Spontaneous pneumothorax in diffuse cystic lung diseases

Diffuse cystic lung diseases (DCLDs) are a group of disorders with different mechanisms that are characterized by the presence of air-filled lung cysts. These cysts are prone to rupture, leading to the development of recurrent spontaneous pneumothoraces. In their new review, Cooley et al., 2017 give an overview of the epidemiology, clinical features, and management of spontaneous pneumothoraces in patients with DCLDs, with a focus on Birt-Hogg-Dube (BHD) syndrome, lymphangioleiomyomatosis (LAM) and pulmonary Langerhans cell histiocytosis (PLCH). The understanding of spontaneous pneumothoraces in DCLDs can help clinicians to find an early diagnosis and appropriately manage the disorders.

BHD syndrome

Patients with BHD have a 50-fold greater likelihood of spontaneous pneumothorax as compared with controls. Recently, studies have shown that BHD can be the underlying cause of 5–10% of apparent primary spontaneous pneumothoraces (Johannesma et al., 2015). The prevalence of spontaneous pneumothorax in BHD varies between reports. Pneumothorax prevalence rates of 22.5-38% are reported by renal/skin-focused centres, while prevalence rates of 42–76% are reported from patients in pulmonary cohorts. Pneumothoraces due to BHD usually occur in the patient’s mid-late 30s; however, they have been reported in pediatric and geriatric patients. Lung cysts and cyst burden are associated with increased risk of pneumothorax. Interestingly, there are reports of pneumothorax in patients without apparent cysts on CT imaging. Family history of pneumothorax is associated with higher risk of pneumothorax but smoking and the presence or severity of renal tumours or fibrofolliculomas are not. Pneumothorax recurrence rates in BHD are estimated to be 75–80%. Patients with BHD-associated pneumothorax experience an average of 3.6 total episodes of pneumothorax. Given the high recurrence rate, pleurodesis should be performed following the first pneumothorax rather than waiting for a recurrence since pleurodesis can halve the recurrence rates of ipsilateral pneumothorax in BHD patients.


55–73% of patients with LAM experience a pneumothorax in their lifetime, with pneumothorax leading to the diagnosis of LAM in approximately 40% of patients. On average, patients experience 2.2 pneumothoraces before the diagnosis of LAM is made. Patients with larger cyst size on HRCT and those with a history of smoking are more likely to develop a pneumothorax. Given the high rates of recurrence, pleurodesis should be performed after the first spontaneous pneumothorax in patients with LAM. In one study, the ipsilateral pneumothorax recurrence rate was 66% if managed conservatively, but this was reduced to 27% with chemical pleurodesis and 32% with surgical pleurodesis.


Approximately 15–20% of patients with PLCH experience a pneumothorax. Nearly 63% of those patients will have more than one pneumothorax. Simultaneous bilateral pneumothorax can occur and may be fatal. Due to high rate of recurrence, pleurodesis should be performed following the initial episode of pneumothorax. A study found a recurrence rate of 58% when managed conservatively, compared with 0% following surgical pleurodesis. Treatment of PLCH must involve smoking cessation. The effect of steroids or chemotherapeutics on disease course, and occurrence of pneumothorax, is unclear.


Air travel and diving may lead to cyst expansion and predispose patients with DCLDs to a higher risk of pneumothorax. The risk of pneumothorax associated with air travel has been studied in LAM and, more recently, BHD as discussed in a previous blog. The risk of flight-related pneumothorax is currently being evaluated for patients with PLCH. Studies have concluded that it is safe for most patients with DCLDs to undertake air travel but patients should be educated about the signs and symptoms of a pneumothorax and counselled prior to boarding an airplane. Limited data are available regarding the safety of diving in patients with DCLDs but the authors recommend that patients with DCLDs avoid diving in accordance with the British Thoracic Society guidelines.

Recent data suggest that BHD, LAM, and PLCH likely cause approximately 10% of apparent primary spontaneous pneumothoraces. Current guidelines do not recommend screening CT for first-time pneumothoraces. However, in a recent study, performing a screening HRCT to facilitate early diagnosis of LAM, BHD, or PLCH followed by pleurodesis was found to be cost-effective. Based on this, the authors recommend that all patients with an apparent primary spontaneous pneumothorax be screened with HRCT for the presence of underlying DCLDs.

In summary, DCLDs are being recognized as the cause of spontaneous pneumothoraces. The impact of pneumothoraces on these diseases, the efficacy of alternative techniques to reduce recurrence risk, the impact of targeted pharmacologic therapy on pneumothoraces and the molecular pathways behind them, are some of the major unanswered questions to be addressed.

Pneumothorax BHD Awareness campaign

BHD Syndrome is the genetic cause of 10% of primary spontaneous pneumothorax. Unfortunately, it is not widely known that spontaneous pneumothorax can be a symptom of BHD. Therefore, many people presenting with pneumothorax go undiagnosed for BHD syndrome. An accurate diagnosis would allow patients to be screened for kidney cancer enabling them to receive timely treatment, and would also give their family the chance to be tested for Folliculin mutation.

The BHD Foundation (with the Myrovlytis Trust) is promoting an awareness campaign that tackles this issue.

Pneumothorax BHD Awareness campaign is a campaign to raise awareness among pulmonologists, radiologists, thoracic surgeons, respiratory nurses, technicians and medical students of the fact that pneumothorax can be a symptom of BHD syndrome, and that BHD should be considered as a diagnosis for patients presenting with pneumothorax. Another aim of the campaign is to inform as many people as possible, who have had an unexplained pneumothorax, of BHD as a potential cause.

Initially, we are aiming to have a presence (personally and in combination with oral or poster presentations, leaflets and exhibition stands) at respiratory conferences, and possibly submitting a collaborative multi-author piece to a respiratory journal.

This week we are at the JointCHESTSGP2017 in Basel, Switzerland, a joint congress organised by the American College of Chest Physicians (CHEST) and the Swiss Pneumology Society (SGP) with almost 1000 chest specialists. We have been talking to doctors, students, nurses and imaging experts about BHD Syndrome, unknown to most of them.

A social media engagement plan is also being developed to create an online movement around the campaign. We hope that patients, professional and patient societies will help to promote the campaign through their social channels and we will provide social media content to support with this.

In the future, we are considering contacting radiology, pulmonary, thoracic clinics/hospital services with a questionnaire about pneumothorax and BHD and sending informational e-mails about pneumothorax and BHD that is clear, credible and attracts attention. Also, creating an eLearning module about BHD (possibly combined with LAM and other diffuse cystic lung diseases) and producing “Hospital information packs” about BHD to be sent to staff at hospitals.

Help us spread the word! Connect with us on social media and take our Clinical Introduction to BHD and our Lung Symptoms leaflets to your doctor and lung specialist to make them consider BHD.

Three new BHD case reports

Marous et al. (2017) present the case of a 38-year-old white man with confirmed BHD, manifesting cutaneous fibrofolliculomas and trichodiscomas on the face and upper chest, bilateral spontaneous pneumothorax and a choroidal nevus in the left eye. Later, the patient experienced photopsia and decreased visual field in the left eye. Microscopy revealed subtle iris melanocytosis in the left eye. Fundus evaluation in the left eye disclosed a choroidal melanoma arising from patchy inferotemporal sectoral choroidal melanocytosis. In addition, subtle pinpoint retinal pigment epithelium (RPE) microdetachments were noted. Fluorescein angiography confirmed hyperfluorescence of the melanoma and also highlighted multifocal pinpoint RPE defects in each eye.

One ophthalmic study previously reported two patients with BHD with RPE alterations, and another report described choroidal melanoma with no documentation of melanocytosis (Walter et al., 1997; Fontcuberta et al., 2011). The relationship of these findings with BHD remains unclear. It would be interesting to examine a cohort of BHD patients to investigate the prevalence of RPE alterations and choroidal melanocytic features.

Rato et al. (2017) describe two BHD patients with previously-undescribed FLCN mutations, and one of them with a type of adrenal gland tumour associated for the first time with BHD. The first case was a 60-year-old caucasian man with multiple asymptomatic skin lesions of the face, scalp and neck. The patient denied any respiratory signs or symptoms. His medical history was consistent with hypertension, diabetes mellitus, dyslipidemia and benign prostatic hypertrophy. Physical examination revealed multiple, dome-shaped, whitish and erythematous papules scattered over the scalp, face and upper neck. Biopsy of two papules showed features consistent with fibrofolliculoma. A genetic test revealed a new heterozygotic mutation in exon 6 of the FLCN gene (p.Lys192Argfs*31). Computed tomography (CT) scan of the thorax and abdomen showed numerous bilateral lung cysts without suspicious kidneys lesions. Patient had no history of spontaneous pneumothorax. Pulmonary function tests, thyroid ultrasonography and colonoscopy were normal.

The second case was a 39-year-old caucasian man with a history of epilepsy and asthma presenting with multiple asymptomatic skin lesions located on the face and neck. Similar dermatological findings were present in the patient’s family. Physical examination found numerous firm, whitish papules on the face and upper neck. Biopsy of two papules was performed and histopathological analysis was compatible with fibrofolliculoma. The patient was assessed for FLCN gene mutations and a new heterozygotic mutation was detected in exon 9 of the FLCN gene (c.1015C>T), responsible for the introduction of a premature stop codon at position 339 amino acid (p.Gln339*). This mutation has not been described previously but it can be considered pathogenic and so responsible for BHD in the patient. CT scan of the thorax and abdomen showed a lung cyst in the right middle lobe and a nodular formation. Laparoscopic left adrenalectomy was performed and histopathological examination revealed a malignant perivascular epithelioid cell tumour (malignant PEComa). Pulmonary function tests and thyroid ultrasonography were normal. This case represents the first malignant PEComa diagnosed in a patient with BHD. Some types of PEComa are seen at high frequency in tuberous sclerosis complex (TSC), like renal angiomyolipoma, as well as pulmonary lymphangioleiomyomatosis. A possible association between BHD and the occurrence of malignant PEComa in this patient is interesting but purely speculative.

These new cases reinforce the message that the management of Birt-Hogg-Dube patients should be multidisciplinary.

  • Rato M, Monteiro AF, Parente J, Aranha J, & Tavares E (2017). Birt-Hogg-Dubé Syndrome – report of two cases with two new mutations. Journal of dermatological case reports, 11 (1), 12-15 PMID: 28539984

The PI3K/mTOR inhibitor GSK2126458 is effective for treating TSC solid renal tumours

Tuberous sclerosis (TSC) is an inherited tumour syndrome that shares clinical similarities with Birt-Hogg-Dube Syndrome. It is caused by mutations in TSC1 or TSC2 that lead to aberrant activation of mTOR, affecting multiple organs, including the kidney and lung. In the kidney, lesions such as multiple renal cysts and renal cell carcinoma (RCC) can occur. Tumour reduction in TSC patients after treatment with rapamycin, an inhibitor of mTOR, is partial and reversible probably due to feedback activation of Akt. In their new study, Narov et al. (2017) test the efficacy of GSK2126458, an inhibitor of PI3K/mTOR, in comparison to rapamycin, for treatment of renal tumours in genetically engineered Tsc2+/- mice, that spontaneously develop various lesions in the kidneys. Both GSK2126458 and rapamycin caused significant reduction in number and size of solid renal tumours. GSK2126458 inhibited both PI3K and mTOR while rapamycin exerted stronger inhibitory effect on mTORC1 in renal tumours. Both GSK2126458 and rapamycin suppressed proliferation of tumour cells. However, GSK2126458 increased apoptosis of solid tumours but rapamycin did not. Further investigations are needed to test whether rapamycin in combination with GSK2126458 can improve anti-tumour therapy.

The kidney lesions of adult Tsc2+/- mice had aberrant activation of the mTOR complex 1 (mTORC1) and mTORC2. The MAPK pathway was also activated in these lesions. To test the anti-tumour efficacy of GSK2126458, the authors first determined the maximum tolerated dose (MTD) of GSK2126458 in Tsc2+/- mice. After this, adult mice were treated for two months with vehicle, the MTD dose of GSK2126458 or rapamycin. Both GSK2126458 and rapamycin significantly reduced total number, size and cellular area of solid renal tumours and other lesion types. Rapamycin caused greater reduction than GSK2126458 in number, size and cellular area of all types of lesions but the difference in reduction of solid tumour burden was not significant. IHC of kidney sections was used to investigate PI3K/Akt/mTOR signalling. GSK2126458 reduced phosphorylation of Akt at T308 but rapamycin did not. It is not known whether this reduction in Akt phosphorylation contributes to antitumour efficacy. The phosphorylation of a part of the Erk1/2 signalling pathway, was reduced in GSK2126458 treated solid renal tumours. The Erk1/2 signalling was inhibited by rapamycin in solid renal tumours. In contrast, reduced phosphorylation of mTOR at S2481, an indicator of mTORC2 activation, was detected in rapamycin treated solid tumours. Ki67 staining was used to assess proliferation of renal tumour cells on treated mice. Both GSK2126458 and rapamycin markedly reduced the median percentage of Ki67-positive cells. Rapamycin inhibited proliferation of tumour cells to a greater extent than GSK2126458. Active caspase 3 was used to test whether treatment induced apoptosis in tumour cells. Interestingly, GSK2126458 significantly increased the median total area of active caspase 3-positive tumour cells but rapamycin did not. Similar results were observed when other apoptosis marker was analysed. To investigate the mechanism behind the increased apoptosis associated with GSK2126458 treatment, expression of p53 and phosphorylation of MDM2 at S166 were analysed by IHC. Renal lesions had a lower level of p53 and decreased phosphorylation of MDM2 in rapamycin treated mice, but not GSK2126458 treated mice, compared to vehicle treated mice. Similar results were observed by Western analysis.

In conclusion, the authors demonstrated that GSK2126458 was effective for treating solid renal tumours. A Phase I clinical trial has recently reported that GSK2126458 is well tolerated in patients treated for multiple solid malignancy types and tumour responses and disease stabilization were observed (Munster et al., 2016). The authors found that GSK2126458 inhibited both mTORC1 and mTORC2 in all types of renal lesions in Tsc2+/- mice but the inhibitory effect of GSK2126458 on mTORC1 was weaker than that of rapamycin. Both GSK2126458 and rapamycin reduced proliferation of tumour cells. However, GSK2126458 increased apoptosis of solid tumours but rapamycin did not.

The clinical similarities between BHD and TSC suggest that FLCN and TSC proteins may function within a common pathway. It may be worthwhile investigating whether combination of GSK2126458 with rapamycin could improve anti-tumour therapy through increased tumour cell death in different disease models.

  • Narov, K., Yang, J., Samsel, P., Jones, A., Sampson, J., & Hong Shen, M. (2017). The dual PI3K/mTOR inhibitor GSK2126458 is effective for treating solid renal tumours in Tsc2+/- mice through suppression of cell proliferation and induction of apoptosis Oncotarget DOI: 10.18632/oncotarget.17215

Characterization of a FLCN mutation associated with RCC

Mutations in the FLCN gene are the cause of Birt-Hogg-Dubé (BHD) syndrome, a rare disease characterized by renal cell carcinoma (RCC), pneumothorax and fibrofolliculomas. In their new study, Bartram et al. (2017) identify a heterozygous mutation in the FLCN gene in a patient with RCC. DNA from tumour and a metastasis was analysed and the authors demonstrated skipping of exon 11 as the consequence of this mutation leading to a shift in the reading frame and the insertion of a premature stop codon. The FLCN protein was still expressed but it was strongly destabilized and had a different subcellular localization. Both altered protein stability and subcellular localization could be partly reversed by blocking proteasomal and lysosomal degradation.

In this study, a 55-year-old patient presented with weight loss, bilateral kidney cysts and tumours. He and family members had a history of recurrent pneumothorax. Histology after kidney tumour nephrectomy showed elements of a chromophobe RCC (chRCC) and a small cell carcinoma component. In addition, CT scan showed liver and spleen enlargement, and several lung cysts and pulmonary nodules. Open surgery revealed peritoneal metastases.

Histology of one metastasis showed features of the small cellular tumour component, suggesting that these cells might contribute to the aggressive tumour phenotype in the patient. After surgery, hemodialysis was initiated due to renal failure. Unfortunately, the patient died shortly afterwards as a consequence of the advanced stage of the metastatic tumour disease.

The co-occurrence of chromophobe RCC with familial recurrent pneumothorax lead to the suspicion of BHD syndrome.

BHD-syndrome associated RCC normally show a benign nature and rarely metastasize. Here, the patient suffered from metastases and pulmonary lesions. The metastases did not show the classical characteristics of the chRCC but rather a small-cell morphology. Since the chRCC showed different levels of dedifferentiation towards the areas of the small cell tumour component the authors speculate that the small cell carcinoma arose from the chRCC by acquiring further genetic alterations.

FLCN sequencing identified an intronic c.1177-5_-3delCTC alteration that most likely affected the correct splicing of exon 11 of the FLCN gene. In silico analyses by bioinformatic tools predicted this variant to be likely pathogenic. FLCN MLPA analyses were consistent with deletion of the second FLCN allele in both tumour tissues.

The metastasis appears to be linked to BHD since it showed loss-of-heterozygosity in the FLCN gene. It will be interesting to see in future cases whether this entity is associated with BHD syndrome.

To validate the mutation’s impact on splicing of the FLCN transcript the authors generated minigene constructs containing either the FLCN  wild-type (WT) or the mutant sequence. These minigene constructs showed that the deletion indeed abrogated the acceptor splice site of exon 11, leading to skipping of exon 11 and fusion of exon 10 to exon 12 which generates a frameshift and premature stop codon.

To investigate whether the predicted FLCN protein is expressed the authors analysed protein expression in HEK293T cells. Overexpression of a FLAG-tagged cDNA revealed that the mutant protein is expressed, however, at lower levels than the WT protein. A commercially available anti-FLCN antibody detected the overexpressed mutant protein in Western Blots and this antibody was used to analyse the endogenous expression in patient tumour tissue with a clear signal being obtained by immunohistochemistry. To confirm this the authors generated transgenic cell lines using the TALEN technology that expresses GFP-fused versions of either the WT or the mutant protein and mimic the physiological situation. WT FLCN was detected by western blot but the mutant protein again showed markedly lower protein levels. This effect was partially reversed by treatment with MG-132, a proteasome inhibitor and chloroquine, suggesting that inhibition of both lysosomal and proteasomal degradation stabilized the mutant protein.

Additionally, fluorescence imaging revealed an altered subcellular localization of mutant FLCN comparing to the WT protein. WT FLCN localized to both cytoplasm and nucleus and the mutant protein was restricted to the cytoplasm. Treatment with MG-132 not only stabilized the mutant protein but also led to a nuclear localization shift making it more similar to the WT. This may be the consequence of accumulation of ubiquitinated mutant FLCN, since ubiquitination has been shown to be a key regulator of subcellular localization of different proteins. It remains to be elucidated whether a ubiquitination or similar approach may have any therapeutic implication in the treatment of BHD – once stabilized and correctly localized – to fix the molecular function required to prevent tumorigenesis.

While this manuscript was under revision a different group characterized the same mutation in two sisters with RCC and found similar results regarding splicing of WT and mutant FLCN (Rossing et al., 2017).

In summary, this study shows that the functional characterization of the pathogenic mutations in BHD syndrome may shed light into further research for the development of novel diagnostic and therapeutic strategies.

  • Bartram MP, Mishra T, Reintjes N, Fabretti F, Gharbi H, Adam AC, Göbel H, Franke M, Schermer B, Haneder S, Benzing T, Beck BB, & Müller RU (2017). Characterization of a splice-site mutation in the tumor suppressor gene FLCN associated with renal cancer. BMC medical genetics, 18 (1) PMID: 28499369

Findacure workshop – “Engaging your community for Fundraising”

Fundraising helps charities to make a difference for rare disease patients by supporting research, community events and awareness campaigns. At the end of April, Findacure hosted a workshop in London with several speakers sharing their experience of fundraising.

Libbie Read and Mary Rose Roberts, from Findacure, introduced the fundraising theme and gave a talk about how to engage your community for fundraising. They mentioned the benefits of community fundraising: the financial side; unrestricted funding; gift aid; raising awareness; educating; engaging new audiences; building support by establishing new relationships and growing existing ones. They gave examples of fundraising events including ones organised by Findacure  – gala dinners, firewalk, London marathon, pub quizzes, etc., and discussed in detail the planning of a fundraising event, dividing it in different stages and suggesting the steps to follow for each stage:

Initial planning:

  • Outline your fundraising idea
  • Identify your stakeholders
  • Define aims and objectives
  • Consider what is achievable
  • Set a budget – prepare to compromise
  • Remember to check the calendar


  • Assign tasks
  • Set a schedule and make note of deadlines
  • Secure funding
  • Promote – marketing and communication is essential
  • Remember to expect the unexpected.


  • Follow up with participants
  • Collect feedback
  • Evaluate
  • Claim Gift Aid

Robin Marshall, from the AKU Society, talked about putting together special fundraising events. He talked about the things to consider when:

 Planning a budget:

  • Equipment hire
  • Prizes
  • Transport
  • Insurance
  • Fees for licenses and permissions
  • Return of investment
  • Hidden costs (have a contingency plan)

Choosing a venue:

  • Meet objectives
  • Fit the audience
  • Travel costs

Organising a team:

  • Staff/volunteers
  • Time commitments
  • Particular expertise needed?
  • Define roles/responsibilities

Callum Appleby from the Bone Cancer Research Trust gave an exciting talk about fundraising with challenge events. He started by highlighting that challenge events make people achieve more than they think it’s possible. They are events that take people out of their comfort zone, it can be anything and it varies from one person to another. Challenge events can be pre invested or own place events. In pre invested events, the charities buy places in these events from either the event organisers or 3rd party’s acting on behalf of the organisers, the costs can vary, buying places means charities have secured places and can ask people to raise sponsorship in return for a place. In own place events there is no investment by charities. People secure their own places in the event then take part for an organisation and the amount a person raises is completely up to them. Callum mentioned the importance of choosing a successful event by asking questions such as:

  • Is the event well established?
  • Does the event sell out?
  • Does it fit a gap in the market?
  • Is the event popular amongst your target demographic?
  • What makes the event special?
  • What is the cost? – Ensure high return
  • Location and support costs

In special eventsrecruitment can be a challenge, so it is important to build a database of contacts, to offer incentives, to have a call to action, to reach new audiences and to explain clearly the need for support.

Sharmila Nikapota from the Sohana Research Fund gave a presentation about special fundraising events for your community such as gala dinners, auctions, etc., that have a high level of entertainment and that make the participants feel special, have a good experience and reward the organisers.

NKTR-214 therapy study in patients with RCC

Early this year at the ASCO Genitourinary Cancers 2017 meeting, Hurwitz et al. (2017) presented clinical data from a Phase I clinical trial of the oncology agent NKTR-214 in patients with renal cell carcinoma (RCC) showing encouraging evidence of anti-tumour activity, and a favourable safety and tolerability profile. NKTR-214 was developed by Nektar Therapeutics to expand specific cancer-fighting immune cells in the tumour environment and increase expression of the cell surface receptor PD-1 on these immune cells.

NKTR-214 is a CD122-biased cytokine agonist conjugated with multiple releasable chains of polyethylene glycol and designed to provide sustained signalling through the heterodimeric IL-2 receptor pathway (IL-2Rβγ) to preferentially activate and expand effector CD8+ T and natural killer (NK) cells – usually cancer-fighting immune cells – over T regulatory cells (Tregs) – immunosuppressive cells that usually limit anti-tumour response.

A dose escalation trial of NKTR-214 was initiated to assess the safety, tolerability and explore immune changes in the blood and tumour microenvironment in patients with RCC. NKTR-214 was administered IV every 2 or 3 weeks. Pre and post treatment blood and tumour samples were collected and analysed for immune phenotyping, gene expression and changes in the tumour microenvironment by immunohistochemistry.

Among 25 patients dosed, 15 had RCC. Treatment with single-agent NKTR-214 was well tolerated and the maximum tolerated dose (MTD) was not reached. There were no autoimmune-related adverse events or organ related inflammation. 6 out of the 15 RCC patients, with prior tyrosine kinase inhibitor (TKI) treatments, experienced tumour shrinkage. Analysis of the tumour microenvironment revealed several significant immunological changes post treatment, including increase in total and proliferating NK, CD8+, and CD4+ T cells. There was good correlation between increase in activated CD4+ and CD8+ T cells in peripheral blood with an increase in T cell infiltrates within the tumour tissue. There was a greater abundance of CD8+ T cell compared to Treg immune suppressive cells accumulating in the tumour tissue. NKTR-214 also increased cell-surface expression of PD-1 on CD4+ and CD8+ T cells.

NKTR-214 increased immune infiltration in the tumour and anti-tumour activity in patients who previously progressed on TKIs, with a favourable safety profile. The ability to alter the immune environment and increase PD-1 expression on effectors T cells may improve the effectiveness of anti-PD-1 blockade. A trial combination of NKTR-214 and nivolumab is being evaluated. The Phase 1/2 clinical program will enrol up to 260 patients and will evaluate the potential for the combination of Opdivo (nivolumab) and NKTR-214 to show improved and sustained efficacy and tolerability above the current standard of care in melanoma, kidney, triple-negative breast cancer, bladder and non-small cell lung cancer patients.

The NKTR-214 clinical trial is currently recruiting participants, you can find more information here.

Findacure – Medical Research Explained: Clinical research

Following last week’s blog about pre-clinical research, this week we introduce the second part of Findacure’s webinar explaining clinical research.

The subject of clinical research was presented by Sarah Venugopal from Raremark, a company connecting families affected by rare diseases with information on the latest research and treatments in the field.

Clinical research, also known as clinical trials, clinical studies or human trials, are conducted to collect data about the safety and effectiveness of a potential new test, drug or device before it is approved and widely used.

The key players in clinical research are:

  • The sponsor – organization that funds the clinical trial, can be a pharma company, a charity, a hospital or even an individual
  • The ethics review boards – independent groups responsible for the protection of the rights, safety and well-being of people taking part in a trial
  • The principal investigator (PI) – person who leads the trial, usually a specialist doctor or researcher
  • The study coordinator – person who supports the PI and is in charge of the day-to-day running of the trial, there can be more than one
  • The participants – the patients or healthy volunteers taking part in the trial

People take part in clinical trials to help speed up the approval process for new drugs for themselves or others, to access specialist care, to potentially access an experimental treatment and to be monitored closely.

Main types of clinical trial:

  • Diagnostic trials – new tests or procedures for diagnosing diseases
  • Natural history studies – often done for rare diseases, they generate insights into how diseases might progress naturally over time
  • Observational studies – monitor participants without intervening for example whilst they are already on an existing treatment or after surgery
  • Screening trials – evaluate new tests, test the best way to detect certain diseases or medical conditions
  • Treatment trials – the one people are more familiar with, they evaluate the effectiveness and safety of potential new treatments

Four phases of Clinical trials:

Phase I – Typically a small study (5-100) with healthy volunteers assessing safety usually with a very low dose of the drug. This phase usually lasts months to a year. In rare diseases, sometimes this phase is with patients with the condition so that the drug can be approved faster. 70% of the drugs tested on phase I make it to phase II.

Phase II – Assessing safety and efficacy, larger study with people with the condition, can include a placebo. 33% of the drugs tested on phase II make it to phase III.

Phase III – Larger studies, randomized and controlled, people with the condition, lead to a potential approval, looks at dosing. 70-90% of the drugs tested on phase III make it to phase IV.

Phase IV – Drug is approved for use, monitor long-term safety and effectiveness in the real world.

Usually the entire process lasts about 6 to 10 years.

What happens during a trial?

It starts with the sponsor designing the trial with doctors/investigators, then investigators see patients and collected data, the data is entered into a database, the data is analysed and presented in a report and finally the report is used to support approval.

Most large clinical trials are randomized, double-blind placebo-controlled trials. This is a common way to design a trial as it ensures the data is robust, reliable and reduces bias.

Placebo-controlled – a placebo contains no active ingredients and is given to some of the participants in a trial so that the effectiveness of the drug can be accurately seen

Randomized – participants are randomly assigned to either take the active drug or the placebo

Double-blind – to reduce bias, neither the researchers nor the patients know who is taking the experimental drug or the placebo

What happens after a trial?

If the drug is shown to be safe the results are submitted for approval to regulatory bodies (FDA or EMA), this process takes a very long time and the data is reviewed to make sure is reliable, cost-effective and has a positive impact. Additional data might be needed and it important to continue to monitor drug in real world. Usually there is publication of results on PubMed or clinicaltrials.gov but unfortunately, not all sponsors publish their results.

You can watch the entire Findacure webinar here and learn about BHD Syndrome clinical trials here.

Findacure – Medical Research Explained: Pre-clinical research

Last week Findacure hosted a webinar explaining the complex medical research field that introduced the different stages of pre-clinical and clinical research necessary to get a treatment available to patients.

The first talk focused on pre-clinical research and was presented by Oliver Timmis from the AKU Society, a charity that supports patients with the rare condition alkaptonuria.

Oliver explained that assessing a potential treatment of a condition by a drug starts with pre-clinical research. Pre-clinical research in drug development aims to show that a drug is safe and effective in a non-human model before human research is initiated. The drug development timeline is time consuming, taking 16 years on average from the discovery of a drug to having it as an available treatment for patients. The timeline starts with several years of pre-clinical testing and then clinical testing in different phases. Pre-clinical testing is quite variable, compounds are narrowed down at this phase with just a few going to clinical trials – this has very high costs (~$1 billion net cost invested over 15 years).

Pre-clinical development was defined as the testing of promising candidate compounds to ensure that they are:

  • Likely to be effective
  • Safe
  • Sufficiently stable
  • Excreted safely from the body

Around the world there are regulatory guidelines by agencies such as the FDA and the EMA that help in the development of effective pre-clinical and clinical research.

Animal studies are widely used in pre-clinical research, in fact, they are a legal requirement for any human drug developed in Europe.  Animal studies are a standard procedure but controversial, therefore, ethical guidance for appropriate use is in place with systems such as the principles of the 3Rs.

Research can be divided into two categories – Basic and Applied/Clinical. Basic research aims to discover facts about how and why things occur without any relation to clinical outcome; applied research uses information generated by basic research to treat and prevent illness.

You can find BHD-specific basic and clinical research in the BHD Article Library.

Before pre-clinical research there is a period of “pre-pre-clinical research” to establish a target, to choose a molecule that is likely to work, act on symptoms/pathways most important to patients, and work out how to measure if this molecule is having the expected effect.

A classic type of pre-clinical studies is toxicology studies. These studies are usually performed in animals to support human studies. Important considerations are which species and how many animals to use.  It has to be practical and the duration of studies should not be too long since the idea is to get to clinical studies quickly.

Toxicology studies are conducted to assess:

  • Toxic effects following single and multiple dosing
  • Effects on reproduction
  • Potential for teratogenicity
  • Peri/postnatal effects
  • Potential to cause cancer and genetic abnormalities
  • Effects in the immune system
  • Potential to cause skin and eye problems

There are acute/short term toxicity studies which look for effects over short periods using the route of administration intended for humans. Usually, animals are observed for 30 days for eating/drinking habits, weight change, toxic effects and psychomotor changes. Subacute/subchronic studies, uses repeat dosing aligned to intended human usage. Testing over 90-180 days is required to support human administration for 1 week. For a chronic human illness testing over 1 year is required in animals

Definitive animal studies define the No Observable Adverse Effect Level (NOAEL) – the highest amount of drug used with lowest side effects. There is the need to use most sensitive species available and to consider the treatment regime.

An important tool used in medical research is the biomarker. Biomarkers are surrogates for problems in the human body, changes in biological systems that are related to exposure to a toxin. To be useful in pre-clinical studies biomarkers should be chemical specific, quantifiable at low levels, and the ability to be monitored in a non-invasive way is an advantage. Different types of biomarkers include biomarkers of exposure, of response effects and of susceptibility.

In summary, preclinical studies are undertaken to ensure that medicines are safe and effective.  Success in preclinical studies means that a drug has been demonstrated to be probably effective, safe, sufficiently stable, and excreted safely from the body.  Then agreement from the regulators is needed before moving on to clinical research (testing in humans).

Clinical research, the second part of this webinar will be discussed in a future blog.

You can watch the entire webinar here.

DHM attenuates obesity-induced slow-twitch-fiber decrease via FLCN/FNIP1/AMPK pathway

Obesity is often associated with decreases in the proportion of skeletal muscle slow-twitch fibers and insulin sensitivity. Slow-twitch fibers are rich in mitochondria and utilize fatty acid oxidative phosphorylation for energy production. In their new study, Zhou et al. (2017) explore the role of the FLCN/FNIP1/AMPK signalling pathway in obesity-induced reductions in slow-twitch fibers and insulin sensitivity in skeletal muscle using high-fat-diet-induced (HFD) obese mice, ob/ob mutant mice, and palmitate-treated C2C12 myotubes. The authors also assess the effects of dihydromyricetin (DHM) on the obesity-induced decrease in slow-twitch fibers, and the molecular mechanisms responsible for this effect.

AMP-activated protein kinase (AMPK) plays a central role in skeletal muscle oxidative metabolism and fiber-type specification. AMPK activation in skeletal muscle induces expression of its downstream transcriptional regulator PGC-1α. FLCN, responsible for Birt-Hogg Dubé syndrome, interacts with the AMPK signalling pathway by binding to folliculin-interacting protein 1 (FNIP1) (Baba et al., 2006). Folliculin (FLCN) and FNIP1 may regulate skeletal muscle-fiber-type specification through the AMPK/PGC-1α pathway (Hasumi et al., 2012). Although the interaction between FLCN/FNIP1 and AMPK appears to play an important role in skeletal muscle adaptations, its involvement in the obesity-induced decrease in slow-twitch fibers and insulin resistance remains unclear.

Exercise is commonly prescribed for obesity and metabolic diseases, including insulin resistance and diabetes, since it increases AMPK activity, promoting slow-twitch fibers and increasing the use of fatty acids in skeletal muscle (Lantier et al., 2014). The authors have previously reported that the flavonoid DHM enhanced exercise performance (Zou et al., 2014), and improved skeletal muscle insulin resistance by autophagy induction via AMPK (Shi et al., 2015). However, the ability of DHM to increase the proportion of skeletal muscle slow-twitch fibers via the AMPK signalling pathway remains unclear.

In the HFD-fed and ob/ob mice the proportions of slow-twitch fibers, insulin sensitivity (detected by the markers of insulin sensitivity, insulin-stimulated Akt and insulin receptor substrate 1 (IRS-1) phosphorylation) and oxidative metabolism in skeletal muscle were decreased compared with control mice, and this effect was prevented by DHM treatment.

Increased non-esterified fatty acids (NEFA) levels are closely associated with insulin resistance in obesity and type 2 diabetes. In line with these results, the authors found that plasma NEFA levels were significantly increased in HFD-fed and ob/ob mice compared with controls and they negatively correlated with slow-twitch-fiber proportion.

To verify the results obtained in mice, in vitro experiments with C2C12 myotubes were performed. Palmitate, one of the most elevated plasma NEFA in obesity, was used to induce insulin resistance in C2C12 myotubes and shown to decrease expression of slow-fiber specification Myh7 protein, this was inhibited by DHM.

Western blot analysis show decreased phosphorylation of AMPK in both HFD-fed and ob/ob mice, and in palmitate-treated C2C12 myotubes. The similar trends in AMPK activity and changes in slow-twitch fibers and insulin resistance suggest that AMPK might be involved in these obesity-induced changes. FNIP1 and FLCN expression levels were significantly increased in skeletal muscle in HFD-fed and ob/ob mice, and in palmitate-treated C2C12 myotubes and negatively correlated with AMPK activity. These results implicated FNIP1/FLCN in obesity-induced AMPK inactivation, and the subsequent decreases in slow-twitch fibers and insulin sensitivity in skeletal muscle.

The role of the FLCN/FNIP1/AMPK signalling pathway in obesity-induced insulin resistance and the decrease in slow-twitch fibers was further clarified using over-expression and knock-down of FNIP1 and FLCN. Transfection of C2C12 myotubes with FNIP1 resulted in a corresponding increase in FLCN levels. AMPK phosphorylation levels increased following FLCN or FNIP1 knock-down, and decreased following their over-expressions. mRNA levels of the PGC-1α encoding gene were assessed and results showed that its expression was negatively related to FNIP1/FLCN expression, and consistent with AMPK activity.

DHM ameliorated the obesity-induced decrease in slow-twitch-fiber proportion, insulin sensitivity, and AMPK activity. However, it was necessary to verify if these effects of DHM were mediated by FNIP1 and FLCN. FNIP1 and FLCN expression levels were significantly decreased following DHM administration in HFD-fed and ob/ob mice, and in palmitate-induced C2C12 models. Also, the preventive effects of DHM on the palmitate-induced decrease in slow-twitch fibers, AMPK activation, p-Akt and p-IRS-1 expression, were blocked by FLCN over-expression. These results demonstrated that the effects of DHM were mediated by the FNIP1/FLCN/AMPK signalling pathway.


Obtained from Zhou et al. (2017)

Yan et al. recently reported FLCN/AMPK as a novel molecular pathway involved in regulating mitochondrial function and browning of white adipocytes, this was discussed on a previous blog. Here, the results of the study demonstrate that the FNIP1/FLCN complex might play an important role in AMPK/PGC-1α signalling in the obesity-induced decreases in slow-twitch-fibers and insulin sensitivity. Furthermore, DHM acts as a potential exercise mimetic by attenuating these obesity-induced effects via the FNIP1/FLCN/AMPK signalling pathway. These results provide new insights into the FNIP1/FLCN/AMPK signalling pathway, key in BHD research, and novel mechanisms and potential targets for treatments of insulin resistance and type 2 diabetes.

  • Zhou Q, Gu Y, Lang H, Wang X, Chen K, Gong X, Zhou M, Ran L, Zhu J, & Mi M (2017). Dihydromyricetin prevents obesity-induced slow-twitch-fiber reduction partially via FLCN/FNIP1/AMPK pathway. Biochimica et biophysica acta PMID: 28363698