BHD Research Blog: 2014 Annual Review

With the New Year upon us, we thought we would use this week’s blog to review the studies we’ve particularly enjoyed writing about, and to revisit emerging themes.

During the summer-autumn period, there were a number of interesting studies that shed light on the molecular function of FLCN. Firstly, Goncharova et al. reported that FLCN activates AMPK signaling via LKB1 and E-cadherin, in Alveolar Type II (ATII) cells. Conversely, both Yan et al. and Possik et al. showed that FLCN inhibits AMPK signaling in MEFs and C.elegans nematodes, and that loss of FLCN leads to tumorigenic metabolic changes consistent with the Warburg Effect. These conflicting findings were resolved in August, when Khabibullin et al. published data showing that FLCN function is highly cell-specific.

FLCN’s role in autophagy was further elucidated by Dunlop et al. who reported that FLCN modulates autophagy through its interactions with ULK1 and GABARAP; FLCN was shown to be important for cardiomyocyte development (Hasumi et al., 2014); and a hitherto unknown role for FNIP1 in iNKT cell development was reported by Park et al.

The results from the trial of Rapamycin as a treatment for fibrofolliculomas were published this year, and reported that Rapamycin was not effective (Gijezen et al., 2014).

A cohort study of 33 BHD patients with kidney cancer showed that the majority of tumours were of oncocytic, choromophobe or mixed histology, and that the median age at the diagnosis of the first tumour was 46 (Benusiglio et al., 2014). This corresponds well with a study by Schuch et al. which found that patients with a genetic predisposition to kidney cancer developed tumours nearly 25 years earlier than patients with sporadic tumours. Thus, Schuch et al. recommend that doctors should consider germline genetic testing in kidney cancer patients under the age of 46.

A number of papers regarding the lung symptoms of BHD have been published this year. In July, it was reported that BHD can cause pneumothoraces in children in rare cases (Johannesma et al., 2014a), and in September, 1 in 16 BHD patients were found to be at risk of developing a pneumothorax within 30 days of taking a commercial flight (Postmus et al. 2014). Two separate groups hypothesized that defective cell-cell adhesion made cyst walls more likely to burst under mechanical stress, leading to an accumulation of air in the pleural space and potentially causing a pneumothorax (Johannesma et al., 2014b, Kumasaka et al., 2014).

Finally, two case studies have reported that somatic FLCN mutations contributed to the development of sporadic tumours (Sirintrapun et al., 2014, Wagle et al., 2014). These studies indicate that FLCN mutations may play a wider role in disease than just causing BHD Syndrome, suggesting that BHD is a fundamental disease.

These papers are just a selection of those published in 2014, and we at the BHD Foundation are very much looking forward to seeing how the field develops in 2015. We wish all our readers a very Happy New Year.

 

  • Benusiglio, P., Giraud, S., Deveaux, S., Méjean, A., Correas, J., Joly, D., Timsit, M., Ferlicot, S., Verkarre, V., Abadie, C., Chauveau, D., Leroux, D., Avril, M., Cordier, J., & Richard, S. (2014). Renal cell tumour characteristics in patients with the Birt-Hogg-Dubé cancer susceptibility syndrome: a retrospective, multicentre study Orphanet Journal of Rare Diseases, 9 (1) DOI: 10.1186/s13023-014-0163-z
  • 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) PMID: 25126726
  • Gijezen LM, Vernooij M, Martens H, Oduber CE, Henquet CJ, Starink TM, Prins MH, Menko FH, Nelemans PJ, & van Steensel MA (2014). Topical rapamycin as a treatment for fibrofolliculomas in birt-hogg-dubé syndrome: a double-blind placebo-controlled randomized split-face trial. PloS one, 9 (6) PMID: 24910976
  • 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 reports, 7 (2), 412-23 PMID: 24726356
  • Hasumi Y, Baba M, Hasumi H, Huang Y, Lang M, Reindorf R, Oh HB, Sciarretta S, Nagashima K, Haines DC, Schneider MD, Adelstein RS, Schmidt LS, Sadoshima J, & Marston Linehan W (2014). Folliculin (Flcn) inactivation leads to murine cardiac hypertrophy through mTORC1 deregulation. Human molecular genetics PMID: 24908670
  • Johannesma PC, van den Borne BE, Gille JJ, Nagelkerke AF, van Waesberghe JT, Paul MA, van Moorselaar RJ, Menko FH, & Postmus PE (2014a). Spontaneous pneumothorax as indicator for Birt-Hogg-Dubé syndrome in paediatric patients. BMC pediatrics, 14 PMID: 24994497
  • Johannesma PC, Houweling AC, van Waesberghe JH, van Moorselaar RJ, Starink TM, Menko FH, & Postmus PE (2014b). The pathogenesis of pneumothorax in Birt-Hogg-Dubé syndrome: A hypothesis. Respirology (Carlton, Vic.), 19 (8), 1248-50 PMID: 25302759
  • 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. Physiological reports, 2 (8) PMID: 25121506
  • Kumasaka T, Hayashi T, Mitani K, Kataoka H, Kikkawa M, Tobino K, Kobayashi E, Gunji Y, Kunogi M, Kurihara M, & Seyama K (2014). Characterization of pulmonary cysts in Birt-Hogg-Dubé syndrome: histopathologic and morphometric analysis of 229 pulmonary cysts from 50 unrelated patients. Histopathology PMID: 24393238
  • Park H, Tsang M, Iritani BM, & Bevan MJ (2014). Metabolic regulator Fnip1 is crucial for iNKT lymphocyte development. Proceedings of the National Academy of Sciences of the United States of America, 111 (19), 7066-71 PMID: 24785297
  • Possik E, Jalali Z, Nouët Y, Yan M, Gingras MC, Schmeisser K, Panaite L, Dupuy F, Kharitidi D, Chotard L, Jones RG, Hall DH, & Pause A (2014). Folliculin regulates ampk-dependent autophagy and metabolic stress survival. PLoS genetics, 10 (4) PMID: 24763318
  • Postmus PE, Johannesma PC, Menko FH, & Paul MA (2014). In-Flight Pneumothorax: Diagnosis May Be Missed because of Symptom Delay. American journal of respiratory and critical care medicine, 190 (6), 704-5 PMID: 25221882
  • Shuch B, Vourganti S, Ricketts CJ, Middleton L, Peterson J, Merino MJ, Metwalli AR, Srinivasan R, & Linehan WM (2014). Defining early-onset kidney cancer: implications for germline and somatic mutation testing and clinical management. Journal of clinical oncology : official journal of the American Society of Clinical Oncology, 32 (5), 431-7 PMID: 24378414
  • Sirintrapun SJ, Geisinger KR, Cimic A, Snow A, Hagenkord J, Monzon F, Legendre BL Jr, Ghazalpour A, Bender RP, & Gatalica Z (2014). Oncocytoma-like renal tumor with transformation toward high-grade oncocytic carcinoma: a unique case with morphologic, immunohistochemical, and genomic characterization. Medicine, 93 (15) PMID: 25275525
  • 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. The New England journal of medicine, 371 (15), 1426-33 PMID: 25295501
  • 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. The Journal of clinical investigation PMID: 24762438

A shower of second hit mutations causes bilateral, multifocal kidney cancer in TSC patients

Tuberous sclerosis complex (TSC) is caused by autosomal dominant inactivating mutations in either the TSC1 or TSC2 genes, and patients are predisposed to developing tumours in the brain, eyes, heart, skin, lungs and kidneys throughout their lifetime. While more than 80% of TSC patients develop benign renal angiomyolipoma, only 3% of TSC patients develop renal cell carcinoma (Yang et al., 2014).

Tyburczy et al., (2014) describe two TSC patients who presented with bilateral multifocal renal cell carcinoma.

The first patient received a left nephrectomy to remove 12 tumours at the age of 24. Three years later, a right nephrectomy was required to remove three tumours. Histological analysis showed all tumours to be of TSC-associated papillary renal cell carcinoma (RCC) (Yang et al., 2014).

Fresh frozen tissue was available from four left and one right kidney tumour. Direct sequencing and microsatellite marker analysis showed loss of heterozygosity (LOH) at the TSC2 allele in one tumour sample, but not in the other four tumours. Targeted next generation sequencing found different second hit somatic TSC2 mutations in each of the other four tumours: two nonsense, one frameshift and one missense mutation. Whole exome sequencing of DNA from all five tumours showed an average of four somatic mutations in other genes in each tumour, and no gene was mutated in more than one sample. Furthermore, no copy number changes were observed in any tumour. This suggests that biallelic inactivation of TSC2 is the driving tumorigenic mutation in all five tumours.

The second patient was found to have bilateral, multifocal tumours, and received partial nephrectomy of both kidneys at the age of 36. All tumours showed the same TSC-associated papillary RCC histology. Analysis of three tumours showed no evidence of LOH at the TSC2 allele. Only one tumour sample yielded sufficient DNA for sequencing analysis, and showed a somatic splicing mutation in TSC2, again suggesting that somatic second hits in TSC2 are driving tumorigenesis in this patient.

Both patients carried the same missense R905Q TSC2 mutation, which is associated with a milder presentation of TSC (Jansen et al., 2006). As this mutation is present in fewer than 1% of TSC patients, and only 3% of TSC patients develop RCC, it seems likely this this mutation may predispose patients to developing RCC. Indeed, in a cohort of 19 TSC patients who developed RCC, germline sequencing data was available for three patients who also developed TSC-associated papillary RCC. While none carried the R905Q mutation, all three had non-truncating TSC2 mutations – two missense and one in-frame deletion. This suggests that patients with non-truncating mutations may be at a higher risk of developing RCC (Yang et al., 2014).

Tyburczy et al. calculate the likelikhood of the first patient developing 15 tumours through 15 independent second hit events to be 1 in 3.6 trillion. This suggests that all tumours arose following a single event, which led to a shower of second hit TSC2 mutations occuring in these patients’ kidney tissue. However, the disease mechanism that leads to multiple second hit mutations in in a single gene is currently unknown.

 

  • Jansen AC, Sancak O, D’Agostino MD, Badhwar A, Roberts P, Gobbi G, Wilkinson R, Melanson D, Tampieri D, Koenekoop R, Gans M, Maat-Kievit A, Goedbloed M, van den Ouweland AM, Nellist M, Pandolfo M, McQueen M, Sims K, Thiele EA, Dubeau F, Andermann F, Kwiatkowski DJ, Halley DJ, & Andermann E (2006). Unusually mild tuberous sclerosis phenotype is associated with TSC2 R905Q mutation. Ann Neurol, 60 (5), 528-39 PMID: 17120248
  • Tyburczy ME, Jozwiak S, Malinowska IA, Chekaluk Y, Pugh TJ, Wu CL, Nussbaum RL, Seepo S, Dzik T, Kotulska K, & Kwiatkowski DJ (2014). A shower of second hit events as the cause of multifocal renal cell carcinoma in Tuberous Sclerosis Complex. Human molecular genetics PMID: 25432535
  • Yang P, Cornejo KM, Sadow PM, Cheng L, Wang M, Xiao Y, Jiang Z, Oliva E, Jozwiak S, Nussbaum RL, Feldman AS, Paul E, Thiele EA, Yu JJ, Henske EP, Kwiatkowski DJ, Young RH, & Wu CL (2014). Renal cell carcinoma in tuberous sclerosis complex. Am J Surg Pathol, 38 (7), 895-909. PMID: 24832166

Providing written information significantly improves parents’ understanding of TSC

Last week, we launched the new patient information pages on the BHD Foundation website. The information has been rewritten with the principles of health literacy in mind, and we hope these will be of greater use to patients and their families. This week’s blog discusses a study which demonstrates the value of providing well-written patient information.

Tuberous Sclerosis Complex (TSC) is a paediatric genetic syndrome which causes tumours to form in the brain, eyes, heart, skin, lungs and kidneys. There is no cure for TSC, but correct management of the individual symptoms can greatly improve health outcomes. Thus, educating care givers – usually the child’s parents – can improve patients’ quality of life.

Samia et al. (2014) tested whether providing parents with written information in addition to the information given in person at the TSC clinic in Cape Town, South Africa, improved parents’ understanding of TSC. 21 parents took part in the study, their baseline understanding of TSC was determined, and parents were split into two groups at random. The first group received a written leaflet about TSC to take home after their child’s appointment, while the second group did not. Parents’ knowledge of TSC was assessed 3 months later at their child’s next clinic appointment. Leaflets were based on information available on the TS Alliance website, and translated into both Xhosa and Afrikaans.

The study found that parents’ understanding of TSC increased by 20% in the group who received the written leaflet, compared with only 3% in the group who did not. The difference in knowledge was particularly striking in those parents who had completed between 8 and 11 years of education. The level of knowledge improved less in those parents who had over 11 years of education, suggesting that their baseline level of understanding was already high.

However, the study also found that written information was not useful to those parents who had not completed at least 8 years of education, and should be supplemented with verbal counseling. These appointments should be in a more relaxed setting, as previous studies have shown that in the formal setting of a doctor’s appointment, patient retention of information is low.

In this study, the majority of caregivers who took part in the study were female (18/21), and for most the clinic was their primary source of information about TSC. However, there are countries where women do not receive the same level of education as men, but are still likely to be the main caregiver of a disabled child. In these cases, health information providers will need to predominantly cater for a low literacy audience.

Additionally, in the UK, the internet is the primary source of health information for 87% of people, meaning that information providers should predominantly produce online content, and concentrate less on developing hard copy pamphlets to distribute at clinics. Thus, information providers need to understand their audience and have a flexible approach. This is especially important for those writing rare disease information, where fewer information resources are available and information providers are more likely to be generating information that is used internationally.

The link between improved health literacy and health outcomes is well-documented (Berkman et al., 2011): greater understanding of a disease leads people to seek diagnosis earlier and to greater compliance to treatment regimens. In turn this leads to improved health outcomes and reduced healthcare costs. Therefore, providing patients and caregivers with the right information, at the right time, and in the right format is an issue of public health.

 

  • Berkman ND, Sheridan SL, Donahue KE, Halpern DJ, & Crotty K (2011). Low health literacy and health outcomes: an updated systematic review. Ann Intern Med, 155 (2), 97-107. PMID: 21768583
  • Samia P, Donald KA, Schlegel B, & Wilmshurst JM (2014). Parental Understanding of Tuberous Sclerosis Complex. Journal of child neurology PMID: 25414235

Introducing the new BHD patient information pages

Like many rare diseases, there is no cure for BHD. However, appropriate management of symptoms – particularly kidney cancer – can vastly reduce the likelihood of early death due to the disease (Menko et al., 2009, Stamatakis et al., 2013). Therefore, providing BHD patients with clear, easy to understand information can help patients manage their condition and improve their quality of life.

Last year we wrote about how learning about health literacy helped us develop our Introductory Pamphlets for newly diagnosed patients. Whilst doing this project, we realised that some of the patient information on the website was quite technical and could be improved. Today we are pleased to launch the new patient information pages. These have been updated to include new information, and rewritten with the principles of health literacy in mind.

The new patient pages are split into ten sections: What is BHD; a Genetics overview; information about the skin, lung, kidney and other symptoms of BHD; practical considerations; the Science of BHD; FAQs; and Glossary.

What is BHD, simply provides a brief overview of BHD. The “Genetics overview” describes the genetics of BHD; suggests who should be tested for BHD, and why you should consider genetic testing; provides information about the genetic test itself and getting the results; and discusses the next steps are if you do have BHD, including information about how to find a doctor.

Previously, medical information about BHD was split into two sections: symptoms, and treatment & diagnosis. Now, this information is split into the different organs affected by BHD: skin, lung, kidney and other symptoms. For the skin, lung and kidney sections, each has its own introduction, followed by information about the symptoms, diagnosis, treatment options and differential diagnosis.

The “Practical considerations” section has remained largely the same. However, we have added three new resources to “Telling others about BHD”. The Medical Education Kit links to three important papers written by BHD experts, describing the best treatment regimens for patients (Gupta et al., 2013, Menko et al., 2009, Stamatakis et al., 2013). We recommend that patients print out copies of these papers to give to their doctors. This allows patients to educate their doctors about BHD and the best methods of treating patients. We have also written a lay summary of each paper so that patients are aware of the results and recommendations outlined in each.

To help patients tell their families about BHD, we have written a BHD Family Letter, which can be personalised to fit each patient’s circumstances. We have also reinstated the BHD Medicard, which can be folded into the size of a credit card, meaning that patients can easily carry round a handy list of their symptoms and treatments at all times.

There are three brand new sections. Firstly, we have added an FAQs section, which lists the answers to questions we are often asked about BHD. We have also added a glossary of terms, which spells out the more difficult or unfamiliar terms phonetically. And finally, we have written a brief description of the science of BHD specifically for patients. This section does not provide information that is directly relevant to patients’ health or care, but provides an insight into the underlying biology of the disease.

We hope that patients find the new pages informative and are better able to understand and manage their condition as a result. If you would like give any feedback on the new pages, you can email us at contact@BHDSyndrome.org, or fill out our online feedback form.

  • Gupta N, Seyama K, & McCormack FX (2013). Pulmonary manifestations of Birt-Hogg-Dubé syndrome. Familial cancer, 12 (3), 387-96 PMID: 23715758
  • 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. The Lancet. Oncology, 10 (12), 1199-206 PMID: 19959076
  • Stamatakis L, Metwalli AR, Middelton LA, & Marston Linehan W (2013). Diagnosis and management of BHD-associated kidney cancer. Familial cancer, 12 (3), 397-402 PMID: 23703644

 

The natural history of angiomyolipoma in cases of sporadic LAM

Lymphangioleiomyomatosis (LAM) is a cystic lung disease that predominantly affects women. Roughly 90% of cases are sporadic and are caused by somatic mutation of the TSC2 gene, but some patients develop LAM as part of the syndrome Tuberous Sclerosis Complex (TSC), which is caused by germline mutations either TSC1 or TSC2.

Angiomyolipoma (AML) is a type of kidney tumour that develops in nearly 100% of patients with TSC, and 50% of patients with sporadic LAM. AML are typically benign, but if untreated, larger tumours can haemorrhage and require the patient to undergo a nephrectomy. While the natural history of AML is known for TSC patients, it has not been systematically studied in LAM patients, and there are no official clinical recommendations of how to monitor and treat AML in sporadic LAM patients.

Yeoh et al. (2014) recently published a natural history study on a cohort of 107 sporadic LAM patients recruited via the National Centre for LAM in Nottingham, UK. Of these patients, 53 (50%) had had at least 1 AML in their lifetime and the average age of AML diagnosis was 39.1. Ten patients were diagnosed with AML up to 11 years before they were diagnosed with LAM, ten patients were diagnosed with both at the same time, and the remaining patients were diagnosed with AML up to 38 years after their LAM diagnosis. This suggests that sporadic LAM patients are at risk of developing AML throughout their entire lifetime.

Presence of AML was not linked to the severity of the pulmonary symptoms of LAM in these patients. AMLs in sporadic LAM patients were generally smaller, less likely to be bilateral, and the time from diagnosis to renal event – such as haemorrhage or surgical intervention – was 34 years, compared with 25 years in TSC patients. However, there was no difference in the incidence of haemorrhage or the need for medical intervention between this cohort and TSC patients. This suggests that AML in sporadic LAM patients should be managed in a similar fashion to AML in TSC patients.

The authors recommend that abdominal MRI should be performed every 1 to 3 years throughout the lifetime of sporadic LAM patients, and surgical intervention should occur when tumours reach 3 cm, as patients with larger tumours are at significant risk of haemorrhaging. All patients should have an abdominal MRI scan when they are diagnosed with LAM to assess whether they have any AMLs. Patients with tumours smaller than 1 cm in diameter should have follow up scans every two years, patients with tumours between 1-2cm in diameter should be screened annually, and patients with tumours over 3 cm should either have follow up scans after 6 months, or should be referred for intervention.

The authors note that this study only takes tumour size into account and suggest that contrast CT or MRI could be used to detect the vasculature of AML tumours, which may allow patients to be stratified into high risk and low risk groups that are managed differently. Indeed, there were 11 patients in this cohort who underwent nephrectomy to treat AML haemorrhage, and the average age of these patients at the time of surgery was 23. This is significantly younger than the average age of AML diagnosis of the cohort as a whole, which was 39.1, and suggests that these 11 patients did have a more aggressive form of AML.

 

  • Yeoh ZW, Navaratnam V, Bhatt R, McCafferty I, Hubbard RB, & Johnson SR (2014). Natural history of angiomyolipoma in lymphangioleiomyomatosis: implications for screening and surveillance. Orphanet journal of rare diseases, 9 PMID: 25277108

Computational approaches may expedite drug repurposing for rare diseases

Drug repurposing is predicated on the fact that many diseases are caused by the dysregulation of similar signaling pathways, or that drugs may affect several biological targets at once, meaning that a single drug may be able to treat multiple diseases. Repurposing an existing drug is easier, cheaper and faster than developing a brand new drug, as there are comparatively few research and development costs, and the drug is already known to be effective and safe for use in patients.

Currently, the vast majority of additional indications for existing drugs have been found by chance (Liu et al., 2013). However, as technology improves, researchers have developed high throughput in silico methods to analyse the structures and biological activities of existing drugs to identify new indications (Ekins et al., 2011, Liu et al., 2013). A recent study by Gramatica et al. (2014) report an alternative approach, using computational linguistics and graph theory to identify previously unknown links between drugs and diseases.

Gramatica et al. analysed three million publication abstracts relating to 300 rare diseases from the PubMed literature database. Analysing the language in the abstracts allowed the researchers to build visual networks linking different types of node, such as disease, protein, biological process, or drug. To show how these networks were built, the researchers used an example from a paper describing the pathogenesis of lymphangioleimyomatosis (LAM).

Each sentence was analysed independently, the nodes highlighted, and arranged into a network with each node linked pairwise, like so:

Each separate network was then rearranged to form a single, larger network representing the whole paper:

Where multiple links occur between nodes – such as LAM and Lung in this example – these nodes were considered to be more closely linked. Networks were then combined to cover multiple papers, and multiple diseases, building a large network of rare diseases, genes, biological processes, and drugs.

By arranging the results of a large number of studies – in this case three million – into a single network, links between drugs and diseases that were not previously recognized can be identified. Pathways with fewer steps linking a drug and a disease, and where multiple different pathways linked a drug and a disease, were considered to be particularly plausible candidates for drug repurposing.

To test whether this method led to the identification of new drug-disease pairs, the researchers used the network to identify new therapies for the rare lung disease Sarcoidosis, and new disease targets for the cancer drug Imatinib. They found that the peptides Aviptadil, ɑ-Melanocyte Stimulating Hormone (ɑ-MSH) and C-type Natriuretic Peptide (CNP) were possible candidates to treat Sarcoidosis. Indeed, a Phase II clinical trial in 20 patients reports that Aviptadil is an effective treatment for Sarcoidosis (Prasse et al., 2010), suggesting that ɑ-MSH and CNP may also be effective.

The network also predicted that Imatinib may be an effective treatment for spongiform encephalopathies, such as Creutzfeldt-Jakob disease. Imatinib inhibits the c-Abl tyrosine kinase, which has shown to be dysregulated and lead to neuronal cell death in multiple neurodegenerative disorders (Schlatterer et al., 2011) and Imatinib has been shown to clear mis-folded proteins from prion infected cells (Ertmer et al., 2004).

Together, these results suggest that combined computational linguistics and graph theory is able to identify previously unrecognized drug-disease pairs, which will expedite the repurposing of drugs for new indications.

Drug repurposing is a particularly valuable approach for identifying new treatments for rare and neglected diseases, where there is a high unmet medical need, and a number of rare disease organisations such as Findacure, Cures Within Reach and IRDiRC are actively funding and promoting this research. Indeed, there have been a number of successes in this area, for example Rapamycin has been repurposed as a treatment for Autoimmune Lymphoproliferative Syndrome and thalidomide is now an approved treatment for leprosy, multiple myeloma and bone marrow cancer (Teo et al., 2005).

The number of new therapies being approved for rare diseases is at an all-time high, but with only 133 new therapies approved since 2010, at this rate it will still take around 200 years to develop treatments for all rare diseases. Thus, although computational approaches to drug repurposing are in their infancy, the continued refinement of these approaches to expedite the discovery of drug-disease pairs will be of immense value to the field of rare diseases.

 

  • Ekins S, Williams AJ, Krasowski MD, & Freundlich JS (2011). In silico repositioning of approved drugs for rare and neglected diseases. Drug discovery today, 16 (7-8), 298-310 PMID: 21376136
  • Ertmer A, Gilch S, Yun SW, Flechsig E, Klebl B, Stein-Gerlach M, Klein MA, & Schätzl HM (2004). The tyrosine kinase inhibitor STI571 induces cellular clearance of PrPSc in prion-infected cells. The Journal of biological chemistry, 279 (40), 41918-27 PMID:15247213
  • Gramatica R, Di Matteo T, Giorgetti S, Barbiani M, Bevec D, & Aste T (2014). Graph theory enables drug repurposing–how a mathematical model can drive the discovery of hidden mechanisms of action. PloS one, 9 (1) PMID: 24416311
  • Liu Z, Fang H, Reagan K, Xu X, Mendrick DL, Slikker W Jr, & Tong W (2013). In silico drug repositioning: what we need to know.Drug discovery today, 18 (3-4), 110-5 PMID: 22935104
  • Prasse A, Zissel G, Lützen N, Schupp J, Schmiedlin R, Gonzalez-Rey E, Rensing-Ehl A, Bacher G, Cavalli V, Bevec D, Delgado M, & Müller-Quernheim J (2010). Inhaled vasoactive intestinal peptide exerts immunoregulatory effects in sarcoidosis. American journal of respiratory and critical care medicine, 182 (4), 540-8 PMID: 20442436
  • Schlatterer SD, Acker CM, & Davies P (2011). c-Abl in neurodegenerative disease. Journal of molecular neuroscience : MN, 45(3), 445-52 PMID: 21728062
  • Teo SK, Stirling DI, & Zeldis JB (2005). Thalidomide as a novel therapeutic agent: new uses for an old product. Drug discovery today, 10 (2), 107-14 PMID: 15718159

Nephron-sparing surgery reduces the risk of cardiovascular events

Radical nephrectomy is generally the preferred method to treat advanced kidney cancers, while partial nephrectomy is performed when the disease is localised, or if the patient has a genetic predisposition to developing kidney tumours. However, a recent study suggests that wherever possible, partial nephrectomy should be used, as the risk of subsequent cardiovascular events is reduced in these patients (Capitanio et al., 2014).

In order to investigate whether the two surgical approaches led to different outcomes, the researchers performed a retrospective review of 1331 patients who had had surgery for kidney cancer at one of four different hospitals between 1987 and 2013. All patients had tumours below 7 cm in diameter, with no metastasis or lymph node involvement; 462 (34.7%) patients had undergone radical nephrectomy, while 869 (65.3%) had undergone nephron sparing surgery.

The researchers analysed these patients’ medical records for pre- and post-surgical glomerular filtration rate, age, BMI, gender, smoking habits, co-morbidities, and cardiovascular events requiring hospitalisation. Cardiovascular events included coronary artery disease, cardiomyopathy, vasculopathy, hypertension, heart failure, dysrhythmias, and cerebrovascular disease. No patients in this study had been diagnosed with any cardiovascular disease prior to their kidney cancer surgery.

Overall, 21.8% of patients had experienced a cardiovascular event within 10 years following surgery. However, when the two groups of patients were analysed separately, 25.9% of the radical nephrectomy patients experienced a cardiovascular event within 10 years following surgery, compared to only 9.9% of the partial nephrectomy patients. Multivariate analysis accounting for the clinical characteristics and cardiovascular profiles of the two groups showed that patients who underwent nephron-sparing surgery were nearly half as likely to develop cardiovascular symptoms after surgery (hazard ratio = 0.57).

Reduced glomerular filtration (GFR) rate post-surgery was also highly correlated with increased risk of cardiovascular events. As radical nephrectomy significantly reduces GFR compared with partial nephrectomy, it is likely that this is responsible for the increased risk of cardiovascular disease in these patients. Interestingly, year of surgery was also correlated with risk of cardiovascular event, suggesting that as surgical techniques have improved over time, the risk of cardiovascular events has fallen. This could also reflect a trend towards increased use of partial nephrectomy to treat localised tumours.

This study suggests that the risk of having a cardiovascular event following kidney surgery is significant, but that the risk is greatly reduced following nephron-sparing surgery. This underscores the importance of improving diagnosis rates, as identifying tumours at an earlier stage will increase the likelihood that a nephron-sparing approach will be curative with respect to the kidney cancer, and will furthermore protect the patient’s cardiovascular health. It additionally provides evidence of the benefit of active surveillance approaches for benign tumours, as removing these tumours is of little clinical benefit to patients, but may increase their risk of developing cardiovascular disease.

 

  • Capitanio U, Terrone C, Antonelli A, Minervini A, Volpe A, Furlan M, Matloob R, Regis F, Fiori C, Porpiglia F, Di Trapani E, Zacchero M, Serni S, Salonia A, Carini M, Simeone C, Montorsi F, & Bertini R (2014). Nephron-sparing Techniques Independently Decrease the Risk of Cardiovascular Events Relative to Radical Nephrectomy in Patients with a T1a-T1b Renal Mass and Normal Preoperative Renal Function. European urology PMID: 25282367

 

Cohort study describes the kidney tumour characteristics of 33 BHD patients

In order to determine the characteristics of renal cell carcinomas (RCC) in BHD patients, Benusiglio et al. (2014) recruited 124 French BHD patients from Hôpital Bicêtre near Paris, and the Edouard Herriot University Hospital in Lyon.

Of the patients recruited, 33 had had kidney cancer. The median age at the diagnosis of the first tumour was 46, with an age range of 20 – 83. Whilst the majority of patients had a solitary tumour at diagnosis, four patients (12%) had two tumours, and nine patients (27%) presented with multifocal disease. Pathology reports were available for all but five patients, and, as expected, the majority of tumours (23/33, or 70%) were of oncocytic or hybrid oncocytic/ choromophobe histology. However, one patient had a papillary RCC, one had an undifferentiated RCC, and 3 patients (9%) had clear cell RCC.

Four patients had metastatic disease at diagnosis. One patient was found to have a lung metastasis 23 years after his initial diagnosis. The metastatic tumour was removed, and the patient showed no sign of cancer relapse upon his death four years later due to an unrelated condition. Another patient received nephrectomy and adjuvant radiotherapy for multiple metastatic retroperitoneal lymph-nodes, and is alive and well seven years later with no signs of disease progression.

Two patients in this cohort received systemic treatment for metastatic disease. One patient presented with multifocal kidney tumours and liver metastases, and disease stability was achieved following three years of systemic treatment with sunitinib, everolimus and temsirolimus. Eight years after ceasing treatment, this patient is still alive and well, and her tumours are stable. Another patient has survived for 5 years following diagnosis, and has received multiple systemic treatments to control slow growing liver and lung metastases.

The survival time following a diagnosis of metastatic renal cancer is usually between 4 months and 2 years (Manola et al., 2011), and all four of these BHD patients have survived with metastatic kidney cancer for more than five years. This suggests that even when BHD renal cancers do metastasise, they are clinically benign compared to more common metastatic renal cancers. Interestingly, mTOR inhibitors achieved long-term disease stability in one patient, which is consistent with the observation that tumours with somatic FLCN mutations respond well to mTOR inhibitors, and suggest that this class of drug may be particularly effective to control metastatic disease in BHD patients.

The results of this study correspond well with previous cohort studies (Schmidt et al., 2005, Toro et al., 2008), and together suggest that roughly 30% of BHD patients are at risk of developing renal cell carcinoma. Although the median age of diagnosis with kidney cancer was 46, the range was 20 – 83 years, suggesting that abdominal screening should commence at a young age – the current recommendation is to commence screening at 20 – 21 years old (Menko et al., 2009, Stamatakis et al., 2013) – and should continue throughout the patient’s lifetime. Furthermore, while the majority of BHD patients who develop RCC will have oncocytic, chromophobe or hybrid tumours, 10% of patients are at risk of developing clear cell RCC, which is more aggressive. Finally, BHD patients who present with metastatic disease seem to have a significantly better prognosis than patients who present with sporadic forms of metastatic RCC.

 

  • Benusiglio, P., Giraud, S., Deveaux, S., Méjean, A., Correas, J., Joly, D., Timsit, M., Ferlicot, S., Verkarre, V., Abadie, C., Chauveau, D., Leroux, D., Avril, M., Cordier, J., & Richard, S. (2014). Renal cell tumour characteristics in patients with the Birt-Hogg-Dubé cancer susceptibility syndrome: a retrospective, multicentre study Orphanet Journal of Rare Diseases, 9 (1) DOI: 10.1186/s13023-014-0163-z
  • Manola J, Royston P, Elson P, McCormack JB, Mazumdar M, Négrier S, Escudier B, Eisen T, Dutcher J, Atkins M, Heng DY, Choueiri TK, Motzer R, Bukowski R, & International Kidney Cancer Working Group (2011). Prognostic model for survival in patients with metastatic renal cell carcinoma: results from the international kidney cancer working group. Clinical cancer research : an official journal of the American Association for Cancer Research, 17 (16), 5443-50 PMID: 21828239
  • 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. The Lancet. Oncology, 10 (12), 1199-206 PMID: 19959076
  • Schmidt LS, Nickerson ML, Warren MB, Glenn GM, Toro JR, Merino MJ, Turner ML, Choyke PL, Sharma N, Peterson J, Morrison P, Maher ER, Walther MM, Zbar B, & Linehan WM (2005). Germline BHD-mutation spectrum and phenotype analysis of a large cohort of families with Birt-Hogg-Dubé syndrome. American journal of human genetics, 76 (6), 1023-33 PMID: 15852235
  • Stamatakis L, Metwalli AR, Middelton LA, & Marston Linehan W (2013). Diagnosis and management of BHD-associated kidney cancer. Familial cancer, 12 (3), 397-402 PMID: 23703644
  • Toro JR, Wei MH, Glenn GM, Weinreich M, Toure O, Vocke C, Turner M, Choyke P, Merino MJ, Pinto PA, Steinberg SM, Schmidt LS, & Linehan WM (2008). BHD mutations, clinical and molecular genetic investigations of Birt-Hogg-Dubé syndrome: a new series of 50 families and a review of published reports. Journal of medical genetics, 45 (6), 321-31 PMID: 18234728

TSC1 is required for iNKT cell maturation and function

Invariant Natural Killer T (iNKT) cell development is highly regulated, starting at stage 0, where DP thermocytes become committed to the iNKT cell lineage, and ending as fully mature stage 3 iNKT cells, which are capable of illiciting an immune response. iNKT cells are a subtype of T-cells that can recognise bacterial infections, viruses, and even tumours. However, upon repeated exposure, iNKT cells can become anergic and stop responding to stimuli (reviewed by Cianferoni, 2013).

Earlier this year, it was reported that the FLCN interacting protein FNIP1 was required for iNKT cells to complete the stage 2 to 3 transition, that this phenotype was cell autonomous and partially due to mTOR dysregulation (Park et al., 2014). Two studies have found that TSC1, which when mutated causes the related kidney cancer syndrome Tuberous Sclerosis Complex, is required for both iNKT cell development, and mature iNKT cell function.

In order to investigate the role of TSC1 in iNKT cell development, Wu et al. (2014a) specifically deleted TSC1 in murine T-cells using the Cd4Cre allele. They found that although these mice had similar numbers of stage 2 iNKT cells compared with wildtype mice, they had far fewer stage 3 iNKT cells, due to a higher rate of apoptosis in these cells. Therefore, similarly to FNIP1, TSC1 is required to promote the survival of mature stage 3 iNKT cells.

While the overall number of iNKT cells was reduced, a rare subset of iNKT cells which produce IL-17 (iNKT-17 cells) were present in increased numbers in the TSC1fl/fl; Cd4-Cre mice. Expression analysis showed that TSC1 causes cells to preferentially develop into iNKT rather than iNKT-17 cells by promoting the expression of T-bet and inhibiting the expression of RORɣT and ICOS.

Bone marrow transplants into irradiated mice showed using a 1:2.5 mixture of wild-type and TSC1-null bone marrow cells showed stage 1 and 2 iNKT cells were derived from both donor cell types. However, stage 3 iNKT cells were more commonly from wild-type donor cells, indicating that TSC1’s effect on iNKT cell development is cell autonomous. Furthermore, Rapamycin treatment of TSC1fl/fl; Cd4-Cre mice nearly completely reversed the block in iNKT cell development and predominance of iNKT-17 cells, meaning that dysregulated mTOR signaling was responsible for this phenotype. These results are very similar to those seen in FNIP1-null iNKT cells, suggesting that FLCN and TSC1 may co-operate to regulate iNKT cell development.

In a second study from the same team, Wu et al. (2014b) used a Tamoxifen inducible allele to delete TSC1 in mature iNKT cells. They found that, unlike wild-type iNKT cells, TSC1-null iNKT cells did not become anergic upon secondary stimulation with ɑ-galactosylceramide. The expression of the anergy promoting genes PD-1, Egr2, Egr3, Grail and p27kip were all reduced in TSC1-null iNKT cells. This suggests that TSC1 normally promotes an anergic response by activating the expression of these genes. Given the close overlap between TSC1 and FNIP1’s roles in iNKTcell development, it would also be of interest to determine whether FNIP1 or FLCN also regulate iNKT cell anergy.

Anti-cancer vaccines, like the anti-PD1 vaccine, are currently being tested in clinical trials, and preventing T-cell anergy is a significant area of interest to ensure the continued efficacy of vaccinations (Pal et al., 2014). Mice carrying TSC1-null iNKT cells developed fewer tumour nodules than wild-type mice when injected with B16F10 melanoma cells, meaning that reducing the anergic response increased iNKT cells’ ability to target tumours. These results suggest that inhibiting TSC1 in T-cells might prevent them becoming anergic, and so increase the effectiveness of cancer vaccines.

     

  • Cianferoni A (2014). Invariant Natural Killer T Cells. Antibodies, 3 (1), 16-36 doi: 10.3390/antib3010016
  • Park H, Tsang M, Iritani BM, & Bevan MJ (2014). Metabolic regulator Fnip1 is crucial for iNKT lymphocyte development. Proceedings of the National Academy of Sciences of the United States of America, 111 (19), 7066-71 PMID: 24785297
  • Pal SK, Hu A, Chang M, & Figlin RA (2014). Programmed death-1 inhibition in renal cell carcinoma: clinical insights and future directions. Clinical advances in hematology & oncology : H&O, 12 (2), 90-9 PMID: 24892254
  • Park H, Tsang M, Iritani BM, & Bevan MJ (2014). Metabolic regulator Fnip1 is crucial for iNKT lymphocyte development. Proceedings of the National Academy of Sciences of the United States of America, 111 (19), 7066-71 PMID: 24785297
  • Wu J, Yang J, Yang K, Wang H, Gorentla B, Shin J, Qiu Y, Que LG, Foster WM, Xia Z, Chi H, & Zhong XP (2014). iNKT cells require TSC1 for terminal maturation and effector lineage fate decisions. The Journal of clinical investigation, 124 (4), 1685-98 PMID: 24614103
  • Wu J, Shin J, Xie D, Wang H, Gao J, & Zhong XP (2014b). Tuberous sclerosis 1 promotes invariant NKT cell anergy and inhibits invariant NKT cell-mediated antitumor immunity. Journal of immunology (Baltimore, Md. : 1950), 192 (6), 2643-50 PMID: 24532578

 

Somatic mutations in FLCN can cause cancer

The majority of research on FLCN is within the context of BHD syndrome, which is caused by heterozygous germline mutations in the FLCN gene. However, two recent papers have reported that somatic FLCN mutations may be a factor in the development of sporadic tumours.

Sirintrapun et al. (2014) describe the case of a 74 year old man who presented with metastatic renal cancer. Molecular analysis showed that roughly a third of the primary tumour consisted of benign oncocytic cells while the remaining two thirds of the tumour was a high grade oncocytic carcinoma. The patient showed an extended 20 month progression free survival with Temsirolimus treatment.

Genetic and expression profiling revealed that both parts of the tumour had a common origin, indicating that the benign oncocytoma gave rise to the high grade tumour. A number of genomic rearrangements were present in the high grade cells only, including heterozygous loss of 17p, which contains the FLCN gene.

Thus, the authors report a rare case of somatic FLCN deletion contributing to a sporadic case of renal cell carcinoma, and the first known case of a benign oncocytoma transforming to a high grade carcinoma. However, there were additional oncogenic genomic rearrangements present in the high grade oncocytic cells, such as loss of 8p and gain of 8q, suggesting that loss of FLCN was only partly responsible for the disease progression in this patient.

In the second study, Wagle et al. (2014) describe the case of a 57 year old woman with anaplastic thyroid cancer. The patient was enrolled in a phase II clinical trial testing everolimus and had a sustained response for 18 months, at which point her tumour became resistant to treatment. Whole exome sequencing of germline, pretreatment, and resistant tumour DNA revealed that the pretreatment tumour had somatic inactivating mutations in FLCN, TSC2, and TP53, which likely lead to increased mTOR signaling in tumour cells, making the tumour particularly sensitive to treatment with mTOR inhibitiors.

The resistant tumour had developed a missense mutation (mTORF2108L) which prevents everolimus binding to mTOR. In vitro studies show that mTORF2108L is still sensitive to kinase inhibitors, such as Torin1, suggesting that kinase inhibiton may be a suitable follow up treatment for this patient. The authors suggest that sequencing tumour DNA before and during treatment may suggest the most effective treatment regimen for the patient.

These studies contain a number of interesting findings. Firstly, that somatic mutations in the FLCN gene can cause not just kidney cancer, but other tumour types when combined with additional genetic lesions. Indeed, somatic mutations in FLCN have been found in rare cases of sporadic renal cell carcinoma, colorectal cancer, and thyroid oncocytoma (Gad et al., 2007, Kahnoski et al., 2003, Khoo et al., 2003, Pradella et al., 2013). This suggests that BHD is a fundamental disease, and research insights on BHD will yield relevant results for other types of cancer.

Secondly, that FLCN mutations – and other mutations which lead to increased mTOR signaling – may indicate that the tumour will respond well to treatment with mTOR inhibitors, and indeed both patients in these studies had particularly long responses to mTOR inhibitors (20 and 18 months respectively). This is particularly exceptional in the second patient’s case, as the median survival time for anaplastic thyroid cancer is five months.

Thirdly, that a personalised medicine approach – where treatment is based on the underlying metabolic abnormalities present within that tumour – will likely improve cancer survival rates. Additionally, ongoing monitoring of how tumours evolve resistance to treatment will suggest the most effective follow up treatments, even further extending lifespan following a cancer diagnosis.

When considered as a whole, these studies demonstrate the utility of genetic sequencing of tumours to determine how tumours develop and evolve, and suggest that in the future, tumours may be classified and treated according to the mutations they carry, rather than by tumour site.

 

  • Gad S, Lefèvre SH, Khoo SK, Giraud S, Vieillefond A, Vasiliu V, Ferlicot S, Molinié V, Denoux Y, Thiounn N, Chrétien Y, Méjean A, Zerbib M, Benoît G, Hervé JM, Allègre G, Bressac-de Paillerets B, Teh BT, & Richard S (2007). Mutations in BHD and TP53 genes, but not in HNF1beta gene, in a large series of sporadic chromophobe renal cell carcinoma. British journal of cancer, 96 (2), 336-40 PMID: 17133269
  • Kahnoski K, Khoo SK, Nassif NT, Chen J, Lobo GP, Segelov E, & Teh BT (2003). Alterations of the Birt-Hogg-Dubé gene (BHD) in sporadic colorectal tumours. Journal of medical genetics, 40 (7), 511-5 PMID: 12843323
  • Khoo SK, Kahnoski K, Sugimura J, Petillo D, Chen J, Shockley K, Ludlow J, Knapp R, Giraud S, Richard S, Nordenskjöld M, & Teh BT (2003). Inactivation of BHD in sporadic renal tumors. Cancer research, 63 (15), 4583-7 PMID: 12907635
  • Pradella LM, Lang M, Kurelac I, Mariani E, Guerra F, Zuntini R, Tallini G, MacKay A, Reis-Filho JS, Seri M, Turchetti D, & Gasparre G (2013). Where Birt-Hogg-Dubé meets Cowden syndrome: mirrored genetic defects in two cases of syndromic oncocytic tumours. European journal of human genetics : EJHG, 21 (10), 1169-72 PMID: 23386036
  • Sirintrapun SJ, Geisinger KR, Cimic A, Snow A, Hagenkord J, Monzon F, Legendre BL Jr, Ghazalpour A, Bender RP, & Gatalica Z (2014). Oncocytoma-like renal tumor with transformation toward high-grade oncocytic carcinoma: a unique case with morphologic, immunohistochemical, and genomic characterization. Medicine, 93 (15) PMID: 25275525
  • 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. The New England journal of medicine, 371 (15), 1426-33 PMID: 25295501