3rd IRDiRC Conference – Trends in Patient Advocacy

As mentioned in last week’s blog, the 3rd International Rare Disease Research Consortium (IRDiRC) conference was held on the 8-9th February in Paris bringing together international experts in the rare disease field to discuss progress and to set new goals.

One of the sessions of the conference focused on global trends in patient advocacy in the rare disease field.

Yann Le Cam from EURORDIS discussed patient engagement in research, product life cycle and healthcare in Europe. Yann introduced EURORDIS and its aim of creating value for rare disease patients. EURORDIS has representation in external networks, organisations and institutions. In research, they act at the policy level creating position papers, advocacy actions and commissioning expert groups. At the operational level, they participate as partners through EU funded projects bringing the patient’s perspective to the table. At a capacity building level, they organise workshops to promote the sharing of information between researchers and patients. In therapy development, EURORDIS dialogues with the pharma industry and gets involved in European Medicines Agency (EMA) committees. They also review the orphan drug status and perform benefit/risk assessments. They also organise the EURORDIS Summer School, a program that allows rare disease patients and researchers to improve their advocacy skills and understand the process of orphan product development. The next Summer School will be held in June 2017 in Barcelona, Spain.

Matt Might from the University of Utah told the inspiring story of his son who had an undiagnosed condition that caused serious development delays and other issues. After 4 years without answers a team performed gene sequencing and found a mutation in the NGLY1 gene that was leading to a lack of enzyme recycling. This was the first and only case reported and therefore it was considered “not actionable”. Matt decided to take action himself, he started researching about the gene and found only 5 papers on Pubmed mentioning the gene mutation. Matt wrote a blog post looking for other patients “Hunting down my son’s killer” to find other cases of NGLY1 deficiency. Since writing the blog post he has found 15 more cases, a NGLY1 community has been formed, biomarkers have been found, conferences organised, assays, drug screening and different models have also been developed .

Yukiko Nishimura from ASrid in Japan presented the current situation of patient groups in Japan. In Japan the term “NANBYO”, which means ‘difficult disease’ and is used for rare, incurable and chronic diseases, started being used to raise awareness in the rare disease field. Yukiko introduced ASrid as an intermediary organisation to connect all stakeholders in the rare disease field to try to improve the current situation in Japan. Currently, the focus in rare disease is almost exclusively on social/welfare and not on R&D, there is a lack of funding and human resources and lack of English skills. Most patient groups are just patient groups and not patient advocacy groups. Other issues that Yukiko raised were the fact that some patient groups are not modern enough with elderly patients being unable to use email/phone and the fact that there is not a philanthropy culture in Japan.

Finally, Hawa Drame from the Fitima Foundation in Burkina Faso talked about the management of patients with rare diseases in the African context. Hawa mentioned that adding to all the “usual” issues in rare diseases, there is the specific economic risk in Africa. The many barriers to progress in the field include social-cultural issues, the weakness of healthcare system with low number of hospitals and doctors, inequality, difficulties accessing medicines, the weakness of social security system, lack of information and the failure to respect the fundamental rights to health. Trying to fight this, the Fitima Foundation was founded in 2003 to provide patients living with rare disease and disabilities (especially children) with improved life. Fitima has a human rights approach and currently works in Burkina Faso and Guinea. The objectives of the foundation include bringing together local doctors and paramedical specialists, organising multidisciplinary national medical consultations and defining pathways of care.

This session clearly showed how patient involvement and advocacy is vital to incentivise progress and research into rare diseases. Patient groups are starting to play a very active role in decision making, acting as a voice for their patients, increasing education of rare diseases and facilitating networking and data sharing.

3rd International Rare Disease Research Consortium Conference

The 3rd International Rare Disease Research Consortium (IRDiRC) conference was held last week in Paris. It brought together international experts in the fields of public health, funding agencies, academic research, patient advocacy and health industry to discuss the progress in the rare disease field and to set new goals for the next decade.

The conference started with an introduction to the IRDiRC history and achievements.

Ruxandra Draghia-Akli from the European Commission introduced the IRDiRC 2010-2020 headline goal of having 200 new therapies and means to diagnose most rare diseases by 2020. The IRDiRC, formally launched in 2012, has over 40 members of several institutions around the world.

Paul Lasko from the McGill University in Canada introduced the IRDiRC basic principles of cooperation at international level to stimulate, coordinate and maximise output of rare disease research efforts around the world. The IRDiRC teams up public and private organisations investing in rare diseases research. The research funders can join and work together but each organisation funds research its own way adhering to a common framework. Diagnostic projects funded by IRDiRC members have been the major factor in the discovery of ~1400 new rare diseases and have led to the optimization of NGS pipelines in a clinical setting.

Christopher Austin, chair of the IRDiRC, explained how the 2010-2020 IRDiRC objectives have already been achieved and the new objectives are being formulated. A consultation process is ongoing to develop new goals to be published in Spring 2017.

The second session of the conference aimed to give a global view of the rare disease field in 2017.

Makoto Suematsu, from AMED in Japan, presented the AMED program – Initiative on Rare and Undiagnosed Diseases (IRUD) – created to improve the life of rare disease patients, to promote global data sharing and the centralization of expensive analysing machines and to create matrices to check quality of projects. The IRUD Regional Alliance Hospital Network includes >200 hospitals in Japan and >2000 registered families with rare diseases. Another successful collaboration promoted by AMED is the SCRUM-Japan, an academia-industrial collaboration program incentivising data sharing among pharma sectors in the rare disease field.

Irene Norstedt and Caroline Hager from the European Commission gave an overview of the rare disease field in Europe. There are ~6000 diseases affecting ~30M EU citizens. So far, 128 orphan medicines have been authorized addressing 142 conditions. Since 2007 over €800M has been invested in rare disease research. EU investment in rare disease research increases every year, for Horizons 2020: to date €200M has been invested into 44 projects. An interesting projected funded by the EU – the RD connect project – is bringing together different platforms providing tools for rare disease online analysis. They also mentioned the Orphanet consortium, the clinical trials projects – ASTERIX, IDeAl, InSPiRE, and the European Reference Networks (ERNs).

Petra Kaufmann from the National Center for Advancing Translational Sciences (NCATS) presented a perspective on North American rare diseases research. Her key points on how to make a difference for rare diseases were the smart use of data, creating a research consortium, partnering for success, engaging the next generation of researchers, integrating care and research and integrating data from multiple sources. Petra also mentioned the importance of continuity of data, data standards and the importance of having ‘end-user’ in mind. She also presented the NCATS Toolkit project that aims to create a source for online educational and informational research tools that patient groups can access along with context, to improve coordination rather than re-create existing resources and to promote continuity across the lifecycle of the drug development process.

Hugh Dawkins from the Western Australia (WA) Department of Health presented the Australian perspective. Australia has a dispersed population and resources and 6-8% of Australian population with undiagnosed diseases are “invisible” in the health system. The WA Department of Health decided to start collecting information about rare diseases. They surveyed patient organisations, explored the existing clinical services, the local information, used genomic policy makers and finally created the WA Rare Disease Strategic Framework 2015-2018.

Anders Olauson from the Agrenska Foundation in Sweden presented the UN NGO Committee for Rare Diseases. This UN meeting started with the need for a global platform to share the knowledge that we have and to make sure that it is applicable for all socio-economic and cultural contexts, to explore what more could be done to advance knowledge of rare diseases at a global level, to connect rare diseases stakeholders across borders, to collaborate with other stakeholders to exchange knowledge and expertise and finally for recognition and attention at the UN level, where rare diseases remain an area little explored, with great social and economic impact. The focus of this UN committee should not be just on research and drugs but also on education, fighting discrimination and helping people cope with living with a rare disease.

Several other sessions focussed on diagnostics and therapeutics research, on trends in patient advocacy and a final session considered the IRDiRC goal-setting for 2017-2027, which we plan to discuss in future blogs.

This kind of event is crucial to promote dialogue and collaboration between stakeholders in a field that should be part of the global health agenda. Everyone who participated left the conference more informed, motivated and empowered to work together to improve the lives of people living with rare diseases globally.

BHD in patients undergoing chest CT and characteristics of BHD in Korea.

To date, there have been no prospective studies attempting to diagnose BHD syndrome or literature reviews on BHD in Korea. Park et al. (2017) address this in their new study that aims to detect BHD prospectively in patients undergoing chest computed tomography (CT) scans and to classify the characteristics of BHD in Korea.

The authors reviewed the chest CT scans obtained from 10,883 patients from Korea. Eighteen of these patients were suspected of BHD and seventeen underwent screening for FLCN mutation. Six were confirmed to have BHD by FLCN gene test.  These patients were classified into a BHD group (n=6) and the remaining (negative for FLCN mutation) into a non-BHD group (n=11). Spontaneous pneumothorax tended to be more frequent in the BHD group than in the non-BHD group. The prevalence of kidney and skin lesions was not significantly different between the groups. None of the patients had a family history of kidney or skin BHD symptoms.

The maximum size of the pulmonary cysts observed on chest CT in the BHD group was significantly larger than that in the non-BHD group. In addition, unlike the non-BHD patients, all the patients in the BHD group had cysts with variable morphology. One third of patients with BHD had multiseptated cysts but no cysts of this type were found in the non-BHD group. However, there was no significant difference in the number of cysts and their location, and costophrenic angle (CPA) involvement between the two groups.

In the BHD group, typical skin and kidney lesions were not observed in any patients. However, some atypical lesions were found. Within the four patients who underwent a skin examination, a fibroma was found on the nose of one patient. Within the four patients with renal imaging available, one had multiple renal cysts, and one clear cell-type renal cell carcinoma (RCC). All patients had normal lung function.

The authors then analysed a total of twelve Korean patients with BHD: six diagnosed in the present study (Patients 1–6), plus six previously diagnosed with BHD in the literature (Patients 7–12). The male to female ratio was 5:7, and the mean age was 50 years. Nine patients had a history of pneumothorax. None of the nine patients who underwent renal imaging had typical lesions, although atypical renal cyst or clear cell RCC was present in three patients. Two of the ten patients who underwent skin examination had typical skin lesions and three had atypical lesions (fibromas or papules with lymphocyte infiltration). Patient three had lung adenocarcinoma, however, lung pathology related to BHD was not found.

The most frequently observed FLCN mutations were c.1285dupC in four patients with two of them presenting typical skin lesions followed by c.1557delT. Patients 11 and 12 were related to patient 10 and the same mutation, c.882_884delTTC. In addition, c.1215C>G and c.1285delC mutations were each detected in one patient. There was no relationship between genotype and phenotype.

The prevalence and incidence of BHD in this study could not be determined because at least 2 years are needed to define this, the research was conducted at a single institution, and the patients were not representative of the general population.

BHD is considered to be the cause in 5–10% of spontaneous pneumothorax cases (Johannesma et al., 2015). However, the present study included patients with BHD who did not have pneumothorax history (50%). Therefore, careful review of the chest CT scans of patients with pulmonary cysts, even in patients without pneumothorax, might be helpful in identifying patients with BHD and their family members and radiologists should be aware of this.

Typical skin lesions are observed in ~90% of patients with BHD in the USA and Europe, but less frequently in Asia (Murakami et al., 2014; Toro et al., 2008; Furuya et al., 2013). This study found typical skin lesions in only 20% of Korean cases. In addition, the authors did not identify typical renal lesions in the Korean cases. In the USA and Europe, typical renal lesions are present in 15–35% of patients with BHD, but less frequently in Asia (Ding et al., 2015). In the USA and Europe, spontaneous pneumothorax has been reported to occur in 24–41% of cases (Skolnik et al., 2016; Toro et al., 2007), but has been reported in 68% of cases in China (Ding et al., 2015), in accordance with the also high prevalence of spontaneous pneumothorax (75%) in this study. However, it is important to address that the detection rate of skin, renal lesions and pneumothorax depends on the design of the study.

The small number of patients with BHD in the study does not allow reaching conclusions regarding demographic differences. Therefore, further studies comparing the prevalence of BHD symptoms according to nationality are needed.

  • Park HJ, Park CH, Lee SE, Lee GD, Byun MK, Lee S, Lee KA, Kim TH, Kim SH, Yang SY, Kim HJ, & Ahn CM (2017). Birt-Hogg-Dube syndrome prospectively detected by review of chest computed tomography scans. PloS one, 12 (2) PMID: 28151982

AMCR Delving into Digital Conference

Digital technology is important in every sector of society, particularly in health. The potential of the medical charity sector in digital health was the theme of this week’s AMRC Conference at Google DeepMind in London. The event showcased charity projects and partnerships in digital health and explored the potential for digital innovation in medical research.

Aisling Burnand from the AMRC and Nick Partridge from the NHS kicked off the meeting with a warm welcome and introductory remarks about the potential of the charity sector in digital and the main objectives of the meeting: inspiring to explore the potential of digital and enabling networking and collaborations.

  • Julie Dodd from Parkinson’s UK presented their partnership with Global Kinetics to develop a device called PKG that tracks the symptoms of Parkinson’s patients as a control of their medication balance – it has shown a significant impact leading to changes in medication regime in 80% of cases. Julie mentioned that companies will not make much profit out of these devices so charities should step in.
  • Dominic King from DeepMind introduced their company as a research organisation that creates general artificial intelligence algorithms to tackle society issues. They are looking into problems in the NHS such as the moving from papers to digital and the difficulty in booking appointments and changing medication. He also provided insights on digital health such as putting the user at the centre when building a product, taking a robust approach to develop digital health, the importance of good design (“People expect products that delight them.”), of measuring impact and of data security.
  • Michael Seres, a Crohn’s patient from 11Health, talked about the importance of doctors and patients working as a team. Michael started a blog about his experience after a transplant that started being read by other patients and by the transplant team that performed the surgery. Michael mentioned how we could use everyday tools, such as Skype and WhatsApp, in healthcare and talked about the #wearenotwaiting movement created by a group of patients to track their own health using digital tools.
  • Lenny Naar from the Helix Centre gave a presentation about user centred design. He mentioned that the design of a product should engage people into technology. At the Helix Centre multidisciplinary teams that combine design & digital expertise with academic and engineering expertise and patients expertise work to make healthcare better.
  • Tim Parry from the Alzheimer’s Research UK presented their collaboration with Deutsche Telekom to create an app with a game to help dementia patients – SeaHero Quest. Data was collected from 2.4M players regarding visual/motor skills, path integration, wayfinding and memory. Tim mentioned some things to consider when developing such an app: tensions between scientists and gamers; keeping data collection small not to scare people; and ensuring that you show patient benefit.
  • Liam O’Toole from Arthritis Research UK talked about issues of patients with arthritis in obtaining information using conventional helplines: too many questions, very specific and personal, lack of continuity. He suggested that the solution could be to use cognitive computing since it understands human speech and patterns, puts questions in context and it learns and links. A prototype for a cognitive computing ‘helpline’ platform is currently being developed. Liam pointed out that while there is risk involved in creating something new, we also need to measure the risks of not doing it.
  • Jon Spiers from Autistica talked about the problem of severe anxiety in patients with autism. The techniques to approach anxiety are behavioural and simple to follow without a clinician – currently there is a toolkit for anxiety but it is a big book difficult to transport and to navigate. The charity had the idea of converting the toolkit into an app and making it available to families and individuals with autism. They started a partnership with Deutsche Bank that run a hackathon with experts to develop prototypes.
  • Michele Acton from Fight for Sight presented the Portable Eye Examination Kit (PEEK). It can detect serious eye diseases and allows examination of millions of people who do not have access to eye tests. PEEK was developed with the eye surgeon Andrew Bastawrous and you can watch his inspiring TED talk here.

 In summary, it was an inspiring meeting bringing together more than 80 delegates who discussed and shared their knowledge and ground-breaking projects in digital health. Medical charities have a clear interest in adding digital technology to their strategies, in sharing their ideas and in collaborating with each other and with industry partners to promote the development of digital technologies that can improve health.

TSC1 expression is affected by VHL alterations and HIF-1α production in clear-cell RCC

VHL genetic alterations do not affect the production of HIF-α in clear-cell renal cell carcinoma (ccRCC). However, their effects on tuberous sclerosis proteins (TSC1/2) and heat shock protein 90 (Hsp90) expression are currently unknown. In a recent study, Damjanovic et al. (2016) evaluated the impact of VHL genetic alterations and HIF-α production on the expression of TSC proteins and Hsp90 in 47 sporadic ccRCCs and corresponding normal tissues.

The authors found somatic mutations in VHL gene in 31/47 of the ccRCC samples. Most of these mutations were frameshifts caused by intragenic deletions and duplications. Monoallelic inactivation (intragenic mutation or loss of heterozygosity (LOH)) was found in 10/47 and biallelic inactivation (intragenic mutation plus LOH) in 27/47 of ccRCCs. Altogether, 37/47 of ccRCC samples had alteration in VHL region.

mRNA expression of HIF-1α and HIF-2α was similar in tumours and normal tissues and VHL independent. In addition, the abundance of HIF-α mRNAs was similar in HIF-1α positive and negative tumours.

Western blot analysis showed that HIF-1α and HIF-2α were overexpressed in 10/47 and 21/47 of tumours compared to normal tissue. In 30/47 of tumours HIF-1α expression was reduced in comparison to control and in 16/47 of ccRCCs samples HIF-1α was undetectable. Multiple tests revealed that only tumours with loss of HIF-1α overexpressed HIF-2α. Tumorous production of HIF-α proteins was unaffected by alterations in VHL. These results suggest that the expression of these proteins is post-transcriptionally regulated.

TSC1 expression on tumours was affected by both VHL functional status and HIF-1α production. TSC1 expression was reduced in tumours with monoallelic VHL inactivation compared to control and tumours with biallelic inactivation. This lower expression was found more frequently among tumours with loss of HIF-1α. In addition, Fuhrman’s nuclear grade was higher in tumours overexpressing TSC1.

 VHL functional status and HIF-1α production did not affect Hsp90 expression in ccRCCs, suggesting that its role in HIF-α stabilization is maintained.

In addition to the western blot analysis, immunostaining revealed that expression of both TSC1 and TSC2 was reduced in tumours, but only TSC1 expression was dependent on VHL functional status. TSC2 expression was reduced in all tumours while TSC1 only in tumours with VHL alterations. In addition, ccRCCs with loss of HIF-1α showed reduced immunostaining for TSC1. These results suggest that HIF-1α could be involved in positive regulation of TSC1 expression in these tumours and that wild-type pVHL negatively regulates TSC1 expression.

Tumour size positively correlated with tumour grade and TSC1 expression. In VHL altered ccRCCs tumour diameter correlated with nuclear grade, TSC1 and Hsp90 expression and with the HIF-2α expression ratio between control and tumour. In stepwise multivariate linear regression analysis, a significant portion of variability in tumour diameter could be explained by variations in nuclear grade, TSC1 expression and HIF-2α expression ratio. In ccRCCs with VHL alterations, a significant portion of variability in the tumour size can be explained by Fuhrman’s grade, TSC1 expression and HIF-2α overexpression. Because nucleolar size depends on mTORC1 activity (Fuhrman et al., (1982)Li et al., (2009)), the authors results suggest a TSC1 dependency on pVHL.

In summary, while TSC2 is broadly downregulated in ccRCC, TSC1 expression is reduced in two subsets of these tumours, those with monoallelic VHL gene inactivation and those with both low HIF-1α and high HIF-2α expression. The effect of nuclear grade, TSC1 and HIF-2α in the progression of VHL altered tumours, suggests that pVHL regulates TSC1. These results are important for future studies on ccRCC where reduced expression and imbalances in TSC1/TSC2 expression should be considered in developing future therapies.

  • Damjanovic SS, Ilic BB, Beleslin Cokic BB, Antic JA, Bankovic JZ, Milicevic IT, Rodic GS, Ilic DS, Todorovic VN, Puskas N, & Tulic CD (2016). Tuberous sclerosis complex protein 1 expression is affected by VHL Gene alterations and HIF-1α production in sporadic clear-cell renal cell carcinoma. Experimental and molecular pathology, 101 (3), 323-331 PMID: 27845047

RCC: Updates on Guidelines for Adjuvant Therapy and new drug combination

The European Association of Urology (EAU) Renal Cell Carcinoma (RCC) guidelines panel has recently updated its recommendation on adjuvant therapy with sunitinib in non-metastatic RCC after surgical tumour removal (Bex et al., 2016). These clinical guidelines provide urologists with evidence-based information and recommendations for the management of RCC and the panel includes urological surgeons, oncologists, pathologists, radiologists and patient advocates. Based on the conflicting results of two available clinical studies (ASSURE and S-TRAC), the panel rated the quality of the evidence of the trials, the harm-to-benefit ratio, the patient preferences and the costs. As a result, the EAU panel, including representatives from a patient advocate group (International Kidney Cancer Coalition) voted and reached a consensus recommendation that adjuvant therapy with sunitinib for patients with high-risk RCC after nephrectomy should not be given.

The two phase III studies (ASSURE and S-TRAC) recently reported findings on whether adjuvant VEGF-targeted therapy can improve outcomes for patients with renal cell cancer that has been removed by surgery. In these two trials, already mentioned on previous blogs here and here, sunitinib, an oral tyrosine kinase inhibitor (TKI), or a placebo, was given to patients who underwent surgical kidney tumour removal and who presented a high risk for recurrence after surgery. The S-TRAC study, which included more than 670 patients, showed positive results with 1 year of sunitinib therapy resulting in a 1.2-year longer time of disease-free survival (DFS), its primary endpoint.  However, the ASSURE study did not show any benefits with no significant improvements in disease-free survival or overall survival compared to placebo in patients following surgical tumour removal. Many patients remained without recurrence. In addition, the side effects of sunitinib were significant in both studies and included hypertension, hand-foot reaction, fatigue and diarrhoea. The poor benefit-to-harm ratio and the lack of evidence of an overall survival benefit led the EAU RCC Guidelines Panel, including patient representatives, to recommend against using adjuvant sunitinib after tumour removal in these RCC patients.

Meric-Bernstam et al. (2016) presented at the 28th EORTC-NCI-AACR Symposium on Molecular Targets and Cancer Therapeutics, preliminary data from a phase I clinical trial .  The trial gave patients with clear cell and papillary RCC a combination therapy of everolimus and  CB-839, a highly selective inhibitor of glutaminase, which is a key enzyme in the use of glutamine by many cancer types. Ten patients received the combination treatment, which was well tolerated, and a very high percentage of the patients had their tumour controlled by the regimen. Out of eight patients with clear cell or papillary RCC the tumour shrank at least 30% in one patient (partial response) and was stable in the other seven patients (stable disease). In the two patients with non- clear cell or papillary RCC best tumour response was progressive disease– at least a 20% increase in tumour size. These results suggest that CB-839 is a very tolerable drug with significant potential in combination therapy for kidney cancer patients. This study is currently recruiting patients. Researchers who continue to enrol and treat patients in this trial plan to evaluate CB-839 in combination with everolimus in a randomised controlled trial in the future. In addition, in the current trial, the efficacy of CB-839 in kidney cancer in combination with another drug, cabozantinib, is also being assessed.

The development of new mono and combination drug treatments for different types of RCC and the assessment of the efficacy and safety of these drugs is a very active field with several clinical trials currently recruiting patients. The diversity of these studies is encouraging for patients with BHD syndrome that are predisposed to develop histologically diverse RCC.

  • Bex A, Albiges L, Ljungberg B, Bensalah K, Dabestani S, Giles RH, Hofmann F, Hora M, Kuczyk MA, Lam TB, Marconi L, Merseburger AS, Staehler M, Volpe A, & Powles T (2016). Updated European Association of Urology Guidelines Regarding Adjuvant Therapy for Renal Cell Carcinoma. European urology PMID: 27986369
  • Meric-Bernstam, F., Tannir, N., Harding, J., Voss, M., Mier, J., DeMichele, A., Munster, P., Patel, M., Iliopoulos, O., Owonikoko, T., Whiting, S., Orford, K., Bennett, M., Carvajal, R., McKay, R., Fan, A., Telli, M., & Infante, J. (2016). Phase 1 study of CB-839, a small molecule inhibitor of glutaminase, in combination with everolimus in patients (pts) with clear cell and papillary renal cell cancer (RCC) European Journal of Cancer, 69 DOI: 10.1016/S0959-8049(16)32626-0

Nutrient-dependent FNIP degradation regulates FLCN localization and promotes renal cancer progression

Birt-Hogg-Dubé (BHD) syndrome is a rare disorder caused by mutations in FLCN and associated with increased risk of kidney cancer. It has been shown that FLCN-interacting protein 1 and 2 (FNIP1 and FNIP2) double knockout mice, like the FLCN knockout mice, develop renal carcinoma (Hasumi et al., 2015). However, the molecular mechanisms linking FNIP and FLCN remain unknown. In their new study, Nagashima et al. (2016) show that FNIP2 undergoes proteasome-dependent degradation via β-TRCP and Casein Kinase 1 (CK1)-directed ubiquitination in a nutrition-dependent manner. Degradation of FNIP2 leads to lysosomal dissociation of FLCN and association of mTOR, which promotes the proliferation of renal cancer cells.

The authors started by showing that FNIP protein abundance decreases after nutrient stimulation in starved HeLa cells. Refeeding increased mTOR and S6K phosphorylation, suggesting FNIP downregulation in mTORC1 activation. The presence of a proteasome inhibitor (MG132) blocked nutrient-stimulated FNIP downregulation. These results suggest that FNIP stability is negatively regulated by nutrient stimulation in a proteasome-dependent manner.

The knockdown of β-TRCP, a well-characterized F-box protein known to play a role in regulating various signalling pathways, led to an increase in FNIP protein levels without affecting mRNA expression. β-TRCP specifically bound to exogenous and endogenous FNIP2 suggesting that SCFβ-TRCP ubiquitin ligase complex interacts with FNIP to control their stability.

A potential β-TRCP phosphorylation binding motif in FNIP1 and FNIP2 was identified and the authors generated a mutant without this motif (FNIP2-3A) for subsequent analyses. The FNIP2-3A mutant was unable to interact with β-TRCP, indicating that β-TRCP recognizes FNIP2 through this specific motif. To identify a protein kinase responsible for phosphorylation in the β-TRCP binding motif, FNIP2 protein levels following co-expression with various kinases was evaluated. Expression of CK1 led to a decline in FNIP2 and co-expression of CK1 and β-TRCP enhanced FNIP2 poly-ubiquitination indicating that CK1-mediated phosphorylation promoted SCFβ-TRCP-dependent ubiquitination and subsequent degradation of FNIP2.

β-TRCP was depleted in HeLa cells and the FNIP protein levels examined under starved and refed conditions. Following starvation, FNIP protein accumulated in both control and β-TRCP depleted cells. However, upon refeed, FNIP levels decreased in control cells, while β-TRCP knock-down cells maintained high protein levels of FNIP, despite the decrease in mRNA expression, suggesting that β-TRCP regulates the degradation of FNIP in a nutrient-dependent manner.

Starvation promotes FLCN recruitment to lysosomes and FNIP co-expression facilitatesFLCN accumulation at the lysosome via the formation of a complex (Starling et al., 2016). mTORC1 is recruited and activated in the lysosome in a nutrient-dependent manner. The authors assessed how β-TRCP depletion influenced FLCN distribution in a nutrient-dependent manner. β-TRCP depletion led to a partial enrichment of FLCN to the lysosomal membrane under growth conditions, and enhanced enrichment of FLCN to lysosomes was retained even after nutrition stimulation, whereas mTOR continued to be diffusely localized in the cytoplasm. These results suggest a causal relationship of lysosomal localization between FLCN and mTOR complexes.

To exclude possible pleiotropic effects of β-TRCP knockdown, the authors ectopically expressed Flag-FNIP2 WT and FNIP2-3A mutant in HeLa cells to confirm the results that show that FNIP abundance is critical in lysosomal localization of both FLCN and mTOR, suggesting that β-TRCP-mediated FNIP degradation activates mTORC1 signalling in a nutrient-dependent manner.

FNIP levels and mTORC1 activity were also assessed in the FLCN-null BHD cell line UOK-257 starved and refed. Consistent with a previous report (Baba et al., 2006), loss of FLCN led to activated mTORC1 signalling. Loss of FLCN displayed mTOR enrichment to the lysosomal membrane in a nutrient-independent manner and FLCN restoration in UOK-257-2 cells and its association with lysosomes caused the dissociation of mTOR from lysosomes in a nutrient-dependent manner. To assess the role of β-TRCP-mediated FNIP degradation in renal cancer cell progression, FNIP was depleted from UOK-257-2 cells and FNIP2-WT or non-degradable FNIP2- 3A reintroduced. FNIP knockdown in UOK-257-2 cells led to the activation of mTORC1 signalling. Conversely, reintroducing FNIP2-WT diminished the enhanced mTORC1 activity, while introducing the FNIP2-3A mutant further enhanced mTORC1 activity. FLCN expression suppressed colony formation but these effects were reversed by FNIP depletion in UOK-257-2 cells. Reintroducing FNIP2-WT conversely blocked colony formation whereas reintroducing the FNIP2-3A mutant further suppressed cell growth, suggesting a tumour suppressive role of FNIP2 and a contribution of β-TRCP in promoting renal cancer cell proliferation. To validate the in vitro studies, the authors performed mouse xenograft experiments using UOK-257 and UOK-257-2 cell lines. Tumour growth of UOK-257-2 cells was significantly suppressed when compared to that of UOK-257 cells. FNIP depletion enhanced tumorigenesis. Reintroducing FNIP2-WT in FNIP depleted cells resulted in decreased tumour size and FNIP2-3A mutant presented further suppressive effects. In addition, mTORC1 activity was inversely correlated with FNIP2 levels in xenografts. These data indicate a tumour suppressive role for the FLCN complex in BHD-associated renal cancer tumorigenesis.

In summary, the results indicate that SCFβ-TRCP negatively regulates the FLCN complex by promoting nutrient-dependent FNIP degradation and provide molecular insight into the pathogenesis of BHD-associated renal cancer with β-TRCP and CK1 signalling being potential therapeutic targets.

  • Nagashima K, Fukushima H, Shimizu K, Yamada A, Hidaka M, Hasumi H, Ikebe T, Fukumoto S, Okabe K, & Inuzuka H (2016). Nutrient-induced FNIP degradation by SCFβ-TRCP regulates FLCN complex localization and promotes renal cancer progression. Oncotarget PMID: 28039480

H255Y and K508R missense mutations in FLCN promote kidney neoplasia

The germline FLCN missense mutations H255Y (Hasumi et al., 2009) and K508R (Toro et al., 2008) have been identified in patients with bilateral multifocal (BMF) kidney tumours and other clinical symptoms of Birt-Hogg-Dube (BHD) syndrome, or with BMF kidney tumours as the only manifestation. Building on their previous work identifying the H255Y mutation in human BHD kidney tumour, Hasumi et al. (2016) investigated whether these mutations have an impact on FLCN function. The authors evaluated the FLCN missense mutations, H255Y and K508R in genetically engineered mice, and demonstrated that they both are pathogenic mutations that promote aberrant kidney cell proliferation to different degrees, with the potential for malignancy. In addition, the phenotypic effect of the Flcn K508R mutant expression in mice with wild-type Flcn by expressing the Flcn K508R mutant transgene in heterozygous Flcn knockout mice was examined.

The authors reported three patient cases. one patient had germline FLCN missense mutation H255Y andwas clinically diagnosed with BHD; the other two patients had germline FLCN missense mutation K508R and presented with kidney neoplasia but without cutaneous or pulmonary BHD manifestations. To evaluate the pathogenicity of these FLCN missense mutations, the authors looked for a somatic second hit mutation in the remaining FLCN allele in tumour samples from these patients. In a tumour that developed in the patient carrying H255Y mutation, a somatic intron 12 splice site mutation was identified, suggesting that the loss of the wild type FLCN allele led to kidney tumorigenesis. None of the renal tumours that developed in the patients with the FLCN K508R mutation had second hit somatic mutations in FLCN suggesting that haploinsufficiency of FLCN may be sufficient for tumour development in patients with the FLCN K508R mutation.

Using BAC transgenic technology, the authors established genetically engineered mouse models introducing each of the missense mutant transgenes into their kidney-targeted Flcn knockout mouse model, which develops enlarged polycystic kidneys (Baba et al., 2008), in order to determine if expression of the Flcn missense mutant protein could rescue the polycystic kidney phenotype.Expression of the Flcn H255Y mutant in the kidney-targeted Flcn knockout mouse model could not rescue the polycystic kidney phenotype of mice with Flcn deficient kidneys, mirroring the phenotype of kidney-targeted homozygous Flcn deletion.

Expression of the Flcn K508R mutant protein in the kidney-targeted Flcn knockout mouse model partially abrogated the Flcn-deficient phenotype but eventually lead to highly cystic kidneys, suggesting that both Flcn H255Y and K508R mutant proteins were functionally impaired resulting in aberrant kidney cell proliferation. The FLCN K508R missense mutation is responsible for the kidney tumour phenotype in patients with this mutation, but can be classified as ‘weak’ compared to the H255Y mutation.

Heterozygous (Het) Flcn knockout mice begin to develop renal cysts after 10 months of age but these mice do not display any phenotype in other organs (Hasumi et al., 2009). Expression of the Flcn K508R mutant in Het Flcn knockout mice caused the development of renal cystis and cardiac hypertrophy in 26 and 7% of mice, respectively, by 9 months of age, indicating that the Flcn K508R mutant protein may have a potential dominant negative effect on the function of wild-type Flcn to suppress cell growth of kidney cells and cardiomyocytes. These results suggest that increased copy number of FLCN K508R mRNA may predispose to cardiac hypertrophy suggesting that cardiac follow up should be considered in FLCN K508R patients.

In summary, germline missense mutations H255Y and K508R in the FLCN gene were associated with kidney tumours in patients with and without BHD cutaneous manifestations. Based on phenotypes resulting from their expression in kidney-targeted Flcn-deficient mouse models, FLCN H255Y and K508R are predicted to be pathogenic mutations that promote aberrant kidney cell proliferation. The FLCN K508R mutant protein may have a dominant negative effect on wild-type FLCN in kidney and heart. These findings provide further insight into the role of FLCN in regulating kidney tumourigenesis and will add knowledge to the development of novel therapies for kidney cancer.

  • Hasumi H, Hasumi Y, Baba M, Nishi H, Furuya M, Vocke CD, Lang M, Irie N, Esumi C, Merino MJ, Kawahara T, Isono Y, Makiyama K, Warner AC, Haines DC, Wei MH, Zbar B, Hagenau H, Feigenbaum L, Kondo K, Nakaigawa N, Yao M, Metwalli AR, Marston Linehan W, & Schmidt LS (2016). H255Y and K508R missense mutations in tumour suppressor folliculin (FLCN) promote kidney cell proliferation. Human molecular genetics PMID: 28007907

Annual review 2016

2016 has been a busy year for BHD research. With the new year approaching, this week’s blog will review the studies we’ve particularly enjoyed writing about and revisit the year’s highlights.

Iribe et al. (2016) assessed several RCC subtypes from BHD patients to determine if they had similar or distinct patterns of genetic abnormalities. Only a few chromophobe RCC and clear cell RCC samples showed variation in chromosomal copy number. However, all subtypes had various areas of loss of heterogeneity.  Kato et al. (2016) assessed whether variability in chromosomal status could be used to identify BHD-associated RCC: they found that the status of chromosome 17 was useful in distinguishing BHD-associated hybrid oncocytic/chromophobe tumours (HOCT) and chromophobe RCC from sporadic chromophobe RCC.  Furuya et al. (2016) established a new cell line from a BHD patient’s chromophobe RCC.

A very comprehensive review from Kennedy et al. (2016) summarised the current understanding of BHD pulmonary pathology relative to the stretch hypothesis for cyst formation. Gupta et al. (2016b) published a review exploring the key points and research advances in BHD genetics and pathogenesis, clinical manifestations, diagnosis and disease management.

Starling et al. (2016) showed that FLCN controls the dynamic cytoplasmic position of lysosomes. Starvation‐induced FLCN association with lysosomes drives the formation of contact sites between lysosomes and Rab34‐positive peri-nuclear membranes.  Zhong et al. (2016) described FLCN as a ciliary protein that regulates mTORC1 through primary cilia: they showed that FLCN recruits LKB1 to primary cilia for activation of AMPK, causing mTORC1 down-regulation. Yan et al. (2016) and Wada et al. (2016)  showed that FLCN regulates the browning of adipose tissue via a non-canonical mTOR pathway.  Woodford et al. (2016) showed that the stability of FLCN is dependent on the chaperone function of Hsp90. The authors reported that FNIPs act as co-chaperones of Hsp90 by regulating its ATPase activity and chaperoning. Kenyon et al. (2016) examined the role of FLCN in zebrafish development using morpholino oligonucleotides to generate a zebrafish BHD model.

Hoshika et al. (2016) isolated lung fibroblasts from BHD patients. They showed that FLCN is associated with chemotaxis in lung fibroblasts and that, together with reduced TGF-β1 expression by BHD lung fibroblasts, FLCN haploinsufficiency seems to cause lung fibroblast dysfunction, impairing tissue repair.

Dong et al. (2016)  reported two BHD patients also affected with papillary thyroid cancer. The authors recommended neck ultrasound for BHD patients and families and suggest an investigation to evaluate the prevalence of thyroid cancer in patients with BHD. There is currently insufficient evidence to associate thyroid cancer and other thyroid conditions with BHD. However, all the studies mentioned in the post suggest a possible link between the two that should be considered for future research.

A study by Whitworth et al. (2016) reported new cases of multiple germline mutations in inherited cancer syndrome genes involving the combination of mutations in FLCN with mutations in other genes. Authors suggest that clinicians should consider this phenomenon in patients with unexpected inherited cancer syndrome phenotypes.

In the autumn, a new study by Johannesma et al. (2016) evaluated the incidence of spontaneous pneumothorax (SP) in patients with BHD during or shortly after air travel and diving. The data suggested that patients might possibly have an increased risk for pneumothorax in flying and diving.  Gupta et al. (2016) evaluated the cost-effectiveness of high resolution computed tomographic (HRCT) chest imaging for early diagnosis of LAM, BHD, and PLCH in patients presenting with an apparent primary SP. The authors showed that HRCT image screening is cost-effective and suggest that clinicians should consider performing it in these patients to lead to appropriate management approaches.

During the year, several new case studies were reported worldwide. You can find them in our BHD Article Library: Clinical Research.

In addition, we wrote about the updates on clinical trials for BHD and RCC.

We have also attended conferences and workshops including The International RLDC and LAM symposium, the Cilia 2016 and Findacure workshops. These events represent wonderful opportunities to meet researchers, patients and advocates, and to hear about projects helping rare disease research.

Finally, we have redesigned our website. The objectives of the new design were aesthetics, facilitating the navigation and having a responsive layout for all platforms. It also includes an updated and more interactive FLCN signalling pathway.

We at the BHD Foundation are very much looking forward to seeing how the BHD research field develops in 2017. We wish all our readers a very Happy New Year.

  • Gupta, N., Sunwoo, B., & Kotloff, R. (2016). Birt-Hogg-Dubé Syndrome Clinics in Chest Medicine, 37 (3), 475-486 DOI: 10.1016/j.ccm.2016.04.010

Establishment of a new BHD Syndrome cell line

Birt-Hogg-Dubé syndrome is caused by mutations in the FLCN gene. The FLCN protein acts as a tumour suppressor and BHD patients have a high risk of developing renal cell carcinoma (RCC). The mechanisms of tumour formation in BHD have been investigated using mouse models and human RCC tissues. However, in vitro signalling studies of human renal cells with mutant FLCN are still scarce. In a recent study, Furuya et al. (2016) established a new cell line from a BHD patient’s chromophobe RCC. The authors investigated FLCN mutations, chromosome profiles, and cytopathologic characteristics of the cell line to confirm its suitability for functional analysis of the typical phenotype of BHD-associated RCC with impaired FLCN.

The only cell line from a BHD patient’s RCC available and used for functional studies of FLCN in vitro is UOK257. UOK257 was established from a high-grade RCC characterized as predominantly clear cell type with aggressive behaviour. However, most renal tumours developing in BHD patients are the less aggressive subtypes chromophobe RCC and hybrid oncocytic/chromophobe tumours (HOCT). Therefore, the authors established an immortalized cell line derived from a chromophobe RCC in a Japanese BHD patient.

The patient was a 52-year-old Japanese man with repeated episodes of pneumothorax, skin papules, multiple pulmonary cysts, bilateral renal tumours and a family history of pneumothorax. Genetic analysis revealed a pathogenic FLCN mutation – a 3-bp deletion in exon 13 (c.1528_30delGAG) confirming the diagnosis of BHD Syndrome. The patient underwent nephrectomy, and multiple tumours were resected. All tumours were histologically classified as chromophobe RCCs or HOCTs.

The immortalized cell line named BHD-F59RSVT was established from one of the resected chromophobe RCCs by transfecting SV40 large T antigen. BHD-F59RSVT had granular eosinophilic cytoplasm and demonstrated positive immunostaining for mitochondria. These morphologic characteristics were similar to those of the original RCC. The cells exhibited the same mutation pattern as that in the germ line. The cell line, as well as the original tumour tissue, had neither a second hit nor promotor methylation of FLCN. However, western blot analysis revealed that FLCN expression at the protein level was suppressed. It is apparent that a second hit of FLCN may not be essential for FLCN suppression. FLCN mRNA levels were also suppressed in the cell line and original tumour compared with those in sporadic RCC.

Chromosomal analysis of BHD-F59RSVT revealed a loss of chromosome 16. Other chromosomes demonstrated balanced chromosomal copy numbers. The pattern was distinctively different from that of sporadic chromophobe RCC. Deletion of 16p was detected in one of the primary culture cells suggesting that a subset of primary culture cells had segmental loss of chromosome 16p during proliferation, leading to an allelic loss in the immortalization process. This chromosomal alteration during the immortalization process was not seen in previously reported BHD-associated RCCs (Iribe et al., 2016; Klomp et al., 2010). More than one RCC-associated gene is on chromosome 16, including TSC2 and PKD1. As FLCN and TSC2 can both be negative regulators of mTOR signalling, TSC2 in this cell line might be exposed to continuous stress of accelerated mTOR signalling for cell proliferation and protein synthesis.

There is the possibility of transfection-associated effects. SV40 large T antigen can inactivate several tumour suppressor genes including p53pRB, and SEN6. Even though chromosome segments containing these genes showed no pathologic event the authors do not exclude the possibility of small intragenic mutations and/or functional alterations of these genes. However, in preliminary experiments using nude mice, inoculated BHD-F59RSVT failed to form a tumour mass in 8 weeks. Although longer surveillance is needed to characterize BHD-F59RSVT in vivo, this cell line may reflect a slow-developing and less aggressive nature of the patient’s tumour.

BHD-F59RSVT may be a suitable model to study molecular pathways in the presence of impaired FLCN. BHD-F59RSVT was established from an RCC with a typical chromophobe phenotype reflecting the nature of RCCs occurring in most BHD patients. The unique features of BHD-F59RSVT may help clarifying the distinct tumour formation mechanism in BHD syndrome.

  • Furuya, M., Hasumi, H., Baba, M., Tanaka, R., Iribe, Y., Onishi, T., Nagashima, Y., Nakatani, Y., Isono, Y., & Yao, M. (2016). Establishment and characterization of BHD-F59RSVT, an immortalized cell line derived from a renal cell carcinoma in a patient with Birt–Hogg–Dubé syndrome Laboratory Investigation DOI: 10.1038/labinvest.2016.137