Starvation-induced FLCN association with lysosomes via a Rab34–RILP complex

Dynamic positioning of lysosomes in the cytoplasm plays an important role in their function and is, in part, regulated by cellular nutrient status. The FLCN/FNIP complex is known to be active on the lysosome surface, where it interacts with Rag GTPases, supports the nutrient‐dependent recruitment and activation of mTORC1, and regulates the localisation of lysosome associated transcription factors (Petit et al., 2013; Tsun et al., 2013). New research from Starling et al. (2016) now shows that folliculin (FLCN) also controls the dynamic cytoplasmic position of the lysosome itself.

Lysosomal positioning coordinates cellular nutrient responses (Korolchuk et al., 2011), and is affected by several components, including the GTPase Rab34 that can promote lysosome clustering in the peri-nuclear region (Wang et al., 2002). Nutrient starvation, which suppresses mTORC1 activity, can also promote peri-nuclear clustering of lysosomes in HeLa cells, while nutrient-abundance and high mTORC1 activity leads to dispersion and accumulation of lysosomes at the cell periphery. mTORC1 is activated on the lysosomal surface, via a signaling network composed of Rag GTPases, the FLCN/FNIP complex and other protein complexes. FLCN/FNIP complex receives signaling inputs from metabolic pathways via phosphorylation, upon activation of mTORC1 and AMPK (Baba et al., 2006).

Starling et al. (2016) present strong evidence for a model where starvation‐induced FLCN association with lysosomes drives the formation of contact sites between lysosomes and Rab34‐positive peri-nuclear membranes, by promoting the association of Rab34 with its effector RILP. This restricts lysosome motility and thus promotes their retention in the peri-nuclear region of the cell.

Figure obtained from Starling et al. (2016)


The group shows that FLCN/FNIP complex is required for starvation‐induced peri-nuclear lysosome clustering.  Depletion of FLCN or of both FNIP1 and FNIP2 proteins using siRNA strongly affects lysosome positioning under starvation conditions, suggesting a functional connection between FLCN/FNIP-lysosome association and lysosome dynamics. As small GTPases are known to play a role in lysosome dynamics the group considered, among other GTPases, Rab34 and its effector protein RILP. Rab34 itself contributes to starvation‐induced peri‐nuclear clustering of lysosomes. However, in HeLa cells, depletion of FLCN significantly suppresses this ability.  FLCN was shown to associate with mitochondrial targeted Rab34 and the FLCN C‐terminal DENN domain is necessary for this association. Pull down experiments show that the FLCN‐DENN domain directly promotes the formation of an active Rab34–RILP complex.

The same type of experiments examining Rab34 and lysosome distribution were conducted in the BHD kidney cancer cell line UOK257 (FLCN deficient) and UOK2572 (FLCN restored) with results showing reduction in lysosome dynamics in the FLCN expressing cells in a DENN domain‐dependent way through Rab34/RILP.  The results in UOK257 cells are nutrient-independent, which the authors suggest might be due to the complete long term loss of full FLCN compared with acute depletion, or perhaps due to metabolic changes in the UOK257 cells.

Overall, the study shows how, in HeLa cells, FLCN couples the lysosomal nutrient signalling network to the cellular machinery that controls the intracellular distribution of the lysosome itself. The functional relevance of the study is supported by similar results, although nutrient-independent, in the BHD kidney cancer cell line, suggesting that this pathway may play a role in the pathogenesis of BHD syndrome. Since UOK257 cells do not show large deficiencies in mTORC1 activity (Baba et al., 2006), authors suggest that expanding these studies to other BHD-relevant epithelial cell types to understand how FLCN/Rab34‐dependent changes in lysosome motility may contribute to BHD syndrome.

In summary, given the complex relationship between lysosome positioning, autophagy and mTORC1 activity (Korolchuk et al., 2011), and the emerging connections between FLCN and the same pathways (Petit et al., 2013; Tsun et al., 2013), the group suggests that the dysregulation of lysosome dynamics by disruption of FLCN may contribute to the dysregulated autophagy and mTORC1 activity phenotypes found in various BHD model systems studied. The study sheds light on the mechanisms of lysosome dysregulation and can be exploited to develop therapies for kidney cancer therapies.

  • Baba M, Hong SB, Sharma N, Warren MB, Nickerson ML, Iwamatsu A, Esposito D, Gillette WK, Hopkins III RF, Hartley JL, Furihata M, Oishi S, Zhen W, Burke TR, Linehan WM, Schmidt LS, Zbar B. (2006). Folliculin encoded by the BHD gene interacts with a binding protein, FNIP1, and AMPK, and is involved in AMPK and mTOR signaling. Proc Natl Acad Sci U S A. 103(42):15552-7. PMID: 17028174
  • Korolchuk VI, Saiki S, Lichtenberg M, Siddiqi FH, Roberts EA, Imarisio S, Luca Jahreiss L, Sarkar S, Futter M, Menzies FM, O’Kane CJ, Deretic V, Rubinsztein DC. (2011). Lysosomal positioning coordinates cellular nutrient responses. Nat Cell Biol. 13(4): 453–460. PMCID: PMC3071334
  • Petit CS, Roczniak-Ferguson A, Ferguson SM. (2013). Recruitment of folliculin to lysosomes supports the amino acid-dependent activation of Rag GTPases. J Cell Biol. 202(7):1107-22. PMID: 24081491
  • Starling GP, Yip YY, Sanger A, Morton PE, Eden ER, Dodding MP. (2016). Folliculin directs the formation of a Rab34-RILP complex to control the nutrient-dependent dynamic distribution of lysosomes. EMBO Rep. PMID: 27113757
  • Tsun ZY, Bar-Peled L, Chantranupong L, Zoncu R, Wang T, Kim C, Spooner E, Sabatini DM. (2013). The folliculin tumor suppressor is a GAP for the RagC/D GTPases that signal amino acid levels to mTORC1. Mol Cell. 52(4):495-505 PMID: 24095279
  • Wang T, Hong W. (2002). Interorganellar Regulation of Lysosome Positioning by the Golgi Apparatus through Rab34 Interaction with Rab-interacting Lysosomal Protein. Mol Biol Cell. 13(12): 4317–4332. PMCID: PMC138636


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BHD pulmonary cysts: The stretch hypothesis

The majority of BHD patients develop pulmonary cysts and approximately 1 in 3 will suffer a pneumothorax. Although BHD pulmonary cysts have defining characteristics compared to other cystic lung diseases (as discussed in recent reviews), the underlying pathogenesis is not yet clearly understood. A recent review from Kennedy, Khabibullin & Henske (2016) summarises the current understanding of BHD pulmonary pathology relative to the stretch hypothesis for cyst formation.

The stretch hypothesis is based on reports of FLCN interacting with PKP4/p0071 to regulate cell-cell adhesion, with the loss of either protein increasing adhesive strength (Medvetz et al., 2012, Nahorski et al., 2012, Khabibullin et al., 2014). During respiration, due to less negative intrapleural pressure, the alveoli in the basal regions of the lungs undergo a greater change in volume than those in the apical regions. The stretch hypothesis suggests that defects in cell-cell adhesion in the areas of the lung repeatedly subjected to higher inspiration stretch-forces, including anchor points to the pleura, lead to failure of the septal wall and subsequent cyst formation. This is supported by the location of BHD pulmonary cysts, which are predominantly basilar and frequently abut pleura and blood vessels.

The loss of the FLCN-PKP4 complex could be impacting cell-cell adhesion in several ways. Abnormal expression and organisation of E-cadherin and Claudin-1, components of adherens and tight junctions respectively, were seen in mouse IMCD-3 renal epithelial cells after FLCN knockdown (Nahorski et al., 2012), and increased desmosome production was reported in FLCN-null human UOK-257 renal cells (Medvetz et al., 2012). This was associated with reduced transepithelial electrical resistance and disrupted cell polarity, resulting in additional cellular stress. E-cadherin expression was also reduced in primary mouse airway epithelial cells lacking Flcn (Goncharova et al, 2014) suggesting that the same disruptions could be contributing to pulmonary pathology.

It is still unclear whether pulmonary cystogenesis in BHD patients is due to haploinsufficiency or if a loss of the second FLCN allele is required, as with renal tumours. Furuya et al. (2012) reported expression of FLCN in BHD patient cysts, but a heterozygous Flcn+/- mouse model showed no airspaces enlargement at five months (Khabibullin et al. 2014).  Interestingly alveolar enlargement was seen in mice where total Flcn loss was induced only in SP-C+ epithelial lung cells (Goncharova et al, 2014). This suggests a specific role for these cells in cyst formation, further supported by reports of SP-C+ epithelial cells lining BHD patient cysts (Furuya et al., 2012, Furuya & Nakatani, 2013) – however these cells still expressed some FLCN protein.

There are several other aspects of pulmonary pathogenesis in BHD patients that also require more research: when and how rapidly the cysts develop; why the risk of pneumothorax decreases with age; whether cystogenesis is due to inflammatory destruction, aberrant proliferation or both; and whether it is due to defects only in epithelial cells or if there is a role for mesenchyml cells. A clearer understanding of the complete pathogenic basis of cystogenesis in BHD patients could enable development of a treatment to reduce cyst formation thereby reducing the risk of pneumothorax.

  • Furuya M, Tanaka R, Koga S, Yatabe Y, Gotoda H, Takagi S, Hsu YH, Fujii T, Okada A, Kuroda N, Moritani S, Mizuno H, Nagashima Y, Nagahama K, Hiroshima K, Yoshino I, Nomura F, Aoki I, Nakatani Y (2012). Pulmonary cysts of Birt-Hogg-Dubé syndrome: a clinicopathologic and immunohistochemical study of 9 families. Am J Surg Pathol. 36(4):589-600. PMID: 22441547.
  • Furuya M, Nakatani Y (2013). Birt-Hogg-Dube syndrome: clinicopathological features of the lung. J Clin Pathol. 66(3):178-86. PMID: 23223565.
  • Goncharova EA, Goncharov DA, James ML, Atochina-Vasserman EN, Stepanova V, Hong SB, Li H, Gonzales L, Baba M, Linehan WM, Gow AJ, Margulies S, Guttentag S, Schmidt LS, Krymskaya VP (2014). Folliculin controls lung alveolar enlargement and epithelial cell survival through E-cadherin, LKB1, and AMPK. Cell Rep. 7(2):412-23. PMID: 24726356.
  • Kennedy JC, Khabibullin D, & Henske EP (2016). Mechanisms of Pulmonary Cyst Pathogenesis in Birt-Hogg-Dube Syndrome: The Stretch Hypothesis. Seminars in cell & developmental biology PMID: 26877139.
  • Khabibullin D, Medvetz DA, Pinilla M, Hariharan V, Li C, Hergrueter A, Laucho Contreras M, Zhang E, Parkhitko A, Yu JJ, Owen CA, Huang H, Baron RM, Henske EP (2014). Folliculin regulates cell-cell adhesion, AMPK, and mTORC1 in a cell-type-specific manner in lung-derived cells. Physiol Rep. 2(8). pii: e12107. PMID: 25121506.
  • Medvetz DA, Khabibullin D, Hariharan V, Ongusaha PP, Goncharova EA, Schlechter T, Darling TN, Hofmann I, Krymskaya VP, Liao JK, Huang H, Henske EP (2012). Folliculin, the product of the Birt-Hogg-Dube tumor suppressor gene, interacts with the adherens junction protein p0071 to regulate cell-cell adhesion. PLoS One. 7(11):e47842. PMID: 23139756.
  • Nahorski MS, Seabra L, Straatman-Iwanowska A, Wingenfeld A, Reiman A, Lu X, Klomp JA, Teh BT, Hatzfeld M, Gissen P, Maher ER (2012). Folliculin interacts with p0071 (plakophilin-4) and deficiency is associated with disordered RhoA signalling, epithelial polarization and cytokinesis. Hum Mol Genet. 21(24):5268-79. PMID: 22965878.
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Rare Disease Day 2016: The Patient Voice

International Rare Disease Day is celebrated on the last day of February to raise awareness of rare diseases amongst the general public, researchers, healthcare professionals and policymakers. Now in its ninth year Rare Disease Day is celebrated in over 80 different countries with events in hundreds of cities. The theme for Rare Disease Day 2016 is the Patient Voice with a campaign inviting a wider audience to join us in making the voice of rare diseases heard.

Increasing awareness of rare diseases in the public and political domains ensures they remain an international health priority, helping patients access high quality care. Patients and their advocacy groups have crucial roles in raising awareness and can help others understand patient needs. In addition they can help instigate changes in policy and healthcare that can improve the lives of patients, family members and carers.

Patients and family members are often experts in their own disease and care, which can be very important when there is a lack of general medical knowledge. Patients can offer their personal experiences and identify their most pressing needs to help researchers and industry develop effective treatments and care strategies. Additionally expert patient involvement in regulatory reviews can help ensure patients can access the most critical treatments and interventions earlier.

Several training opportunities are provided by major rare disease organisations to support patients and advocacy groups that want to be representatives at a national and international level. In the UK, Findacure supports both established and developing patient advocacy groups, running regular training workshops and providing online guides. Similar patient advocacy support is available in other countries from organisations including NORD in the USA and CORD in Canada. Patient advocates interested in training related to clinical research and regulatory affairs can also attend the EURORDIS ExPRESS summer school.

The BHD Foundation provides support to BHD patients and researchers from around the world, and represents the BHD community at numerous international conferences helping to raise awareness. As well as promoting research, we encourage patients to share their stories and expertise through the website and forums including a patient-run Facebook group. Being able to seek advice and reassurance from others in a similar situation can help rare disease patients feel less isolated and more confident to discuss their condition. The organisations above can offer assistance for those wishing to create new support groups but communities can also be create on global platforms like RareConnect.

It is estimated that 1 in 17 people will be affected by a rare disease in their lifetime, meaning that cumulatively rare diseases are not actually uncommon. Rare disease day is a chance to further raise awareness of the impact of rare diseases and the need for representation in research and healthcare. Find out how you can help spread awareness by visiting the Rare Disease Day website, joining the Thunderclap campaign on social media and sharing your own story. You can also find events near you and share the details of your own events online.

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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

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