Maintained disomic chromosome 17 as a diagnostic marker for BHD-associated chromophobe RCC

Renal cell carcinomas (RCCs) can be life-threatening and although mostly sporadic, approximately 5% are associated with genetic conditions such as BHD. Early identification of families carrying cancer-predisposing mutations enables access to regular screening and earlier treatment. However, it can be difficult to distinguish between sporadic and inherited RCC based on standard immunohistological analysis. New research from Kato et al. (2016) assessed whether variability in the chromosomal status of chromosomes 17, 2, and 6 could be used to identify BHD-associated RCC.

The BHD gene FLCN is a tumour suppressor located on chromosome 17, with tumour growth associated with second hit mutations or loss of heterozygosity (Vocke et al., 2005). The most common tumour types in BHD patients are chromophobe RCC (chRCC) and hybrid oncocytoma/ chromophobe tumours (HOCTs) although other subtypes are seen. Sporadic chRCC tumours frequently exhibit variable chromosomal losses including loss of chromosome 17, whereas papillary (papRCC) tumours more often gain copies of chromosomes 7 and 17. Kato et al. decided to determine if these variations in the status of chromosome 17 could be used to help distinguish BHD-associated and sporadic tumours.

Kato et al. used CEN17q fluorescent and chromogenic in situ hybridisation (FISH and CISH respectively) to assess the status of chromosome 17 in BHD-associated tumours (8 chRCC, 7 HOCT and 3 papRCC) and sporadic tumours (14 chRCC and 5 papRCC). The BHD-tumours came from ten genetically confirmed BHD patients with additional somatic FLCN mutations identified in 9/13 samples available for genetic testing. The sporadic RCC patients were not tested for FLCN mutations but had no family history of BHD-associated pulmonary or dermatological pathologies. Where possible samples of normal renal tissue were assessed for comparison.

In each sample the FISH/CISH signal was counted in 100 nuclei with comparison to non-tumour samples used to establish if a tumour sample was monosomic, disomic and trisomic. All of the BHD-HOCT and 7/8 BHD-chRCC samples were disomic compared to 12/14 sporadic chRCC samples being monosomic (p=0.0008). The status of chromosome 17 could therefore be useful in distinguishing BHD-associated HOCTS and chRCC from sporadic chRCC. However, it was insufficient to distinguish between BHD-associated and sporadic forms of papRCC (1 disomic and 2 trisomic compared to 2 disomic and 3 trisomic) or clear cell RCC (ccRCC; data not shown but listed as disomic in all cases).

One BHD-chRCC was judged to be monosomic for CEN17q, but when reassessed with a second probe, CEP17, was found to be disomic. As false monosomy/polysomy could occur with any sample additional markers would be beneficial. Kato et al. found statistically significant differences in the FISH/CISH analysis of chRCC and HOCT samples with CEN2p and CEN6p probes; whilst the majority of sporadic chRCCs were monosomic at these loci the BHD-associated tumours retained disomy.

Previous work from this group has identified variations in sporadic chRCC marker expression (Iribe et al., 2015), as well as changes in FLCN and GPNMB expression (Fuyura et al., 2015) that can help distinguish between some sporadic and BHD-associated RCC subtypes. These markers, along with the findings from Kato et al., are summarised in the table below. Currently no diagnostic markers have been identified than can distinguish between sporadic and BHD-associated ccRCC nor oncocytomas. Although further work is needed to develop a robust marker panel for all RCC subtypes, the identification of distinguishing features of the most common BHD-tumour types is encouraging. Hopefully this and future work can be used by pathologists to identify new BHD patients and families.

Sporadic RCC subtype BHD-associated RCC subtype Distinguishing markers
Chromophobe RCC Chromophobe RCC ↓FLCN, ↑GPNMB, 17q/2p/6p disomy
Chromophobe RCC HOCT ↓CK7, ↓FLCN, ↑GPNMB, 17q/2p/6p disomy
Oncocytoma HOCT ↑Ksp-Cadherin, ↑CD82, ↓FLCN, ↑GPNMB
Papillary RCC Papillary RCC ↓FLCN, ↑GPNMB


  • Furuya M, Hong SB, Tanaka R, Kuroda N, Nagashima Y, Nagahama K, Suyama T, Yao M, Nakatani Y (2015). Distinctive expression patterns of glycoprotein non-metastatic B and folliculin in renal tumors in patients with Birt-Hogg-Dubé syndrome. Cancer Sci. 106(3):315-23. PMID: 25594584.
  • Kato, I., Iribe, Y., Nagashima, Y., Kuroda, N., Tanaka, R., Nakatani, Y., Hasumi, H., Yao, M., & Furuya, M. (2016). Fluorescent and Chromogenic in situ Hybridization of CEN17q as a Potent Useful Diagnostic Marker for Birt-Hogg-Dubé Syndrome-associated Chromophobe Renal Cell Carcinomas Human Pathology DOI: 10.1016/j.humpath.2016.01.004.
  • Iribe Y, Kuroda N, Nagashima Y, Yao M, Tanaka R, Gotoda H, Kawakami F, Imamura Y, Nakamura Y, Ando M, Araki A, Matsushima J, Nakatani Y, Furuya M (2015). Immunohistochemical characterization of renal tumors in patients with Birt-Hogg-Dubé syndrome. Pathol Int. 65(3):126-32. PMID: 25597876.
  • Vocke CD, Yang Y, Pavlovich CP, Schmidt LS, Nickerson ML, Torres-Cabala CA, Merino MJ, Walther MM, Zbar B, Linehan WM (2005). High frequency of somatic frameshift BHD gene mutations in Birt-Hogg-Dubé-associated renal tumors. J Natl Cancer Inst, 15;97(12):931-5. PMID: 15956655.
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TSC2 mutations confer everolimus sensitivity in hepatocellular carcinomas

Hepatocellular carcinomas (HCCs) are the third leading cause of cancer deaths globally; frequently diagnosed only in the advanced stages and aggressive in nature. Although enhanced mTOR activity has a key role in HCC tumourigenesis, the EVOLVE-1 clinical trial of mTOR inhibitor everolimus found no associated improvement in overall survival (Zhu et al., 2014). However, everolimus is an effective treatment for tuberous sclerosis complex (TSC) manifestations, a rare disease associated with mutations in TSC1 and TSC2 that result in high mTOR activity. New research from Huynh et al. (2016) assessed the frequency of TSC2  loss in HCC and suggests this could predict a selective response to everolimus.

Expression of TSC2 was found to be markedly reduced in 5/9 HCC cell lines assessed (Cancer Cell line Encyclopedia collection). Huynh et al. confirmed the loss of TSC2 by immunoblot in four of these lines which showed associated increased mTOR activity and decreased AKT phosphorylation. The four TSC-null cell lines showed enhanced sensitivity, with greater inhibition of cell proliferation, to everolimus than four TSC-wildtype cell lines. Everolimus treatment also reversed the enhanced phosphorylation of S6K1, indicative of mTOR inhibition.

Huynh et al. then assessed the frequency of TSC2 mutations in patient derived xenografts. In 8/26 xenografts TSC2 was undetectable with associated increased S6K1 phosphorylation. When tumour-bearing mice where treated with 1mg/kg everolimus significant anti-tumour responses were seen in the TSC2-null tumours compared to those treated with vehicle only. In contrast the everolimus treated TSC-wildtype tumours showed little or no response indicative of selective efficacy rather than general toxicity. Treatment with everolimus also showed a dose-dependent reduction in S6K1 phosphorylation and increase in AKT activity.

Genetic sequencing of the TSC-null HCC cell lines and xenografts identified a range of mutations and deletions in 3/4 and 6/8 samples respectively. Epigenetic gene silencing was hypothesised to explain the loss of TSC2 in the remaining samples. To validate the existence of TSC2 mutations in primary tumours 13 HCC biopsies were also sequenced and three found to carry TSC2 mutations. Although sequencing can identify mutations it cannot always predict protein loss. Instead Huynh et al. developed an immunohistochemistry (IHC) assay, validated in the HCC cell lines and xenografts, where TSC2 protein levels could be quantified, and samples categorised as either TSC-null, TSC-low or TSC-wildtype. .

This IHC assay was then used to assess tumour samples from 139 patients enrolled on the EVOLVE-1 trial; eight samples were found to be TSC-null and seven to be TSC-low. Detailed assessment of overall survival in these patients determined that 6/10 treated with everolimus had an overall survival (OS) greater than the median for the whole trial: 9.53-32.72 months compared to 7.56 months. Three of the other patients receiving everolimus withdrew after 2-6 weeks treatment (OS 0.76-4.63 months). Comparatively those patients with TSC2 loss who received the placebo had a lower OS of 1.25-5.59 months. This prospective analysis supports the previous results suggesting that everolimus is more effective against tumours that lack TSC2.

Tumour heterogeneity can make treatment responses difficult to predict but understanding the molecular basis of tumourigenesis can help identify treatment targets. Everolimus has also been shown to be effective against other tumour types carrying TSC1 or TSC2 mutations including renal tumours (Voss et al., 2014), metastatic bladder cancer (Iyer et al., 2012) and thyroid tumours (Wagle et al., 2014). This highlights a need to design clinical trials, like basket and umbrella trials, that use the advances in genetic sequencing to stratify patients for a greater understanding of treatment efficacy.

This research also demonstrates how greater understanding of a protein associated with a rare inherited disease can affect the treatment of patients with a sporadic condition. Mutations associated with other cancer-predisposition conditions such as BHD and HLRCC have also been reported in sporadic tumours and could progress the development of more effective treatments based on cancer genetics rather than location.

Huynh H, Hao HX, Chan SL, Chen D, Ong R, Soo KC, Pochanard P, Yang D, Ruddy D, Liu M, Derti A, Balak MN, Palmer MR, Wang Y, Lee BH, Sellami D, Zhu AX, Schlegel R, Huang A (2015). Loss of Tuberous Sclerosis Complex 2 (TSC2) Is Frequent in Hepatocellular Carcinoma and Predicts Response to mTORC1 Inhibitor Everolimus. Mol Cancer Ther. 14(5):1224-35. PMID: 25724664.

Iyer G, Hanrahan AJ, Milowsky MI, Al-Ahmadie H, Scott SN, Janakiraman M, Pirun M, Sander C, Socci ND, Ostrovnaya I, Viale A, Heguy A, Peng L, Chan TA, Bochner B, Bajorin DF, Berger MF, Taylor BS, Solit DB (2012). Genome sequencing identifies a basis for everolimus sensitivity. Science. 338(6104):221 PMID: 22923433.

Voss MH, Hakimi AA, Pham CG, Brannon AR, Chen YB, Cunha LF, Akin O, Liu H, Takeda S, Scott SN, Socci ND, Viale A, Schultz N, Sander C, Reuter VE, Russo P, Cheng EH, Motzer RJ, Berger MF, Hsieh JJ (2014). Tumor genetic analyses of patients with metastatic renal cell carcinoma and extended benefit from mTOR inhibitor therapy. Clin Cancer Res. 20(7):1955-64. PMID: 24622468.

Wagle N, Grabiner BC, Van Allen EM, Amin-Mansour A, Taylor-Weiner A, Rosenberg M, Gray N, Barletta JA, Guo Y, Swanson SJ, Ruan DT, Hanna GJ, Haddad RI, Getz G, Kwiatkowski DJ, Carter SL, Sabatini DM, Jänne PA, Garraway LA, Lorch JH (2014). Response and acquired resistance to everolimus in anaplastic thyroid cancer. N Engl J Med. 371(15):1426-33. PMID: 25295501.

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A role for dermatologists in diagnosing BHD earlier

Birt-Hogg-Dubé (BHD) syndrome was initially described as a heritable dermatological condition based on the presence of multiple fibrofolliculomas, trichodiscomas and acrochordons in a Canadian kindred (Birt et al., 1977). Now it is known that BHD patients can also develop pulmonary cysts, with an associated risk of pneumothorax, and bilateral, multifocal renal tumours. Due to the risk of tumour development it is important that patients are diagnosed early, enabling them to access regular screening and earlier treatment if required.

The pale and skin-coloured growths characteristic of BHD predominantly develop on patients’ faces and necks, and whilst some patients develop only a small number, others develop hundreds. They usually begin to appear in early adulthood, worsening with age, and are therefore often the first sign that a patient has a condition associated with cancer-predisposition. As such it is important that dermatologists are aware of BHD as a potential cause of dermal lesions when patients are referred for assessment and treatment.

Benign hair follicle tumours (BHFTs) can be associated with various conditions and their histology is often key to differential diagnosis. Tallechea et al., (2015) recently reviewed the histology of several BHFTs including fibrofolliculomas and trichodiscomas. These BHD-associated BHFTs have distinct characteristics and can both occur on the same patient, even sometimes in very close proximity suggestive of a shared pathogenesis. If BHD is suspected it is important to take punch biopsies, due to the deep nature of these lesions, rather than shave biopsies to increase the accuracy of diagnosis.

The presence of multiple pale or skin coloured lesions on a patient could be caused by conditions other than BHD – some of which are discussed here. Suspicion should however be higher in patients with a family history of similar dermatological growths, pneumothoraces or renal cancer. The presence of at least five adult onset fibrofolliculomas or trichodiscomas, one of which has been histologically confirmed, can be used as the basis of a diagnosis of BHD (Menko et al., 2009). However, it is recommended that patients undergo genetic testing to identify mutations in the FLCN gene for a firm diagnosis.

Two recent case reports highlight the potential of dermatologists to identify BHD patients carrying previously undetected renal cysts or tumours. Menzies et al. (2016) describe a 42-year-old woman with multiple skin papules that developed in her late 30s and a family history of similar lesions. Punch bioposies and genetic testing confirmed BHD and she was found to have a benign renal cyst in a follow-up ultrasound. Ge and Lowe (2016) describe a 52-year-old man with multiple facial lesions, a personal history of bilateral pneumothorax and whose sons also experienced dermatological growths and recurrent pneumothorax. BHD was confirmed by genetic testing, and subsequently a renal tumour was identified on his left kidney – after removal this was determined to be a hybrid oncocytoma/chromophobe tumour (HOCT), a subtype characteristic of BHD. These patients and their families will now be regularly monitored for tumour growth, ensuring timely treatment.

Fibrofolliculomas, although benign and of little or no health risk, are a visible sign of the disease and their development can therefore be distressing. Dermatologists should therefore be willing to discuss treatment options with patients; although patients should be aware that none of the current treatments can permanently remove the growths. Continuing research into the underlying pathology of fibrofolliculomas could identify new therapeutic targets enabling more efficient treatments to be developed.

  • Birt AR, Hogg GR, Dubé WJ (1977). Hereditary multiple fibrofolliculomas with trichodiscomas and acrochordons. Arch Dermatol. 113(12):1674-7. PMID: 596896.
  • Ge L, Lowe P (2016). Not just a cosmetic problem: facial papules in Birt-Hogg-Dubé syndrome. Med J Aust. 204(1):28-9. PMID: 26763815.
  • Menko FH, van Steensel MA, Giraud S, Friis-Hansen L, Richard S, Ungari S, Nordenskjöld M, Hansen TV, Solly J, Maher ER; European BHD Consortium (2009). Birt-Hogg-Dubé syndrome: diagnosis and management. Lancet Oncol. 10(12):1199-206. PMID: 19959076.
  • Menzies S, Fabre A, Lally A (2016). Birt-Hogg-Dubé syndrome: identifying patients at risk of renal cell carcinoma, pulmonary cysts and pneumothoraces. BMJ Case Rep. Jan 13;2016 pii: bcr2015213865 PMID: 26762352.
  • Tellechea O, Cardoso JC, Reis JP, Ramos L, Gameiro AR, Coutinho I, Baptista AP (2015). Benign follicular tumors. An Bras Dermatol. 90(6):780-98. PMID: 26734858.
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Genome-wide genetic abnormalities in BHD-RCCs

Renal cell carcinoma (RCC) can be sporadic or associated with inherited mutations. These tumours frequently carry additional genetic abnormalities including copy number variations, deletions and amplifications. Although not all tumours have an altered genetic profile, sporadic RCC subtypes have common cytogenetic patterns. New research from Iribe et al. (2016) assessed several RCC subtypes from BHD patients to determine if they have similar or distinct patterns of genetic abnormalities.

In a previous study Klomp et al. (2010) found that six BHD-RCC tumours, which morphologically resembled sporadic chromophobe RCC (chRCC) and oncocytomas, did not share the typical gross chromosomal abnormalities seen in sporadic tumours. However, advances in technology make it possible to detect smaller deletions and amplifications across the genome. Iribe et al. assessed genomic alterations in BHD-associated chRCC, clear cell RCC (ccRCC), and hybrid oncocytotic/ chromophobe tumours (HOCTs).

Nineteen RCC samples were obtained from 10 genetically confirmed BHD patients; 9/19 tumours carried an identifiable additional FLCN mutation. Cytogenetic profiling was completed for 11 tumours including three HOCTs, six chRCC and two ccRCC samples. Sporadic chRCC, ccRCC and oncocytoma samples came from patients who, although not genetically tested for FLCN, had no clinical history of fibrofolliculomas, pulmonary cysts or pneumothoraces.

The common cytogenetic profiles for sporadic RCCs are varied: clear cell RCC (ccRCC) tumours often show loss of chromosome 3q and 19q, and gain of 5q (as discussed in last week’s blog); chRCC tumours contain a wider range of losses in chromosomes 1, 2, 6, 10, 13, 17 and 21 (Davis et al., 2014); and, although less frequently seen, renal oncocytomas can have losses of chromosomes 1 and Y (Lindgren et al., 2004). All of the BHD patient HOCT samples (n=3) and 4/6 BHD-chRCC had a broadly balanced chromosome number, but the remaining two BHD-chRCC samples (from different patients) showed gain of chromosome 3q. The two BHD-ccRCC samples were more unbalanced with gains and losses of several chromosomes – a wider range than sporadic ccRCC samples – and neither carried a VHL mutation. This supports BHD tumours having distinct cytogenetic profiles compared to sporadic tumours.

Loss of heterogeneity (LOH) analysis in the BHD-RCC samples detected numerous LOH regions in all tumour types across all chromosomes – excluding Y as only one sample was male. Allele-specific analysis revealed that most of the LOH regions were the result of uniparental disomy (UPD), with several common UPD regions on chromosomes 3, 8, 16 and X. Although UPD has been reported in other malignancies, levels are not usually very high. Therefore a series of UPD regions, common across BHD-RCC subtypes, could be a cytogenetic marker for tumours associated with BHD.

UPD in cancer cells can lead to altered gene expression; NETO2 at 16q11.2 is up-regulated

  • Davis CF, Ricketts CJ, Wang M, Yang L, Cherniack AD, Shen H, Buhay C, Kang H, Kim SC, Fahey CC, Hacker KE, Bhanot G, Gordenin DA, Chu A, Gunaratne PH, Biehl M, Seth S, Kaipparettu BA, Bristow CA, Donehower LA, Wallen EM, Smith AB, Tickoo SK, Tamboli P, Reuter V, Schmidt LS, Hsieh JJ, Choueiri TK, Hakimi AA; Cancer Genome Atlas Research Network, Chin L, Meyerson M, Kucherlapati R, Park WY, Robertson AG, Laird PW, Henske EP, Kwiatkowski DJ, Park PJ, Morgan M, Shuch B, Muzny D, Wheeler DA, Linehan WM, Gibbs RA, Rathmell WK, Creighton CJ (2014). The somatic genomic landscape of chromophobe renal cell carcinoma. Cancer Cell 26(3):319-30. PubMed PMID: 25155756.
  • Klomp JA, Petillo D, Niemi NM, Dykema KJ, Chen J, Yang XJ, Sääf A, Zickert P, Aly M, Bergerheim U, Nordenskjöld M, Gad S, Giraud S, Denoux Y, Yonneau L, Méjean A, Vasiliu V, Richard S, MacKeigan JP, Teh BT, Furge KA (2010). Birt-Hogg-Dubé renal tumors are genetically distinct from other renal neoplasias and are associated with up-regulation of mitochondrial gene expression. BMC Med Genomics 3:59. PMID: 21162720.
  • Iribe Y, Yao M, Tanaka R, Kuroda N, Nagashima Y, Nakatani Y, Furuya M (2016). Genome-Wide Uniparental Disomy and Copy Number Variations in Renal Cell Carcinomas Associated with Birt-Hogg-Dubé Syndrome. Am J Pathol. 186(2):337-46. PMID: 26776076.
  • Lindgren V, Paner GP, Omeroglu A, Campbell SC, Waters WB, Flanigan RC, Picken MM (2004). Cytogenetic analysis of a series of 13 renal oncocytomas. J Urol. 171(2 Pt 1):602-4. PMID: 14713769.
  • Zaravinos A, Pieri M, Mourmouras N, Anastasiadou N, Zouvani I, Delakas D, Deltas C (2014). Altered metabolic pathways in clear cell renal cell carcinoma: A meta-analysis and validation study focused on the deregulated genes and their associated networks. Oncoscience. 1(2):117-31. PMID: 25594006.
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Increased HIF-1α activity in RCC modulated by p62

Clear cell Renal Cell Carcinoma (ccRCC) is frequently associated with the loss of VHL, but gain of chromosome 5q35.3 – which contains the oncogene SQSTM1 – was also found in approximately 70% of tumours (Li et al., 2013). The SQSTM1 protein, p62, is a scaffold protein involved in cellular processes and signalling pathways related to malignant growth. New research from Chen et al., (2015) determined a role for p62 in regulating glucose metabolism, through HIF-1α signalling.

Expression of p62 is upregulated in various malignancies and can promote tumourigenic cell proliferation and growth by enhancing NF-kB, mTORC1 and NRF2 signalling. Additionally, p62 binds both ubiquitinated proteins and LC3 to promote autophagosome formation. Dunlop et al., (2014) identified increased levels of p62 in BHD tumours and cell lines associated with the dysregulation of autophagy. An additional trademark of cancer cells is altered metabolism; the Warburg effect. Chen et al. assessed glycolytic enzyme expression, glucose uptake and lactate production following sh-RNA knockdown of p62 in ACHN cells: all were reduced and the cells showed reduced cell growth and viability.

In various forms of inherited and sporadic RCC changes in metabolism are regulated by HIF-1α signalling. Chen et al., report that knockdown of p62 results in decreased HIF-1α, but not HIF-2α, expression which was rescued when p62 was restored. In addition to regulating HIF-1α expression, overexpression of p62 also increases HIF-1α transcriptional activity. HIF signalling can be altered in different ways: in VHL and HLRCC there is an increase in HIF-1α stability (Bratslavsky et al., 2007, Isaacs et al., 2005); in TSC HIF signalling is upregulated via mTOR; and in BHD increased AMPK activity, via PGC1A-mediated ROS production, results in increased HIF-1α activity (Yan et al., 2014). Overexpression of p62, commonly seen in these tumours, could also be contributing.

Both mTORC1 signalling and NF-kB signalling can upregulate HIF-1α signalling. Inhibition of either mTOR signalling using rapamycin, or NF-kB by knockdown of the p65 subunit, reduced HIF-1α levels in cells expressing p62. Additionally, p62 has previously been shown to coimmunoprecipitate with VHL E3 ligase complex component CUL2. Chen et al. determined that p62 also coimmunoprecipitates with the other complex components including pVHL, but not with HIF-1α. Using deletion mutations this interaction was mapped to the TRAF6 binding domain in p62 and the intact β-domain in pVHL – the same domain that binds HIF proteins. Chen et al. propose that p62 competes with HIF-1α at this site, blocking HIF-1α ubquitination and reducing protein degradation. VHL E3 ligase activity is further reduced due to p62 blocking the neddylation of CUL2.

To confirm whether p62-promoted aerobic glycolysis due to increased HIF-1α signalling Chen et al. used shRNAs to knockdown either p62 or HIF-1α. Knockdown of HIF-1α impaired p62-mediated increases in glucose uptake and lactate production, but HIF-1α overexpression could reverse the reduced uptake caused by p62 silencing. p62 therefore promotes enhanced HIF-1α signalling both by increasing expression and reducing degradation, which leads to altered cellular metabolism.

Altered mTORC1, NF-kB and NRF2 signalling has been reported in several tumour type and the loss of VHL E3 ligase activity is characteristic of ccRCC. If increased p62 can disrupt VHL E3 ligase in cells with functional pVHL then the same pathways could be perturbed in other tumour types, such as those associated with BHD and FLCN mutations, contributing to the enhanced HIF-1α activity. Understanding how all these different signalling pathways interact is important to further elucidate RCC tumourigenesis and develop more effective treatments that can target the key pathways.

  • Bratslavsky G, Sudarshan S, Neckers L, Linehan WM (2007). Pseudohypoxic pathways in renal cell carcinoma. Clin Cancer Res. 13(16):4667-71. PMID: 17699843.
  • Chen K, Zeng J, Xiao H, Huang C, Hu J, Yao W, Yu G, Xiao W, Xu H, Ye Z (2016). Regulation of glucose metabolism by p62/SQSTM1 through HIF1α. J Cell Sci. Jan 7. pii: jcs.178756. [Epub ahead of print] PMID: 26743088.
  • Dunlop EA, Seifan S, Claessens T, Behrends C, Kamps MA, Rozycka E, Kemp AJ, Nookala RK, Blenis J, Coull BJ, Murray JT, van Steensel MA, Wilkinson S, Tee AR (2014). FLCN, a novel autophagy component, interacts with GABARAP and is regulated by ULK1 phosphorylation. Autophagy 10(10):1749-60. PMID: 25126726.
  • Isaacs JS, Jung YJ, Mole DR, Lee S, Torres-Cabala C, Chung YL, Merino M, Trepel J, Zbar B, Toro J, Ratcliffe PJ, Linehan WM, Neckers L (2005). HIF overexpression correlates with biallelic loss of fumarate hydratase in renal cancer: novel role of fumarate in regulation of HIF stability. Cancer Cell. 8(2):143-53. PMID: 16098467.
  • Li L, Shen C, Nakamura E, Ando K, Signoretti S, Beroukhim R, Cowley GS, Lizotte P, Liberzon E, Bair S, Root DE, Tamayo P, Tsherniak A, Cheng SC, Tabak B, Jacobsen A, Hakimi AA, Schultz N, Ciriello G, Sander C, Hsieh JJ, Kaelin WG Jr. SQSTM1 is a pathogenic target of 5q copy number gains in kidney cancer. Cancer Cell. 2013 Dec 9;24(6):738-50. doi: 10.1016/j.ccr.2013.10.025. PMID: 24332042.
  • Yan M, Gingras MC, Dunlop EA, Nouët Y, Dupuy F, Jalali Z, Possik E, Coull BJ, Kharitidi D, Dydensborg AB, Faubert B, Kamps M, Sabourin S, Preston RS, Davies DM, Roughead T, Chotard L, van Steensel MA, Jones R, Tee AR, Pause A (2014). The tumor suppressor folliculin regulates AMPK-dependent metabolic transformation. J Clin Invest 124(6):2640-50. PMID: 24762438.
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HIF-2α regulates PD-L1 expression in RCC

Tumour cells can create immunosuppressive microenvironments by hijacking natural mechanisms such as PD-L1 expression to impair T-cell function. Several new immunotherapy treatments target the PD-1/PD-L1 pathway and have produced some long-lasting responses in patients (Motzer et al., 2015) but not all patients respond. High expression of PD-L1 in clear cell RCC (ccRCC) has been shown to correlate with metastasis and poor outcome (Thompson et al., 2007). New research from Messai et al. (2015) has assessed PD-L1 expression in ccRCC samples and cell lines carrying VHL mutations to identify meaningful correlations.

Messai et al. initially assessed PD-L1 expression in 32 ccRCC samples – 11 from VHL patients and 21 sporadic tumours – which had been sequenced for VHL gene mutations, loss of function (LOF) and loss of heterogeneity (LOH). Significantly higher PD-L1 expression was found in the tumours which had two altered VHL alleles (21/32), complete LOF (14/32) or had LOH at the VHL locus (23/32). This suggests a link between VHL status and PD-L1 expression in ccRCCs.

To further investigate the role of VHL in regulating PD-L1 expression Messai et al. reintroduced various mutated VHL constructs into VHL-deficient 786-O cells – these cells overexpress HIF-2α but not HIF-1α.  The different pVHL mutants resulted in a gradient expression of HIF-2α which positively correlated with PD-L1 mRNA and protein expression levels. In cells retransfected with wildtype VHL the expression of PD-L1 was significantly lower than those carrying VHL-mutants or no VHL constructs. This further confirms the correlation between VHL mutations and PD-L1 expression seen in the tumour samples.

Increased HIF-2α signalling resulting from VHL loss plays a role in tumourigenesis altering cellular metabolism and increasing angiogenesis. To determine the role of HIF-2α in the correlation between VHL and PD-L1 expression Messai et al. used siRNA to knockdown HIF-2α in 786-0 and A498 cells (also VHL-deficient); resulting in reduced PD-L1 mRNA, protein and surface expression. In addition overexpression of HIF-2α in cells with functional VHL results in increased PD-L1 expression. This suggests that PD-L1 expression is being regulated by HIF-2α signalling, which is mediated by VHL status.

Analysis of the PD-L1 promoter identified several putative Hypoxia Response Elements (HRE). Based on ChIP and luciferase assays, HIF-2α specifically binds to HRE4 and is transcriptionally active. Previously HIF-1α was shown to regulate PD-L1 expression in tumour infiltrating MDSCs, also through binding to HRE4 (Norman et al., 2014). To determine if HIF-1α has a similar role in ccRCC cells Messai et al. assessed PD-L1 expression in RRC4 cells which overexpress both HIF-1α and HIF-2α.  Knockdown of either HIF-1α or HIF-2α, or both resulted in a decrease in PD-L1 expression supporting a direct role for HIF signalling in PD-L1 regulation.

Further studies will be needed to determine the validity of VHL mutation status as a biomarker for PD-L1 expression and if there is any correlation to immunotherapy responses. Increased HIF signalling is generally associated with tumourigenesis and is also seen in BHD tumours and cell lines (Preston et al., 2011, Nishii et al., 2013). Greater understanding of the role of HIF signalling and PD-L1 expression in response to various treatments including immunotherapies could help identify the optimal combination or sequential treatment plans for a range of RCC patients.

  • Messai Y, Gad S, Noman MZ, Le Teuff G, Couve S, Janji B, Kammerer SF, Rioux-Leclerc N, Hasmim M, Ferlicot S, Baud V, Mejean A, Mole DR, Richard S, Eggermont AM, Albiges L, Mami-Chouaib F, Escudier B, Chouaib S (2015). Renal Cell Carcinoma Programmed Death-ligand 1, a New Direct Target of Hypoxia-inducible Factor-2 Alpha, is Regulated by von Hippel-Lindau Gene Mutation Status. Eur Urol. Dec 17. pii: S0302-2838(15)01200-2. PMID: 26707870.
  • Motzer RJ, Rini BI, McDermott DF, Redman BG, Kuzel TM, Harrison MR, Vaishampayan UN, Drabkin HA, George S, Logan TF, Margolin KA, Plimack ER, Lambert AM, Waxman IM, Hammers HJ (2015). Nivolumab for Metastatic Renal Cell Carcinoma: Results of a Randomized Phase II Trial. J Clin Oncol. 33(13):1430-7. PMID: 25452452.
  • Nishii T, Tanabe M, Tanaka R, Matsuzawa T, Okudela K, Nozawa A, Nakatani Y, Furuya M (2013). Unique mutation, accelerated mTOR signaling and angiogenesis in the pulmonary cysts of Birt-Hogg-Dubé syndrome. Pathol Int. 63(1):45-55. PMID: 23356225.
  • Noman MZ, Desantis G, Janji B, Hasmim M, Karray S, Dessen P, Bronte V, Chouaib S (2014). PD-L1 is a novel direct target of HIF-1α, and its blockade under hypoxia enhanced MDSC-mediated T cell activation. J Exp Med. 211(5):781-90. PMID: 24778419.
  • Preston RS, Philp A, Claessens T, Gijezen L, Dydensborg AB, Dunlop EA, Harper KT, Brinkhuizen T, Menko FH, Davies DM, Land SC, Pause A, Baar K, van Steensel MA, Tee AR (2011). Absence of the Birt-Hogg-Dubé gene product is associated with increased hypoxia-inducible factor transcriptional activity and a loss of metabolic flexibility. Oncogene. 30(10):1159-73. PMID: 21057536.
  • Thompson RH, Dong H, Kwon ED (2007). Implications of B7-H1 expression in clear cell carcinoma of the kidney for prognostication and therapy. Clin Cancer Res. 13(2 Pt 2):709s-715s. Review. PMID: 17255298.
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Annual review 2015

With the end of the year fast approaching, we thought we would use this week’s blog to review the studies we’ve particularly enjoyed writing about.

In the spring Iribe et al. characterised the expression patterns in BHD renal tumours with Furuya et al. focusing on FLCN and GPNMB expression. The development of a screening panel for inherited tumours associated with FLCN mutations, rather than sporadic tumours, would enable pathologists to identify BHD patients earlier. This would help ensure they receive optimal future monitoring and treatment.

In June Chen et al. published details of a new tissue specific FLCN-deficient mouse model which develops bilateral renal cysts and tumours within one year. These mice live significantly longer than other kidney-specific FLCN knockout mice enabling longer term study of tumourigenesis. The tumourigenic potential of renal cyst cells from another mouse models was confirmed by Wu et al.  this year; after in vitro culture cells formed into sarcomatoid RCCs.

Tumours derived in both of these mouse models responded to treatment with the mTOR inhibitor rapamycin. Based on studies like these a stage II clinical trial of Everolimus (a rapamycin derivative) in BHD patients with RCC was announced in July.

In the autumn studies in the C. elegans BHD model identified a role for FLCN-1 in modulating resistance to hyperosmotic stress. Possik et al. determined that flcn-1 knockout worms could resist hyperosmotic stress due to increased glycogen accumulation and rapid osmolyte production. As such glycogen deposits could have dual roles in tumourigenesis as an energy source and protection from stress.

Towards the end of the year two groups reported on the structure and function of the yeast orthologues of FLCN and FNIP1/2 – Lst7 and Lst4. Lst4 was confirmed as a DENN-family protein by Pacitto et al. who used X-ray crystallography to solve the 3D structure. The role of Lst7-Lst4 in stimulating TORC1 activity was further elucidated by Peli-Gulli et al. – Lst7-Lst4 complexes recapitulate the reported function of FLCN-FNIP2 at the lysosomal membrane where they act as a GAP to stimulate mTOR signalling.

A recurring theme in BHD research this year has been using CT screening for pulmonary cysts to identify potential BHD patients in pneumothorax and RCC patients. Several reviews of imaging in cystic lung diseases including BHD have also helped raise awareness (Ha et al., 2015, Gupta et al., 2015, Richards et al., 2015, Ferreira Francisco et al., 2015).

Early in the year Johannesma et al. identified FLCN mutations in 7.5% of primary spontaneous pneumothorax (PSP) patients – all of whom had pulmonary cysts below the carina. Ding et al. also identified and mapped large intragenic FLCN deletions in PSP families with characteristic pulmonary cysts. More recently Johannesma et al. screened RCC patients for pulmonary cysts to determine if they could identify BHD patients; no new patients were identified but the presence of multiple cysts in the lower regions of RCC patient lungs, especially those with a family history, could still be key in differential diagnosis.

In addition to the research in September Professor Gennady Bratslavsky and Dr Mehdi Mollapour organised and ran the very successful Sixth BHD Symposium and First International Upstate Kidney Cancer Symposium in New York. Summaries of the scientific & clinical and patient & family member sessions are available online.

These topics are just a selection of those published in 2015, and we at the BHD Foundation are very much looking forward to seeing how the field develops in 2016. We wish all our readers a very Happy New Year.

  • Chen J, Huang D, Rubera I, Futami K, Wang P, Zickert P, Khoo SK, Dykema K, Zhao P, Petillo D, Cao B, Zhang Z, Si S, Schoen SR, Yang XJ, Zhou M, Xiao GQ, Wu G, Nordenskjöld M, Tauc M, Williams BO, Furge KA, Teh BT. Disruption of tubular Flcn expression as a mouse model for renal tumor induction. Kidney Int. 2015 Nov;88(5):1057-69. PMID: 26083655.
  • Ding Y, Zhu C, Zou W, Ma D, Min H, Chen B, Ye M, Pan Y, Cao L, Wan Y, Zhang W, Meng L, Mei Y, Yang C, Chen S, Gao Q, Yi L. FLCN intragenic deletions in Chinese familial primary spontaneous pneumothorax. Am J Med Genet A. 2015 May;167A(5):1125-33. PMID: 25807935.
  • Ferreira Francisco FA, Soares Souza A Jr, Zanetti G, Marchiori E. Multiple cystic lung disease. Eur Respir Rev. 2015 Dec;24(138):552-64. PMID: 26621970.
  • Furuya M, Hong SB, Tanaka R, Kuroda N, Nagashima Y, Nagahama K, Suyama T, Yao M, Nakatani Y. Distinctive expression patterns of glycoprotein non-metastatic B and folliculin in renal tumors in patients with Birt-Hogg-Dubé syndrome. Cancer Sci. 2015 Mar;106(3):315-23. PMID: 25594584.
  • Gupta N, Vassallo R, Wikenheiser-Brokamp KA, McCormack FX. Diffuse Cystic Lung Disease. Part II. Am J Respir Crit Care Med. 2015 Jul 1;192(1):17-29. Review. PMID: 25906201.
  • Ha D, Yadav R, Mazzone PJ. Cystic lung disease: systematic, stepwise diagnosis. Cleve Clin J Med. 2015 Feb;82(2):115-27. Review. PMID: 25897602.
  • Iribe Y, Kuroda N, Nagashima Y, Yao M, Tanaka R, Gotoda H, Kawakami F, Imamura Y, Nakamura Y, Ando M, Araki A, Matsushima J, Nakatani Y, Furuya M. Immunohistochemical characterization of renal tumors in patients with Birt-Hogg-Dubé syndrome. Pathol Int. 2015 Mar;65(3):126-32. PMID: 25597876.
  • Johannesma PC, Reinhard R, Kon Y, Sriram JD, Smit HJ, van Moorselaar RJ, Menko FH, Postmus PE; Amsterdam BHD working group. Prevalence of Birt-Hogg-Dubé syndrome in patients with apparently primary spontaneous pneumothorax. Eur Respir J. 2015 Apr;45(4):1191-4. PMID: 25537564.
  • Johannesma PC, Houweling AC, Menko FH, van de Beek I, Reinhard R, Gille JJ, van Waesberghe JT, Thunnissen E, Starink TM, Postmus PE, van Moorselaar RJ. Are lung cysts in renal cell cancer (RCC) patients an indication for FLCN mutation analysis? Fam Cancer. 2015b Nov 24. [Epub ahead of print] PMID: 26603437.
  • Schmidt LS, & Linehan WM (2015). Clinical Features, Genetics and Potential Therapeutic Approaches for Birt-Hogg-Dubé Syndrome. Expert opinion on orphan drugs, 3 (1), 15-29 PMID: 26581862
  • Pacitto A, Ascher DB, Wong LH, Blaszczyk BK, Nookala RK, Zhang N, Dokudovskaya S, Levine TP, Blundell TL. Lst4, the yeast Fnip1/2 orthologue, is a DENN-family protein. Open Biol. 2015 Dec;5(12). pii: 150174. PMID: 26631379.
  • Péli-Gulli MP, Sardu A, Panchaud N, Raucci S, De Virgilio C. Amino Acids Stimulate TORC1 through Lst4-Lst7, a GTPase-Activating Protein Complex for the Rag Family GTPase Gtr2. Cell Rep. 2015 Oct 6;13(1):1-7. PMID: 26387955.
  • Possik E, Ajisebutu A, Manteghi S, Gingras MC, Vijayaraghavan T, Flamand M, Coull B, Schmeisser K, Duchaine T, van Steensel M, Hall DH, Pause A. FLCN and AMPK Confer Resistance to Hyperosmotic Stress via Remodeling of Glycogen Stores. PLoS Genet. 2015 Oct 6;11(10):e1005520. PMID: 26439621.
  • Richards JC, Lynch DA, Chung JH. Cystic and nodular lung disease. Clin Chest Med. 2015 Jun;36(2):299-312, ix. Review. PMID: 26024606.
  • Wu M, Si S, Li Y, Schoen S, Xiao GQ, Li X, Teh BT, Wu G, Chen J. Flcn-deficient renal cells are tumorigenic and sensitive to mTOR suppression. Oncotarget. 2015 Oct 20;6(32):32761-73. PMID: 26418749.
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