Ammonium regulates mTOR signalling

mTORC1 and mTORC2 are two distinct mammalian TOR (target of rapamycin) complexes that regulate cell growth and metabolism. In cancer, genetic alterations lead to activation of mTOR signalling impacting tumour metabolism. Upregulated glutaminolysis is part of the metabolic reaction occurring in cancer that liberates high levels of ammonium, a toxic waste product. Although the importance of glutamine as a tumour nutrient is recognized, little is known about the potential effects of ammonium produced by glutaminolysis in tumours. In their new study,  Merhi et al., 2017 identify ammonium as a dose-dependent regulator of mTORC2, mTORC1 and of proliferation in cancer cells.

To study potential signalling pathways responding to ammonium, the authors performed a kinase array with lysates of MCF-7 breast cancer cells and detected an increase in the phosphorylation of the kinases AKT-S473 and ERK1/2 upon ammonium addition in a time and dose dependent manner. This was confirmed by western blot. Similarly, the authors observed AKT phosphorylation in other cancer and fibroblast cell lines. The upstream pathway leading to the induction of AKT phosphorylation was investigated. Pre-treatment of MCF-7 cells with a PI3K inhibitor impaired AKT phosphorylation and prevented its induction by ammonium supplementation. mTORC2 has been shown to promote cell proliferation and survival by phosphorylation and activation of the AKT and SGK and the phosphorylation of AKT-S473 is a good readout for mTORC2 activity (Oh et al., 2011). Merhi et al. show that siRNA-mediated knockdown of RICTOR (a subunit of mTORC2), leads to reduction of basal and ammonium-induced AKT-S473 phosphorylation. In addition, ammonium also induced NDRG1-T346 phosphorylation, another readout of mTORC2 activity. Knockdown of YES1 kinase and pharmacological inhibition of the focal adhesion kinase (FAK) decreased ammonium-induced AKT-S473 and NDRG1-T346 phosphorylation. The authors also addressed if integrins, known regulators of FAK signalling, was involved in the ammonium-induced AKT-S473 phosphorylation. Results showed that knockdown of ITGβ1 decreased the basal and the ammonium-induced AKT-S473 phosphorylation. Collectively the data indicates that ammonium-induced activation of mTORC2 involves ITGβ1, FAK, YES1, and PI3K signalling.

Ammonium treatment has been shown to induce a transient increase in calcium in cultured astrocytes (Rose et al., 2005). To explore this, the authors assessed the role of calcium in ammonium-induced mTORC2 activation. Pre-treatment of cells with a calcium chelator decreased basal and ammonium-induced AKT-S473 and NDRG1-T346 phosphorylation. In addition, an increase in cytoplasmic calcium concentration induced a rapid increase in AKT-S473 phosphorylation in a mTORC2- dependent way, suggesting that ammonium-induced mTORC2-dependent AKT phosphorylation is modulated by intracellular calcium mobilization.

The impact of ammonium on the activity of mTORC1 remains unclear. The kinase array revealed a decreased phosphorylation of p70S6K-T389, an mTORC1 readout, after treatment with ammonium, suggesting ammonium potentially inhibits mTORC1. Western blot analysis confirmed this mild decrease in phosphorylation. However, this weak dephosphorylation appeared transient, suggesting that the inhibition of mTORC1 was quickly compensated by a reactivation. AKT activation has been shown to promote mTORC1 by inhibiting the TSC complex. In addition, AKT-mediated phosphorylation of another negative regulator of mTORC1, PRAS40, prevents its inhibitory role (Dibble et al., 2015). Merhi et al. found that ammonium induced the rapid phosphorylation of TSC2-T1462, PRAS40-T246 and of 4EBP1 – another readout of mTORC1 activity – suggesting consequent rapid mTORC1 activation. This shows an additional regulatory process upon ammonium addition that transiently counteracts the mTORC1-mediated stimulation of p70S6K-T389 phosphorylation. Treatment with an inhibitor of AKT, not only prevented the ammonium-induced phosphorylation of AKT, but also reduced the phosphorylation of TSC2, PRAS40 and 4EBP1, consistent with ammonium-induced activation of mTORC1 being AKT-dependent.

The impact of ammonium on the proliferation of MCF-7 cells was also assessed. Adding high concentrations of ammonium (~15 mM) resulted in significant cell growth inhibition while low concentrations stimulated growth both in the presence or absence of glutamine supplementation.

In summary, the authors show that ammonium triggers mTORC2-dependent phosphorylation of AKT-S473 in cancer cells. The mTORC2 activation occurs via the PI3K pathway and relies on YES1 and FAK kinases, on integrin ITGβ1 and on intracellular calcium stores mobilization. In addition, ammonium also leads to an AKT-dependent stimulation of mTORC1 signalling and to a dose-dependent stimulation of proliferation. This study identifies that ammonium, a waste product of cancer cells, impacts both mTORC2 and mTORC1 signalling and brings insights into the molecular mechanism of the ammonium-mediated regulation and tumour growth.

FLCN, the gene responsible for BHD Syndrome, has been shown to regulate mTOR signalling. Our interactive folliculin signalling diagram illustrates the relationship between Folliculin (FLCN) and several proteins and signalling pathways including mTORC1 and mTORC2.

  • Merhi A, Delrée P, & Marini AM (2017). The metabolic waste ammonium regulates mTORC2 and mTORC1 signaling. Scientific reports, 7 PMID: 28303961

Shire Rare Disease Summit

In the field of rare diseases one of the key points of progress is to ensure that rare disease patients benefit from advances in medical sciences. To promote this, the global biotechnology company Shire hosted a Rare Disease Summit yesterday in London, which focused on the policy environment for patient access to orphan drugs and how this can be improved. The Summit was also marked by the launch of a report: “Equity and Access – Making the UK a Rare Disease Leader”, funded by Shire and developed by an expert steering group which includes recommendations to improve access to orphan drugs in the UK.

Segolene Ayme from the French Institute of Health and Medical Research (Inserm) and Bertram Haussler from the IGES Institute presented French and German policy developments in improving the quality of care and access to orphan drugs for rare disease patients.

Josie Godfrey, a former NICE director, talked about the creation of the Advisory Group for National Specialised Services (AGNSS) in the UK and how before there had been no systematic approach to look into rare and ultra rare diseases. Josie focused on the point that everyone should get access to the health system with the same prices and same effectiveness.

Christine Lavery from the MPS Society discussed issues with funding for rare diseases, mentioning that it is important to recognise affordability and budget impact but that the Pharmaceutical Price Regulation Scheme (PPRS) in the UK does not put any money into rare diseases and shows no benefits for these patients.

Trevor Cole from Birmingham Women’s Hospital talked about the “perceived difficulties” in the rare disease field such as the small numbers of patients, the surrogate end points, the fact that there is often no possibility of comparison with other conditions and the high variability between diseases and discussed how we can benefit from these difficulties. For example, small numbers keep the costs down, patients can be followed in a smaller number of centres, there is more detailed observation and therefore it is easier to pick up more nuanced changes in each patient. The endpoints can be considered more critically with expertly designed and run studies and a better understanding of the disease process allowing the identification of better surrogate markers.

Ulf Staginnus from Shire gave an industry perspective mentioning that alternative pricing is needed along with engaging with a broader perspective – thinking about how can rare disease research and drug discovery benefit society – and finding alternative models.

Virginia Acha from the Association of the British Pharmaceutical Industry (ABPI) talked about the importance of raising public consciousness of  rare diseases and how partnerships are key since most innovations in rare disease drugs or devices happen by combining different fields such as biology, engineering and nanotechnology.

Neil Churchill from NHS England shared his experience as director of patient experience dealing with the difficulties patients have with the system such as lack of involvement and poor communication between patients and physicians. Neil emphasised that good experience of care makes patients safer and gives them better outcomes.

Nick Meade from Genetic Alliance UK gave a talk about the patient voice in rare disease decision making and how patients should play a role as policy makers, decision makers, information providers and how they should be able to make informed, private and individual choices about personal care provision. Nick discussed how NHS England is failing to bring the patients into policymaking and the lack of transparency and communication that exists around patients as decision makers as members of Clinical Reference Groups.

Tom Fowler from Genomics England discussed the 100,000 Genomes project that has been discussed in a previous blog. He highlighted the impressive amount of data gathered – 21 petabytes – and how this will bring benefits for NHS England patients.

The meeting ended with some discussion and closing remarks and all delegates left with the idea that there is a long way to go to ensure that rare disease patients receive the care they deserve and how debate and collaboration between all stakeholders in the UK, in Europe and worldwide is essential.

Spontaneous Pneumothorax and air travel in BHD Syndrome

Previous studies show that BHD syndrome causes spontaneous pneumothorax (SP) in 24-38% of patients, with a recurrence rate of up to 75% (Toro et al., 2007; Toro et al., 2008; Houweling et al., 2011). A common preventative strategy used following an initial SP in patients with BHD is pleurodesis, however, its efficacy in preventing recurrent episodes is not well known. Due to the pressure changes during air travel, cystic air spaces expand and compress in the thorax possibly leading to cyst rupture and pneumothorax. In their new study, Gupta et al. (2017) evaluate the risk of pneumothorax, the efficacy of pleurodesis, and the safety of air travel in patients with BHD.

104 BHD patients were recruited from the Rare Lung Diseases Clinic Network and the BHD Foundation, and surveyed about pneumothorax and air travel experiences. This study classified any pneumothorax occurring either during a flight or within 24 hours after as a flight related pneumothorax. Differently, considering symptom delay, a recent study discussed on a previous blog classified any pneumothorax that happened within one month of air travel as flight related pneumothorax (Johannesma et al., 2016). Here, the limit to 24 hours meant to distinguish between a flight related SP and an unrelated SP that happened to occur after a flight.

 The survey results showed that the average age at diagnosis of BHD was 47 years, with an average delay from first symptoms of 13 years. Pulmonary cysts were the most frequent phenotypic manifestation of BHD, in 85% of patients. Spontaneous pneumothorax was the presenting manifestation that led to the diagnosis in 65% of patients, typically after the second episode. Mild symptomatic dyspnea was reported by 50% of the patients. 76% of patients had at least one spontaneous pneumothorax during their lifetime, and 82% had multiple. Of the patients with a sentinel pneumothorax, 73% had an ipsilateral recurrence. The mean age at first and second pneumothorax was 36.5 and 37 years, respectively and the average number of recurrent episodes was 3.6.

Spontaneous pneumothorax was mainly diagnosed by chest radiograph, with computed tomographic imaging used as a diagnostic modality for the first episode in only 8 patients. 62% of patients had at least one pleurodesis to prevent recurrent pneumothoraces. Pleurodesis was generally performed after the second pneumothorax and reduced the recurrence by half – 63% of recurrence rate of SP managed without pleurodesis compared to 33% following pleurodesis. Similar conclusions regarding efficacy of pleurodesis have been previously published (Johannesma et al., 2014).

Air travel

96% of the patients in the study had flown at least once in their lifetime and the average number of flights per patient was 25. There was no difference in the average number of flights taken by patients with a history of SP versus patients without a SP. Patients frequently experienced adverse effects during air travel, including chest pressure, anxiety, headache, shortness of breath and chest pain. 11 episodes of spontaneous pneumothorax occurred in 8 patients either during or within 24 hours of air travel. The authors calculated a flight related pneumothorax risk of 8% per patient, and 0.12% per flight. 8 of these 11 episodes represented recurrent SP and the majority had not undergone prior pleurodesis. Prior pleurodesis reduced the occurrence of a subsequent flight-related pneumothorax.

24% of patients changed their flight frequency after the diagnosis of BHD was established, either by avoiding or reducing air travel. The recommendations that patients were given by physicians regarding the safety of air travel after spontaneous pneumothorax were variable and more than half of the patients were given no specific recommendations. Clear recommendations are currently not available but studies emphasize that patients with a current closed pneumothorax should avoid air travel and recommend flight restrictions between 1 and 4 weeks after resolution of pneumothorax (Hu et al., 2014, Ahmedzai et al., 2011).

A significant proportion of the patients were recruited from pulmonary clinics, representing an ascertainment bias, perhaps causing the higher prevalence of spontaneous pneumothorax (76%) observed in this study compared with the 24-38% previously reported in the literature.

Similarly to Johannesma et al., 2016, the present study indicates that patients with BHD have a risk of spontaneous pneumothorax during flight that is most likely less than 1%, and even lower for patients with a history of pleurodesis. Patients with BHD should be advised about the risks of pneumothorax and benefits of pleurodesis and get medical advice regarding air travel.

  • Gupta N, Kopras EJ, Henske EP, James LE, El-Chemaly S, Veeraraghavan S, Drake MG, & McCormack FX (2017). Spontaneous Pneumothoraces in Patients with Birt-Hogg-Dubé Syndrome. Annals of the American Thoracic Society PMID: 28248571

Rare Disease Day – Findacure Scientific Conference: Drug Repurposing for Rare Diseases

This year’s Findacure Scientific Conference took place in London on Rare Disease Day and was again focused on Drug Repurposing for Rare Diseases. The conference brought together over 100 representatives from patient groups, researchers and members of the healthcare industry to discuss the importance and the latest developments in drug repurposing for rare diseases.

Rick Thompson from Findacure started the conference with an introduction to drug repurposing for rare diseases and its advantages. Currently from the ~7000 rare diseases only 400 have treatments. Finding new uses for existing drugs to treat a new population offers several advantages such as being faster and cheaper, the known safety profile, side effects and pathways of action of the drug and the reduction in early phase trials. To support drug repurposing Findacure is creating a Social Impact Bond (SIB) program to enable investment in drug repurposing projects for rare diseases that have high costs for the NHS by reducing the burden of the current care costs, generating returns from healthcare savings. If this model is successful this will be a new way to quickly develop new treatments for rare diseases. Findacure is promoting an open call for new repurposing ideas.

Tim Barrett from the University of Birmingham introduced drug repurposing for Wolfram syndrome, a very rare disease with no cure, no treatment and little research. p21cip1 was identified as a drug target and a drug screen for repurposed drugs took place. The screening identified sodium valproate, a drug used for epilepsy, as a potential candidate. The hypothesis was that sodium valproate administration would slow the rate of progression of neurodegeneration in Wolfram syndrome compared with current standard of care. Funding for a phase II efficacy study was awarded to pursue this idea. Tim ended his talk suggesting the possibility of having several arms in a clinical trial with different diseases with similar mechanisms or phenotypes being tested with a same drug.

Bev and Georgia Hart talked about their experience as a family affected by Friedreich’s ataxia. They specifically focused on their experience participating in an IFNγ clinical trial in the US highlighting how being able to act is so important for patients and how patients should be involved in deciding the outcomes of clinical trials.

Richard Wyse from The Cure Parkinson’s Trust (CPT) presented Parkinson’s as a series of rare diseases, unique to each patient. Drug repositioning represents one of CPT’s major therapeutic initiatives. They are currently promoting a Linked Clinical Trials initiative to evaluate, prioritise and repurpose existing regulatory-approved medications that may also have direct therapeutic benefits for patients with Parkinson’s.

Christine Charman from the company Takeda presented the TAK-celerator, a new business model to accelerate development of therapies in rare diseases. The TAK-celerator focus is to establish the capacity to execute rare diseases projects with external partners, to bridge the gap between academia and industry providing a mechanism to accelerate early stage programs to preclinical development and to reposition assets from the Takeda pipeline in addition to de novo opportunities

Daniel O’Connor from the MHRA and Jonathan Underhill from NICE talked about regulation, implementation and optimisation in drug repurposing. Daniel mentioned the regulatory incentives to support repurposing such as a new therapeutic indication for a well-established substance, Paediatric use marketing authorisation and Orphan drug designation. Jonathan presented the medicines optimisation approach, a person-centred approach to ensure people obtain the best possible outcomes from their medicines. This approach sees shared decision-making as an essential part of evidence-based medicine and is seeking to use the best available evidence to guide decisions about the care of the individual patient, considering their needs, preferences and values. Jonathan remembered that NICE offers guidelines not tramlines and that informed, shared decision making is key in drug repurposing for rare diseases.

This was followed by six lightning talks with five minutes presentations on mobilising information resources for rare diseases, on bringing the clinical trials to the patients in an in-home service, and on cases studies of drug repurposing in polyglucosan body disease, MdDS and mast cell and on accelerating the UK drug repurposing pipeline.

The last talk was given by Pan Pantziarka from the Anticancer Fund who shared their projects on repurposing non-cancer drugs for new indications in oncology in rare cancers.

BHD is a rare disease with no cure or preventative treatments available.  Greater understanding of the molecular pathways in BHD and their roles in pathology will enable researchers to identify drug target candidates since the BHD clinical trial with Rapamycin, an mTOR inhibitor, did not show promising results (Gijezan et al., 2014). Repositioning of known drugs could then be useful in discovering treatments for BHD and other rare diseases faster and more cost effectively than conventional drug development.

  • 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

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