Cryptic splice mutation in the fumarate hydratase gene in patients with clinical manifestations of Hereditary Leiomyomatosis and Renal Cell Cancer.

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is an autosomal dominant condition characterized by the development of cutaneous and uterine leiomyomas and risk for development of an aggressive form of papillary renal cell cancer. HLRCC is caused by germline inactivating pathogenic variants in the fumarate hydratase (FH) gene, which encodes the enzyme that catalyzes the interconversion of fumarate and L-malate. We utilized enzyme and protein mobility assays to evaluate the FH enzyme in a cohort of patients who showed clinical manifestations of HLRCC but were negative for known pathogenic FH gene variants. FH enzyme activity and protein levels were decreased by 50% or greater in three family members, despite normal FH mRNA expression levels as measured by quantitative PCR. Direct Nanopore RNA sequencing demonstrated 57 base pairs of retained intron sequence between exons 9 and 10 of polyadenylated FH mRNA in these patients, resulting in a truncated FH protein. Genomic sequencing revealed a heterozygous intronic alteration of the FH gene (chr1: 241498239 T/C) resulting in formation of a splice acceptor site near a polypyrimidine tract, and a uterine fibroid obtained from a patient showed loss of heterozygosity at this site. The same intronic FH variant was identified in an unrelated patient who also showed a clinical phenotype of HLRCC. These data demonstrate that careful clinical assessment as well as biochemical characterization of FH enzyme activity, protein expression, direct RNA sequencing, and genomic DNA sequencing of patient-derived cells can identify pathogenic variants outside of the protein coding regions of the FH gene.

A diagnosis of Birt-Hogg-Dubé syndrome in individuals with Smith-Magenis syndrome: Recommendation for cancer screening.

We report a series of four unrelated adults with Smith-Magenis syndrome (SMS) and concomitant features of Birt-Hogg-Dubé (BHD) syndrome based upon haploinsufficiency for FLCN and characteristic renal cell carcinomas and/or evidence of cutaneous fibrofolliculomas. Three of the cases constitute the first known association of histopathologically verified characteristic BHD-associated renal tumors in adults with SMS; the fourth was identified to have histologically confirmed skin fibrofolliculomas. Molecular analysis documented second-hit FLCN mutations in two of the three cases with confirmed BHD renal pathology. These cases suggest the need to expand management recommendations for SMS to include kidney cancer surveillance starting at 20 years of age, as per the screening recommendations for BHD syndrome.

A germline 1;3 translocation disrupting the VHL gene: a novel genetic cause for von Hippel-Lindau.

Von Hippel-Lindau (VHL) disease is an autosomal dominant hereditary tumour susceptibility disease caused by germline pathogenic variation of the VHL tumour suppressor gene. Affected individuals are at risk of developing multiple malignant and benign tumours in a number of organs.In this report, a male patient in his 20s who presented to the Urologic Oncology Branch at the National Cancer Institute with a clinical diagnosis of VHL was found to have multiple cerebellar haemangioblastomas, bilateral epididymal cysts, multiple pancreatic cysts, and multiple, bilateral renal tumours and cysts. The patient had no family history of VHL and was negative for germline VHL mutation by standard genetic testing. Further genetic analysis demonstrated a germline balanced translocation between chromosomes 1 and 3, t(1;3)(p36.3;p25) with a breakpoint on chromosome 3 within the second intron of the VHL gene. This created a pathogenic germline alteration in VHL by a novel mechanism that was not detectable by standard genetic testing.Karyotype analysis is not commonly performed in existing genetic screening protocols for patients with VHL. Based on this case, protocols should be updated to include karyotype analysis in patients who are clinically diagnosed with VHL but demonstrate no detectable mutation by existing genetic testing.

Characterization of genetically defined sporadic and hereditary type 1 papillary renal cell carcinoma cell lines.

Renal cell carcinoma (RCC) is not a single disease but is made up of several different histologically defined subtypes that are associated with distinct genetic alterations which require subtype specific management and treatment. Papillary renal cell carcinoma (pRCC) is the second most common subtype after conventional/clear cell RCC (ccRCC), representing ~20% of cases, and is subcategorized into type 1 and type 2 pRCC. It is important for preclinical studies to have cell lines that accurately represent each specific RCC subtype. This study characterizes seven cell lines derived from both primary and metastatic sites of type 1 pRCC, including the first cell line derived from a hereditary papillary renal carcinoma (HPRC)-associated tumor. Complete or partial gain of chromosome 7 was observed in all cell lines and other common gains of chromosomes 16, 17, or 20 were seen in several cell lines. Activating mutations of MET were present in three cell lines that all demonstrated increased MET phosphorylation in response to HGF and abrogation of MET phosphorylation in response to MET inhibitors. CDKN2A loss due to mutation or gene deletion, associated with poor outcomes in type 1 pRCC patients, was observed in all cell line models. Six cell lines formed tumor xenografts in athymic nude mice and thus provide in vivo models of type 1 pRCC. These type 1 pRCC cell lines provide a comprehensive representation of the genetic alterations associated with pRCC that will give insight into the biology of this disease and be ideal preclinical models for therapeutic studies.

Comprehensive characterization of Alu-mediated breakpoints in germline VHL gene deletions and rearrangements in patients from 71 VHL families.

Von Hippel-Lindau (VHL) is a hereditary multisystem disorder caused by germline alterations in the VHL gene. VHL patients are at risk for benign as well as malignant lesions in multiple organs including kidney, adrenal, pancreas, the central nervous system, retina, endolymphatic sac of the ear, epididymis, and broad ligament. An estimated 30%-35% of all families with VHL inherit a germline deletion of one, two, or all three exons. In this study, we have extensively characterized germline deletions identified in patients from 71 VHL families managed at the National Cancer Institute, including 59 partial (PD) and 12 complete VHL deletions (CD). Deletions that ranged in size from 1.09 to 355 kb. Fifty-eight deletions (55 PD and 3 CD) have been mapped to the exact breakpoints. Ninety-five percent (55 of 58) of mapped deletions involve Alu repeats at both breakpoints. Several novel classes of deletions were identified in this cohort, including two cases that have complex rearrangements involving both deletion and inversion, two cases with inserted extra Alu-like sequences, six cases that involve breakpoints in Alu repeats situated in opposite orientations, and a "hotspot" PD of Exon 3 observed in 12 families that involves the same pair of Alu repeats.

Novel renal medullary carcinoma cell lines, UOK353 and UOK360, provide preclinical tools to identify new therapeutic treatments.

Renal medullary carcinoma (RMC) is a rare, aggressive disease that predominantly afflicts individuals of African or Mediterranean descent with sickle cell trait. RMC comprises 1% of all renal cell carcinoma diagnoses with a median overall survival of 13 months. Patients are typically young (median age-22) and male (male:female ratio of 2:1) and tumors are characterized by complete loss of expression of the SMARCB1 tumor suppressor protein. Due to the low incidence of RMC and the disease's aggressiveness, treatment decisions are often based on case reports. Thus, it is critical to develop preclinical models of RMC to better understand the pathogenesis of this disease and to identify effective forms of therapy. Two novel cell line models, UOK353 and UOK360, were derived from primary RMCs that both demonstrated the characteristic SMARCB1 loss. Both cell lines overexpressed EZH2 and other members of the polycomb repressive complex and EZH2 inhibition in RMC tumor spheroids resulted in decreased viability. High throughput drug screening of both cell lines revealed several additional candidate compounds, including bortezomib that had both in vitro and in vivo antitumor activity. The activity of bortezomib was shown to be partially dependent on increased oxidative stress as addition of the N-acetyl cysteine antioxidant reduced the effect on cell proliferation. Combining bortezomib and cisplatin further decreased cell viability both in vitro and in vivo that single agent bortezomib treatment. The UOK353 and UOK360 cell lines represent novel preclinical models for the development of effective forms of therapy for RMC patients.

H255Y and K508R missense mutations in tumour suppressor folliculin (FLCN) promote kidney cell proliferation.

Germline H255Y and K508R missense mutations in the folliculin (FLCN) gene have been identified in patients with bilateral multifocal (BMF) kidney tumours and clinical manifestations of Birt-Hogg-Dubé (BHD) syndrome, or with BMF kidney tumours as the only manifestation; however, their impact on FLCN function remains to be determined. In order to determine if FLCN H255Y and K508R missense mutations promote aberrant kidney cell proliferation leading to pathogenicity, we generated mouse models expressing these mutants using BAC recombineering technology and investigated their ability to rescue the multi-cystic phenotype of Flcn-deficient mouse kidneys. Flcn H255Y mutant transgene expression in kidney-targeted Flcn knockout mice did not rescue the multi-cystic kidney phenotype. However, expression of the Flcn K508R mutant transgene partially, but not completely, abrogated the phenotype. Notably, expression of the Flcn K508R mutant transgene in heterozygous Flcn knockout mice resulted in development of multi-cystic kidneys and cardiac hypertrophy in some mice. These results demonstrate that both FLCN H255Y and K508R missense mutations promote aberrant kidney cell proliferation, but to different degrees. Based on the phenotypes of our preclinical models, the FLCN H255Y mutant protein has lost it tumour suppressive function leading to the clinical manifestations of BHD, whereas the FLCN K508R mutant protein may have a dominant negative effect on the function of wild-type FLCN in regulating kidney cell proliferation and, therefore, act as an oncoprotein. These findings may provide mechanistic insight into the role of FLCN in regulating kidney cell proliferation and facilitate the development of novel therapeutics for FLCN-deficient kidney cancer.

Comprehensive Molecular Characterization of Papillary Renal-Cell Carcinoma.

BACKGROUND: Papillary renal-cell carcinoma, which accounts for 15 to 20% of renal-cell carcinomas, is a heterogeneous disease that consists of various types of renal cancer, including tumors with indolent, multifocal presentation and solitary tumors with an aggressive, highly lethal phenotype. Little is known about the genetic basis of sporadic papillary renal-cell carcinoma, and no effective forms of therapy for advanced disease exist. METHODS: We performed comprehensive molecular characterization of 161 primary papillary renal-cell carcinomas, using whole-exome sequencing, copy-number analysis, messenger RNA and microRNA sequencing, DNA-methylation analysis, and proteomic analysis. RESULTS: Type 1 and type 2 papillary renal-cell carcinomas were shown to be different types of renal cancer characterized by specific genetic alterations, with type 2 further classified into three individual subgroups on the basis of molecular differences associated with patient survival. Type 1 tumors were associated with MET alterations, whereas type 2 tumors were characterized by CDKN2A silencing, SETD2 mutations, TFE3 fusions, and increased expression of the NRF2-antioxidant response element (ARE) pathway. A CpG island methylator phenotype (CIMP) was observed in a distinct subgroup of type 2 papillary renal-cell carcinomas that was characterized by poor survival and mutation of the gene encoding fumarate hydratase (FH). CONCLUSIONS: Type 1 and type 2 papillary renal-cell carcinomas were shown to be clinically and biologically distinct. Alterations in the MET pathway were associated with type 1, and activation of the NRF2-ARE pathway was associated with type 2; CDKN2A loss and CIMP in type 2 conveyed a poor prognosis. Furthermore, type 2 papillary renal-cell carcinoma consisted of at least three subtypes based on molecular and phenotypic features. (Funded by the National Institutes of Health.).

Comprehensive genomic and phenotypic characterization of germline FH deletion in hereditary leiomyomatosis and renal cell carcinoma.

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is a familial cancer syndrome associated with the development of cutaneous and uterine leiomyomas, and an aggressive form of type 2 papillary kidney cancer. HLRCC is characterized by germline mutation of the FH gene. This study evaluated the prevalence and clinical phenotype of FH deletions in HLRCC patients. Patients with phenotypic manifestations consistent with HLRCC who lacked detectable germline FH intragenic mutations were investigated for FH deletion. A series of 28 patients from 13 families were evaluated using a combination of a comparative genomic hybridization (CGH) array and/or CLIA-approved FH deletion/duplication analyses. Thirteen distinct germline deletions were identified in the 13 UOB families, including 11 complete FH gene deletions and 2 partial FH gene deletions. The size of eight evaluated complete FH deletions varied from ∼4.74 Mb to 249 kb, with all deletions resulting in additional gene losses. Two partial FH gene deletions were identified, with one resulting in loss of exon 1 and the upstream region of the FH gene only. Kidney cancer was diagnosed in 9 (32%) of 28 patients and 7 (54%) of 13 families possessing either complete or partial FH deletions. Cutaneous and uterine leiomyomas were observed at similar rates to those in FH point mutation families. Complete or partial FH gene alterations in HLRCC families are associated with all of the canonical HLRCC manifestations, including type 2 papillary kidney cancer and should be screened for in any patient at-risk for this disorder.

Genomic and metabolic characterization of a chromophobe renal cell carcinoma cell line model (UOK276).

Chromophobe renal cell carcinoma (ChRCC) represents 5% of all RCC cases and frequently demonstrates multiple chromosomal losses and an indolent pattern of local growth, but can demonstrate aggressive features and resistance to treatment in a metastatic setting. Cell line models are an important tool for the investigation of tumor biology and therapeutic drug efficacy. Currently, there are few ChRCC-derived cell lines and none is well characterized. This study characterizes a novel ChRCC-derived cell line model, UOK276. A large ChRCC tumor with regions of sarcomatoid differentiation was used to establish a spontaneously immortal cell line, UOK276. UOK276 was evaluated for chromosomal, mutational, and metabolic aberrations. The UOK276 cell line is hyperdiploid with a modal number of 49 chromosomes per cell, and evidence of copy-neutral loss of heterozygosity, as opposed to the classic pattern of ChRCC chromosomal losses. UOK276 demonstrated a TP53 missense mutation, expressed mutant TP53 protein, and responded to treatment with a small-molecule therapeutic agent, NSC319726, designed to reactivate mutated TP53. Xenograft tumors grew in nude mice and provide an in vivo animal model for the investigation of potential therapeutic regimes. The xenograft pathology and genetic analysis suggested that UOK276 was derived from the sarcomatoid region of the original tumor. In summary, UOK276 represents a novel in vitro and in vivo cell line model for aggressive, sarcomatoid-differentiated, TP53 mutant ChRCC. This preclinical model system could be used to investigate the novel biology of aggressive, sarcomatoid ChRCC and evaluate the new therapeutic regimes.

Mitochondrial DNA mutations distinguish bilateral multifocal renal oncocytomas from familial Birt-Hogg-Dubé tumors.

Oncocytomas are mostly benign tumors characterized by accumulation of defective mitochondria, and in sporadic cases, are associated with disruptive mitochondrial DNA (mtDNA) mutations. However, the role mtDNA mutations have in renal tumors of Birt-Hogg-Dubé (BHD) patients and other renal oncocytomas with an apparent genetic component has not been investigated to date. Here we characterize the mitochondrial genome in different renal tumors and investigate the possibility of employing mtDNA sequencing analyses of biopsy specimens to aid in the differential diagnosis of oncocytomas. The entire mitochondrial genome was sequenced in 25 samples of bilateral and multifocal (BMF) renal oncocytomas, 30 renal tumors from BHD patients and 36 non-oncocytic renal tumors of different histologies as well as in biopsy samples of kidney tumors. mtDNA sequencing in BMF oncocytomas revealed that all tumors carry disruptive mutations, which impair the assembly of the NADH-ubiquinone oxidoreductase. Multiple tumors from a given BMF oncocytoma patient mainly harbor the same somatic mutation and the kidneys of these patients display diffuse oncocytosis. In contrast, renal oncocytomas of patients with BHD syndrome and renal tumors with different histologies do not show disruptive mtDNA mutations. Moreover, we demonstrate that it is feasible to amplify and sequence the entire mtDNA in biopsy specimens, and that these sequences are representative of the tumor DNA. These results show that pathogenic mtDNA mutations affecting complex I of the respiratory chain are strongly correlated with the oncocytoma phenotype in non-BHD-related renal tumors and that mtDNA sequences from biopsies are predictive of the tumor genotype. This work supports a role for mtDNA mutations in respiratory chain complexes as diagnostic markers for renal oncocytomas.

Adrenal nodular hyperplasia in hereditary leiomyomatosis and renal cell cancer.

PURPOSE: The condition hereditary leiomyomatosis and renal cell carcinoma is characterized by cutaneous leiomyomas, uterine fibroids and aggressive papillary renal cell carcinoma. A number of our patients with hereditary leiomyomatosis and renal cell carcinoma had atypical adrenal nodules, which were further evaluated to determine whether these nodules were associated with hereditary leiomyomatosis and renal cell carcinoma. MATERIALS AND METHODS: Patients with hereditary leiomyomatosis and renal cell carcinoma underwent a comprehensive clinical and genetic evaluation. We reviewed the clinical presentation, anatomical and functional imaging, endocrine evaluation, pathological examination and germline mutation testing results. RESULTS: Of 255 patients with hereditary leiomyomatosis and renal cell carcinoma 20 (7.8%) had primary adrenal lesions, including 4 with bilateral adrenal lesions and 4 with multiple nodules. Two patients had adrenocorticotropic hormone independent hypercortisolism. A total of 27 adrenal lesions were evaluated. The imaging characteristics of 5 of these lesions (18.5%) were not consistent with adenoma by noncontrast computerized tomography criteria. Positron emission tomography was positive in 7 of 10 cases (70%). A total of 12 nodules were surgically resected from 10 adrenal glands. Pathological examination revealed macronodular adrenal hyperplasia in all specimens. CONCLUSIONS: Unilateral and bilateral adrenal nodular hyperplasia was detected in a subset of patients with hereditary leiomyomatosis and renal cell carcinoma. A functional endocrine evaluation is recommended when an adrenal lesion is discovered. Imaging frequently reveals lesions that are not typical of adenomas and positron emission tomography may be positive. To date no patient has had adrenal malignancy, and active surveillance of hereditary leiomyomatosis and renal cell carcinoma adrenal nodules appears justified.

Germline PTEN mutation Cowden syndrome: an underappreciated form of hereditary kidney cancer.

PURPOSE: Cowden syndrome is a hereditary cancer syndrome associated with a germline mutation in PTEN. Patients are predisposed to multiple malignancies including renal cell carcinoma. MATERIALS AND METHODS: Patients with Cowden syndrome were evaluated as part of a clinical protocol. Those with a history of renal cell carcinoma underwent review of clinical features, tumor characteristics and family history. Renal tumors were evaluated for loss of heterozygosity. RESULTS: Among 24 patients with Cowden syndrome 4 were identified with renal cell carcinoma (16.7%). Three patients had solitary tumors, 2 with papillary type I histology and 1 with clear cell histology. The fourth patient had bilateral, synchronous chromophobe tumors. No patients had a prior family history of renal cell carcinoma. All patients with renal cell carcinoma had dermatologic manifestations of Cowden syndrome and had macrocephaly. Loss of heterozygosity at the PTEN mutation was identified in 4 tumors (80%). No genotype-phenotype association was found, as the same mutation was identified in different renal cell carcinoma histologies. CONCLUSIONS: Renal cell carcinoma is an underappreciated feature of Cowden syndrome. As most patients lack a prior family history or a distinctive renal cell carcinoma histology, recognition of the associated nonrenal features should target referral for genetic counseling. PTEN loss of heterozygosity is common in Cowden syndrome renal tumors. Because loss of PTEN can activate mTOR and mTOR inhibitors are Food and Drug Administration approved to treat renal cell carcinoma, these agents have clinical potential in renal cell carcinoma associated with Cowden syndrome.

Rilotumumab Resistance Acquired by Intracrine Hepatocyte Growth Factor Signaling.

Drug resistance is a long-standing impediment to effective systemic cancer therapy and acquired drug resistance is a growing problem for molecularly-targeted therapeutics that otherwise have shown unprecedented successes in disease control. The hepatocyte growth factor (HGF)/Met receptor pathway signaling is frequently involved in cancer and has been a subject of targeted drug development for nearly 30 years. To anticipate and study specific resistance mechanisms associated with targeting this pathway, we engineered resistance to the HGF-neutralizing antibody rilotumumab in glioblastoma cells harboring autocrine HGF/Met signaling, a frequent abnormality of this brain cancer in humans. We found that rilotumumab resistance was acquired through an unusual mechanism comprising dramatic HGF overproduction and misfolding, endoplasmic reticulum (ER) stress-response signaling and redirected vesicular trafficking that effectively sequestered rilotumumab and misfolded HGF from native HGF and activated Met. Amplification of MET and HGF genes, with evidence of rapidly acquired intron-less, reverse-transcribed copies in DNA, was also observed. These changes enabled persistent Met pathway activation and improved cell survival under stress conditions. Point mutations in the HGF pathway or other complementary or downstream growth regulatory cascades that are frequently associated with targeted drug resistance in other prevalent cancer types were not observed. Although resistant cells were significantly more malignant, they retained sensitivity to Met kinase inhibition and acquired sensitivity to inhibition of ER stress signaling and cholesterol biosynthesis. Defining this mechanism reveals details of a rapidly acquired yet highly-orchestrated multisystem route of resistance to a selective molecularly-targeted agent and suggests strategies for early detection and effective intervention.

PRDM10 RCC: A Birt-Hogg-Dubé-like Syndrome Associated With Lipoma and Highly Penetrant, Aggressive Renal Tumors Morphologically Resembling Type 2 Papillary Renal Cell Carcinoma.

OBJECTIVE: To characterize the clinical manifestations and genetic basis of a familial cancer syndrome in patients with lipomas and Birt-Hogg-Dubé-like clinical manifestations including fibrofolliculomas and trichodiscomas and kidney cancer. METHODS: Genomic analysis of blood and renal tumor DNA was performed. Inheritance pattern, phenotypic manifestations, and clinical and surgical management were documented. Cutaneous, subcutaneous, and renal tumor pathologic features were characterized. RESULTS: Affected individuals were found to be at risk for a highly penetrant and lethal form of bilateral, multifocal papillary renal cell carcinoma. Whole genome sequencing identified a germline pathogenic variant in PRDM10 (c.2029 T>C, p.Cys677Arg), which cosegregated with disease. PRDM10 loss of heterozygosity was identified in kidney tumors. PRDM10 was predicted to abrogate expression of FLCN, a transcriptional target of PRDM10, which was confirmed by tumor expression of GPNMB, a TFE3/TFEB target and downstream biomarker of FLCN loss. In addition, a sporadic papillary RCC from the TCGA cohort was identified with a somatic PRDM10 mutation. CONCLUSION: We identified a germline PRDM10 pathogenic variant in association with a highly penetrant, aggressive form of familial papillary RCC, lipomas, and fibrofolliculomas/trichodiscomas. PRDM10 loss of heterozygosity and elevated GPNMB expression in renal tumors indicate that PRDM10 alteration leads to reduced FLCN expression, driving TFE3-induced tumor formation. These findings suggest that individuals with Birt-Hogg-Dubé-like manifestations and subcutaneous lipomas, but without a germline pathogenic FLCN variant, should be screened for germline PRDM10 variants. Importantly, kidney tumors identified in patients with a pathogenic PRDM10 variant should be managed with surgical resection instead of active surveillance.

TFEB and TFE3 drive kidney cystogenesis and tumorigenesis.

Birt-Hogg-Dubé (BHD) syndrome is an inherited familial cancer syndrome characterized by the development of cutaneous lesions, pulmonary cysts, renal tumors and cysts and caused by loss-of-function pathogenic variants in the gene encoding the tumor-suppressor protein folliculin (FLCN). FLCN acts as a negative regulator of TFEB and TFE3 transcription factors, master controllers of lysosomal biogenesis and autophagy, by enabling their phosphorylation by the mechanistic Target Of Rapamycin Complex 1 (mTORC1). We have previously shown that deletion of Tfeb rescued the renal cystic phenotype of kidney-specific Flcn KO mice. Using Flcn/Tfeb/Tfe3 double and triple KO mice, we now show that both Tfeb and Tfe3 contribute, in a differential and cooperative manner, to kidney cystogenesis. Remarkably, the analysis of BHD patient-derived tumor samples revealed increased activation of TFEB/TFE3-mediated transcriptional program and silencing either of the two genes rescued tumorigenesis in human BHD renal tumor cell line-derived xenografts (CDXs). Our findings demonstrate in disease-relevant models that both TFEB and TFE3 are key drivers of renal tumorigenesis and suggest novel therapeutic strategies based on the inhibition of these transcription factors.

High-throughput and targeted drug screens identify pharmacological candidates against MiT-translocation renal cell carcinoma.

BACKGROUND: MiT-Renal Cell Carcinoma (RCC) is characterized by genomic translocations involving microphthalmia-associated transcription factor (MiT) family members TFE3, TFEB, or MITF. MiT-RCC represents a specific subtype of sporadic RCC that is predominantly seen in young patients and can present with heterogeneous histological features making diagnosis challenging. Moreover, the disease biology of this aggressive cancer is poorly understood and there is no accepted standard of care therapy for patients with advanced disease. Tumor-derived cell lines have been established from human TFE3-RCC providing useful models for preclinical studies. METHODS: TFE3-RCC tumor derived cell lines and their tissues of origin were characterized by IHC and gene expression analyses. An unbiased high-throughput drug screen was performed to identify novel therapeutic agents for treatment of MiT-RCC. Potential therapeutic candidates were validated in in vitro and in vivo preclinical studies. Mechanistic assays were conducted to confirm the on-target effects of drugs. RESULTS: The results of a high-throughput small molecule drug screen utilizing three TFE3-RCC tumor-derived cell lines identified five classes of agents with potential pharmacological efficacy, including inhibitors of phosphoinositide-3-kinase (PI3K) and mechanistic target of rapamycin (mTOR), and several additional agents, including the transcription inhibitor Mithramycin A. Upregulation of the cell surface marker GPNMB, a specific MiT transcriptional target, was confirmed in TFE3-RCC and evaluated as a therapeutic target using the GPNMB-targeted antibody-drug conjugate CDX-011. In vitro and in vivo preclinical studies demonstrated efficacy of the PI3K/mTOR inhibitor NVP-BGT226, Mithramycin A, and CDX-011 as potential therapeutic options for treating advanced MiT-RCC as single agents or in combination. CONCLUSIONS: The results of the high-throughput drug screen and validation studies in TFE3-RCC tumor-derived cell lines have provided in vitro and in vivo preclinical data supporting the efficacy of the PI3K/mTOR inhibitor NVP-BGT226, the transcription inhibitor Mithramycin A, and GPNMB-targeted antibody-drug conjugate CDX-011 as potential therapeutic options for treating advanced MiT-RCC. The findings presented here should provide the basis for designing future clinical trials for patients with MiT-driven RCC.

Succinate dehydrogenase kidney cancer: an aggressive example of the Warburg effect in cancer.

PURPOSE: Recently, a new renal cell cancer syndrome has been linked to germline mutation of multiple subunits (SDHB/C/D) of the Krebs cycle enzyme, succinate dehydrogenase. We report our experience with the diagnosis, evaluation and treatment of this novel form of hereditary kidney cancer. MATERIALS AND METHODS: Patients with suspected hereditary kidney cancer were enrolled on a National Cancer Institute institutional review board approved protocol to study inherited forms of kidney cancer. Individuals from families with germline SDHB, SDHC and SDHD mutations, and kidney cancer underwent comprehensive clinical and genetic evaluation. RESULTS: A total of 14 patients from 12 SDHB mutation families were evaluated. Patients presented with renal cell cancer at an early age (33 years, range 15 to 62), metastatic kidney cancer developed in 4 and some families had no manifestation other than kidney tumors. An additional family with 6 individuals found to have clear cell renal cell cancer that presented at a young average age (47 years, range 40 to 53) was identified with a germline SDHC mutation (R133X) Metastatic disease developed in 2 of these family members. A patient with a history of carotid body paragangliomas and an aggressive form of kidney cancer was evaluated from a family with a germline SDHD mutation. CONCLUSIONS: SDH mutation associated renal cell carcinoma can be an aggressive type of kidney cancer, especially in younger individuals. Although detection and management of early tumors is most often associated with a good outcome, based on our initial experience with these patients and our long-term experience with hereditary leiomyomatosis and renal cell carcinoma, we recommend careful surveillance of patients at risk for SDH mutation associated renal cell carcinoma and wide surgical excision of renal tumors.

Identification of intragenic deletions and duplication in the FLCN gene in Birt-Hogg-Dubé syndrome.

Birt-Hogg-Dubé syndrome (BHDS), caused by germline mutations in the folliculin (FLCN) gene, predisposes individuals to develop fibrofolliculomas, pulmonary cysts, spontaneous pneumothoraces, and kidney cancer. The FLCN mutation detection rate by bidirectional DNA sequencing in the National Cancer Institute BHDS cohort was 88%. To determine if germline FLCN intragenic deletions/duplications were responsible for BHDS in families lacking FLCN sequence alterations, 23 individuals from 15 unrelated families with clinically confirmed BHDS but no sequence variations were analyzed by real-time quantitative PCR (RQ-PCR) using primers for all 14 exons. Multiplex ligation-dependent probe amplification (MLPA) assay and array-based comparative genomic hybridization (aCGH) were utilized to confirm and fine map the rearrangements. Long-range PCR followed by DNA sequencing was used to define the breakpoints. We identified six unique intragenic deletions in nine patients from six different BHDS families including four involving exon 1, one that spanned exons 2-5, and one that encompassed exons 7-14 of FLCN. Four of the six deletion breakpoints were mapped, revealing deletions ranging from 5688 to 9189 bp. In addition, one 1341 bp duplication, which included exons 10 and 11, was identified and mapped. This report confirms that large intragenic FLCN deletions can cause BHDS and documents the first large intragenic FLCN duplication in a BHDS patient. Additionally, we identified a deletion "hot spot" in the 5'-noncoding-exon 1 region that contains the putative FLCN promoter based on a luciferase reporter assay. RQ-PCR, MLPA and aCGH may be used for clinical molecular diagnosis of BHDS in patients who are FLCN mutation-negative by DNA sequencing.

Kidney tumors associated with germline mutations of FH and SDHB show a CpG island methylator phenotype (CIMP).

Germline mutations within the Krebs cycle enzyme genes fumarate hydratase (FH) or succinate dehydrogenase (SDHB, SDHC, SDHD) are associated with an increased risk of aggressive and early metastasizing variants of renal cell carcinoma (RCC). These RCCs express significantly increased levels of intracellular fumarate or succinate that inhibit 2-oxoglutarate-dependent dioxygenases, such as the TET enzymes that regulate DNA methylation. This study evaluated the genome-wide methylation profiles of 34 RCCs from patients with RCC susceptibility syndromes and 11 associated normal samples using the Illumina HumanMethylation450 BeadChip. All the HLRCC (FH mutated) and SDHB-RCC (SDHB mutated) tumors demonstrated a distinct CpG island methylator phenotype (CIMP). HLRCC tumors demonstrated an extensive and relatively uniform level of hypermethylation that showed some correlation with tumor size. SDHB-RCC demonstrated a lesser and more varied pattern of hypermethylation that overlapped in part with the HLRCC hypermethylation. Combined methylation and mRNA expression analysis of the HLRCC tumors demonstrated hypermethylation and transcription downregulation of genes associated with the HIF pathway, HIF3A and CITED4, the WNT pathway, SFRP1, and epithelial-to-mesenchymal transition and MYC expression, OVOL1. These observations were confirmed in the TCGA CIMP-RCC tumors. A selected panel of probes could identify the CIMP tumors and differentiate between HLRCC and SDHB-RCC tumors. This panel accurately detected all CIMP-RCC tumors within the TCGA RCC cohort, identifying them as HLRCC -like, and could potentially be used to create a liquid biopsy-based screening tool. The CIMP signature in these aggressive tumors could provide both a useful biomarker for diagnosis and a target for novel therapies.