, which may fuel FLCN-deficient tumor cells to acquire a growth advantage driving tumor progression. Redox regulations including pentose phosphate pathway and glutathione synthetic pathway were upregulated in BHD-associated kidney cancer, which may protect FLCN-deficient tumor cell from reactive oxygen species produced from abundant mitochondria with increased respiratory capacity. Conclusions BHD-associated kidney cancer displays unique molecular characteristics which are completely different from sporadic kidney cancer. These data provides mechanistic insight into tumorigenesis under FLCN deficiency as well as a foundation for the development of novel therapeutics for kidney cancer. Funded in part under NCI,NIH contract HHSN261200800001. Citation Format: Ryosuke Jikuya, Mitsuko Furuya, Masaya Baba, Hiroyuki Aburatani, Adam R. Metwalli, Laura S. Schmidt, W. Marston Linehan, Masahiro Yao, Hisashi Hasumi. BHD-associated kidney cancer exhibits unique molecular characteristics and a wide variety of mutations in chromatin remodeling genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1244.