BHD causes pneumothoraces during childhood in rare cases

Birt-Hogg-Dubé Syndrome is caused by inactivating mutations in the FLCN gene, characterised by skin lesions on the face and upper body; lung cysts and predisposition to pneumothorax; and kidney cancer. Although symptoms typically appear in the third and fourth decade of life, Johannesma et al. (2014) suggest that pneumothorax in patients under the age of 18 might be underdiagnosed.

In two large cohort studies, 24% were found to have had one or more episodes of pneumothorax, with a predicted lifetime risk of29% of developing a pneumothorax (Houweling et al., 2011, Toro et al., 2007). The age range of patients’ initial pneumothorax episodes was 18 – 74 years, with a median age of 36 – 38 years, suggesting that the age of onset of this symptom can vary widely (Houweling et al., 2011, Toro et al., 2007). However, the majority of families in both of these studies were recruited via dermatology clinics, meaning that there might be some ascertainment bias in these cohorts.

Johannesma et al. report the cases of two unrelated Dutch BHD patients who suffered their first pneumothorax at the age of 14. Both patients were given chest CT scans, as they suffered recurrent episodes as teenagers, and revealed the presence of lung cysts in the lower lobes of the lungs. Genetic testing confirmed each had a pathogenic FLCN mutation, confirming a diagnosis of BHD.

This report follows three other reports of BHD patients suffering pneumothoraces as teenagers: a 7 year old French boy (Bessis et al., 2006); a 16 year old Japanese girl (Gunji et al., 2007); and an 18 year old Dutch girl (Houweling et al., 2011). Thus, although pneumothorax during childhood is rare in BHD patients, both clinicians and BHD patients with children should be aware that it is a possibility.

It is possible that BHD is underdiagnosed in cases of paediatric spontaneous pneumothorax as BHD is normally considered to be an adult-onset disease. Additionally, CT scans – which would help diagnose BHD if lung cysts were found – are not normally administered to paediatric patients, unless deemed to be absolutely necessary, due to the low dose of radiation patients are exposed to during the scan (Balfour-Lynn et al., 2005).

Given that both patients reported by Johannesma et al. were found to have lung cysts as teenagers but neither had yet developed skin or kidney symptoms, it is possible that lung cysts are the first symptom of BHD to develop, but are not normally found unless the patient receives a chest CT for some other reason. Indeed, pneumothorax risk is strongly correlated with increased number and size of lung cysts (Toro et al., 2007), and it has subsequently been hypothesised that lung collapses are caused by air-filled cysts rupturing and releasing air into the chest cavity (Furuya and Nakatani, 2013). Thus it is likely that all the reported paediatric patients also developed lung cysts in childhood.

Four of the five paediatric BHD patients did have recurrent episodes, with one patient having four recurrent lung collapses before the age of 18 (Johannesma et al. 2014), and another having six episodes between the ages of 16 and 38 (Gunji et al., 2007). Given that the average number of pneumothoraces per patient was found to be two in the cohort studied by Toro et al. this does suggest that BHD patients who have their first pneumothorax in childhood might be at higher risk of recurrent episodes than other BHD patients.

Thus, although BHD patients suffer pneumothoraces during childhood only in rare cases, as childhood pneumothorax is itself rare, only affecting 4 in 100,000 boys and 1 in 100,000 girls, Johannesma et al. recommend that a thorough family history, a low dose CT scan, and genetic testing should be considered for paediatric patients who experience repeated episodes of spontaneous pneumothorax.

 

  • Balfour-Lynn IM, Abrahamson E, Cohen G, Hartley J, King S, Parikh D, Spencer D, Thomson AH, Urquhart D, & Paediatric Pleural Diseases Subcommittee of the BTS Standards of Care Committee (2005). BTS guidelines for the management of pleural infection in children. Thorax, 60, i1-21 PMID: 15681514
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  • Houweling AC, Gijezen LM, Jonker MA, van Doorn MB, Oldenburg RA, van Spaendonck-Zwarts KY, Leter EM, van Os TA, van Grieken NC, Jaspars EH, de Jong MM, Bongers EM, Johannesma PC, Postmus PE, van Moorselaar RJ, van Waesberghe JH, Starink TM, van Steensel MA, Gille JJ, & Menko FH (2011). Renal cancer and pneumothorax risk in Birt-Hogg-Dubé syndrome; an analysis of 115 FLCN mutation carriers from 35 BHD families. British journal of cancer, 105 (12), 1912-9 PMID: 22146830
  • Johannesma PC, van den Borne BE, Gille JJ, Nagelkerke AF, van Waesberghe JT, Paul MA, van Moorselaar RJ, Menko FH, & Postmus PE (2014). Spontaneous pneumothorax as indicator for Birt-Hogg-Dubé syndrome in paediatric patients. BMC pediatrics, 14 PMID: 24994497
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 www.bhdsyndrome.org – the primary online resource for anyone interested in BHD Syndrome.

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Intratumoral peripheral small papillary tufts might be a diagnostic clue of BHD

Contrary to other hereditary kidney cancer conditions, BHD-associated renal tumours are known to be histologically diverse and discordant within families, meaning that members of the same family do not necessarily get the same type of kidney tumour (Pavlovich et al., 2005). Kuroda et al. (2014) recently reported on the histology of tumours from six BHD patients, and found that, despite being histologically diverse, all had intratumoral peripheral small papillary tufts – clusters of papillary cells – suggesting that these microscopic lesions might be a hallmark of BHD.

The authors selected BHD kidney tumours from six patients who were clinically diagnosed with BHD at Kochi Red Cross Hospital in Japan, between January 2010 and October 2013. These six cases correspond to five families and three of these cases have been previously reported (Furuya et al., 2012, Nagashima et al., 2012). Germline DNA samples were available for genetic analysis for five cases, and a pathogenic  FLCN mutation was found in all five. No sample was available for the sixth patient, so a genetic diagnosis could not be made for this patient, although she has been clinically diagnosed with BHD.

The series consisted of three men and three women, and the average age at diagnosis was 60. One patient had a history of spontaneous pneumothorax, three patients had skin lesions, and all six had lung cysts. All patients are alive without disease following treatment, and show no signs of recurrence or disease in a follow up period ranging between 10 and 46 months.

Five patients had multi-focal kidney tumours, and two had bilateral kidney tumours. Only one patient had a solitary tumour, which may be because she was the youngest patient in this cohort at 46 years old.  The tumours analysed in this study consisted of three hybrid oncocytic/chromophobe tumours; a tumour of unclassified histology, but with features resembling hybrid chromophobe/ clear cell histology; a collision tumour consisting of chromophobe, clear cell and papillary primary tumours; a chromophobe tumour; and a clear cell tumour. This wide range of tumour histologies in such a small sample series supports previous observations that BHD renal tumours are highly variable.

However, small papillary tufts were found in all samples. These papillary tufts were present mainly at the interface between the tumour and normal kidney tissue, or in the case of the collision tumour, at the interface between the chromophobe and clear cell tumours. Given their histology and their presence at the periphery of tumours, the authors termed these microscopic lesions intratumoral peripheral small papillary tufts (ITPSPTs). The authors hypothesise that these microscopic lesions could be precursor lesions that subsequently develop into tumours.

Although ITPSPTs have not been previously reported, similar lesions were been reported in patient with acquired renal cystic disease (Cheuk et al., 2002). Although cytogenetic analysis was performed on these papillary tufts, germline or tumour DNA sequencing for FLCN mutations was not performed. Thus it is possible this patient was an undiagnosed BHD patient, as they had multiple kidney cysts and a tumour with both clear cell and papillary histology.

As ITPSPTs were found in all tumours in their cohort, Kuroda et al. suggest that the presence of ITPSPTs might be a hallmark of BHD tumours, and therefore a diagnostic clue. Analysing ITPSPTs in a larger series of BHD renal tumours and control non-BHD kidney tumours, is required to confirm that ITPSPTs are specific to BHD tumours. They also suggest that patients who present with collision tumours or multiple tumours of different histologies may have BHD, and that these patients should be referred for genetic testing.

 

  • Cheuk W, Lo ES, Chan AK, & Chan JK (2002). Atypical epithelial proliferations in acquired renal cystic disease harbor cytogenetic aberrations. Human pathology, 33 (7), 761-5 PMID: 12196929
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  • Nagashima Y, Furuya M, Gotohda H, Takagi S, Hes O, Michal M, Grossmann P, Tanaka R, Nakatani Y, & Kuroda N (2012). FLCN gene-mutated renal cell neoplasms: mother and daughter cases with a novel germline mutation. International journal of urology : official journal of the Japanese Urological Association, 19 (5), 468-70 PMID: 22211584
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www.bhdsyndrome.org – the primary online resource for anyone interested in BHD Syndrome.

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FLCN is important for cardiomyocyte development

Cardiac hypertrophy is an adaptive response that occurs following increased stress on the heart wall, and can be caused by strenuous exercise, hypertension, heart attack or heart valve disease. In some cases, this can lead to heart failure.

Although the biological mechanism underlying cardiac hypertrophy is not fully elucidated, dysregulation of the AMPK-mTOR signalling pathway is known to play a role (Maillet et al., 2013). Hasumi et al. (2014) decided to investigate the role of FLCN in cardiac hypertrophy, given FLCN’s role in AMPK and mTOR signalling, by generating mice with both copies of FLCN specifically deleted in heart and skeletal muscle using the CKM-Cre driver mouse strain.

Mice lacking FLCN in heart and skeletal muscle had cardiac hypertrophy as shown by enlarged hearts, severe cardiac dysfunction and reduced lifespan. In vitro data from mouse embryonic fibroblasts (MEFs), showed that loss of FLCN led to increased mTOR signalling, increased protein synthesis and decreased autophagy, suggesting that dysregulated mTOR signalling may be responsible for the heart pathology in the FLCN knockout mice. In support of this hypothesis, Rapamycin treatment significantly reduced heart size and improved cardiac function.

However, mice lacking both the FLCN and the PCG1a gene showed no cardiac hypertrophy, indicating that the phenotype was mediated entirely through PCG1a. Increased PGC1a expression lead to increased mitochondrial respiration, with increased the amount of intracellular ATP, which  inhibited AMPK activity, and ultimately led to mTOR dysregulation.

The results of this study correspond with those of several others. Goncharova et al. found that loss of FLCN in alveolar type II cells lead to a decrease in E-cadherin expression, reduced LKB1 signalling, and a consequent reduction in AMPK activity. Interestingly, mice lacking functional LKB1 also develop cardiac hypertrophy (Ikeda et al., 2009). Furthermore, Kumasaka et al. recently suggested that heterozygous loss of FLCN in the lung makes alveoli weaker and susceptible to mechanical stress during breathing, which may cause cysts to develop. Taken together, is it possible that reduced E‑cadherin expression and subsequent reduced AMPK activity via reduced LKB1 activity caused by FLCN depletion make cardiac and lung tissues vulnerable to mechanical stress, thus causing cardiac hypertrophy and lung cysts to develop.

Recently Yan et al. also noted increased PCG1a expression, increased mitochondrial mass, and increased ATP production in FLCN-null cells. Yan et al., however, found constitutive AMPK activity to be upstream of PCG1a hyperactivity, rather than downstream. Furthermore, conversely to the data reported here by Hasumi et al., which suggest that FLCN inhibits mTOR signalling, last year two studies reported that FLCN activates mTOR signalling at the lysosome following amino acid restimulation.

The role of FLCN in AMPK-mTOR signalling has been difficult to define, with a number of contradicting reports. While some of these differences are likely to be due to cell specific effects, due to the role of AMPK, mTOR and FLCN in energy sensing, a concerted effort to investigate different culture conditions on FLCN-null cells may also shed light on this issue. Furthermore, systematic analysis of sub-cellular FLCN localisation and which proteins FLCN interacts with in different cell types, in different sub-cellular locations and under different culture conditions will also be valuable to delineate this pathway.

Cardiac hypertrophy is not a symptom of BHD, and the mice studied by Hasumi et al. had both copies of FLCN deleted, whereas BHD patients are heterozygous for FLCN inactivation. Thus BHD patients should not be concerned about developing cardiac hypertrophy. However, it would be interesting to determine the proportion of cardiac hypertrophy patients with sporadic FLCN inactivation to assess the clinical significance of this finding.

 

  • Goncharova EA, Goncharov DA, James ML, Atochina-Vasserman EN, Stepanova V, Hong SB, Li H, Gonzales L, Baba M, Linehan WM, Gow AJ, Margulies S, Guttentag S, Schmidt LS, & Krymskaya VP (2014). Folliculin controls lung alveolar enlargement and epithelial cell survival through E-cadherin, LKB1, and AMPK. Cell reports, 7 (2), 412-23 PMID: 24726356
  • Hasumi Y, Baba M, Hasumi H, Huang Y, Lang M, Reindorf R, Oh HB, Sciarretta S, Nagashima K, Haines DC, Schneider MD, Adelstein RS, Schmidt LS, Sadoshima J, & Marston Linehan W (2014). Folliculin (Flcn) inactivation leads to murine cardiac hypertrophy through mTORC1 deregulation. Human molecular genetics PMID: 24908670
  • Ikeda Y, Sato K, Pimentel DR, Sam F, Shaw RJ, Dyck JR, & Walsh K (2009). Cardiac-specific deletion of LKB1 leads to hypertrophy and dysfunction. The Journal of biological chemistry, 284 (51), 35839-49 PMID: 19828446
  • Kumasaka T, Hayashi T, Mitani K, Kataoka H, Kikkawa M, Tobino K, Kobayashi E, Gunji Y, Kunogi M, Kurihara M, & Seyama K (2014). Characterization of pulmonary cysts in Birt-Hogg-Dubé syndrome: histopathological and morphometric analysis of 229 pulmonary cysts from 50 unrelated patients. Histopathology, 65 (1), 100-10 PMID: 24393238
  • Maillet M, van Berlo JH, & Molkentin JD (2013). Molecular basis of physiological heart growth: fundamental concepts and new players. Nature reviews. Molecular cell biology, 14 (1), 38-48 PMID: 23258295
  • Yan M, Gingras MC, Dunlop EA, Nouët Y, Dupuy F, Jalali Z, Possik E, Coull BJ, Kharitidi D, Dydensborg AB, Faubert B, Kamps M, Sabourin S, Preston RS, Davies DM, Roughead T, Chotard L, van Steensel MA, Jones R, Tee AR, & Pause A (2014). The tumor suppressor folliculin regulates AMPK-dependent metabolic transformation. The Journal of clinical investigation, 124 (6), 2640-50 PMID: 24762438

www.bhdsyndrome.org – the primary online resource for anyone interested in BHD Syndrome.

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Public awareness of cancer and federal funding of research in the US is affected by how cancer is reported in the American media

The link between having a good understanding of your health and of disease (health literacy) and health outcomes is well documented (Berkman et al., 2011). However, access to accurate and unbiased information is required for the public to be able to accurately assess their health risks. A recent study by Jensen et al. (2014) sought to investigate the link between media coverage of cancer and public perceptions of how common certain cancers were in the US. They further investigated how media coverage of cancer affected federal funding of cancer research.

To do this, researchers recruited 400 members of the public in Indiana, and asked them to rank the top 15 most common cancers in the US from most to least prevalent. Respondents were also asked about how often they read the news, and what proportion was from print, television or online.

While participants ranked most cancers fairly accurately, the prevalence of blood and pancreatic cancer were significantly overestimated, while those of male reproductive, lymphatic and bladder cancers were underestimated. This difference in perception mirrored differences in news coverage of these cancers, and participants who were greater news consumers were more likely to incorrectly rank the list of cancers.

A similar pattern was seen when comparing news coverage with federal funding awarded to the National Cancer Institute in 2011. Breast, blood and brain cancers receive disproportionately high press coverage, and each received more the double the amount of funding than expected if funds were awarded proportionally to incidence rates. Conversely, bladder, kidney, thyroid and stomach cancers receive a disproportionately low amount of press coverage and received between 10-fold and 3-fold less funding than expected by incidence rate.

The most negatively affected cancer was bladder cancer, which is the 6th most common cancer in the US, was ranked least common by the public, receives ten-fold less funding than expected, and was the most under-reported cancer compared to its incidence rate. Furthermore, although the public accurately ranked kidney cancer as being the 9th most common malignancy in the US, it receives 3-fold reduction in federal funding than expected and is disproportionately under-reported in the press. This shows that the media can have a massive effect on both the public perception of cancer and on government funding.

The reasons for disproportional reporting on cancers are myriad. For example, some cancers are inherently more attention-grabbing than others. Comparing bladder and blood cancers, bladder cancers tend to affect older Caucasian males in their 60s and 70s, whereas blood cancers are more likely to affect children and young adults, and are often life-threatening (Jemal et al., 2010, Madeb and Messing, 2004). Simplification of scientific findings for clarity may also be a factor – for example “smoking causes lung, mouth, throat and kidney cancer” becomes “smoking causes lung cancer.”

Certain patient advocacy groups – for example, breast cancer charities – have historically been much better than others at raising awareness and engaging the press, meaning that these cancers will be more likely to be covered in the news. Additionally, an initial small funding distortion in favour of a particular cancer could become amplified by a virtuous circle: increased funding means an increased rate of discovery, which leads to more news coverage and increased public awareness, which leads to further financial investment as this area of research is known to be flourishing.

This may explain why, in 2011, breast cancer alone received a quarter of the total federal funding spent on all of the top 15 cancers put together, and more than double that spent on male reproductive cancers, despite male reproductive cancers being the most common cancer in the US and breast cancer being the second most common.

Although this study was US-specific, it is likely that this effect occurs in other countries too. In a survey of 67 science journalists from the UK, US, Canada, Asia and Europe, 50% stated that public interest was their primary motivation in choosing which cancer story to write, making it the most influential factor in topic choice (Aggarwal et al., 2014).

The media clearly have a lot of power in affecting both the public perception of cancer and federal funding of research. Currently public awareness and/or federal funding of bladder, kidney, thyroid and stomach cancers is low due to disproportionately low coverage in the media. The media have a responsibility to re-dress this balance, as it will increase the amount of funding available for research, and it will make the public more aware of these cancers and their symptoms. This would hopefully lead to more cases being prevented, and improved diagnosis and treatment rates for those cases that are unavoidable.

 

  • Aggarwal A, Batura R, & Sullivan R (2014). The media and cancer: education or entertainment? An ethnographic study of European cancer journalists. Ecancermedicalscience, 8 PMID: 24834118
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  • Madeb R, & Messing EM (2004). Gender, racial and age differences in bladder cancer incidence and mortality. Urologic oncology, 22 (2), 86-92 PMID: 15082003

www.bhdsyndrome.org – the primary online resource for anyone interested in BHD Syndrome.

pf button Public awareness of cancer and federal funding of research in the US is affected by how cancer is reported in the American media

FNIP1 is required for iNKT cell maturation

In 2012, two independent studies found that the FLCN-interacting protein FNIP1 is required for proper β-cell maturation, and were the first studies to suggest a role for FLCN and its partners in the immune system. Following their 2012 discovery that FNIP1 is necessary for β-cell development (Park et al., 2012), the same group decided to further elucidate the role of FNIP1 in the immune system.  Park et al., (2014) analysed the phenotype of multiple immune cells in FNIP1-null mice, and found that FNIP1 is also required for the correct development and maturation of a specific subset of T-cells called invariant Natural Killer T cells (iNKT cells) (reviewed by Cianferoni, 2013).

iNKT cells are a subset of T cells that express an invariant T cell receptor and can recognise lipid bound antigens, such as glycolipids, on the surface of other cells. When activated, these cells produce cytokines to illicit an immune response. iNKT cells can recognise bacterial infections, viruses, and tumours. Their development is highly regulated, alternating between TCR rearrangement, proliferative and maturation phases, starting at stage 0, where DP thymocytes become committed to the iNKT cell lineage, and fully mature stage 3 iNKT cells.

Park et al. found that iNKT cells became arrested during stage 1 and 2, with few mature stage 3 cells found in FNIP1-null mice. Additionally, PLZF – which regulates iNKT cell development – was overexpressed in FNIP1-null iNKT cells. Bone marrow transplants into nude mice using a 1:1 mixture of wild-type and FNIP1-null cells showed that β-cells and iNKT cells were derived from wild-type mice only, whereas CD4 and CD8 thymocytes were derived from both types of donor cell. This suggests that FNIP1-null iNKT cells arrest during development due to cell autonomous effects, rather than due to a defective environment.

BrdU pulse experiments showed that FNIP1-null cells over-proliferated and subsequently died in early stage 3, as shown by an increase in Caspase 3-positive cells. Furthermore, FNIP1-null iNKT cells showed reduced mitochondrial mass in stage 1, decreased levels of ATP, larger cell size and increased mTOR signalling. Together, this suggests that dysregulated mTOR signalling leads to higher energy consumption, meaning that cells do not have the required energy reserves for proliferation and maturation, and die between stage 2 and 3.

However, mTOR dysregulation is not fully responsible for this phenotype, as in vivo treatment of pups, beginning in utero, did not rescue the iNKT cell phenotype. AMPK, which FNIP1 binds, activates the proautophagy gene Vps34, which is also required for iNKT cell development in mice (Parekh et al., 2013). Thus, concomitant loss of autophagy and mTOR dysregulation might explain the loss of metabolic homeostasis in FNIP1-null iNKT cells.

These results are similar to those reported in FNIP1-null β-cells, which were reported to be due a loss of metabolic homeostasis (Park et al., 2012) and increased apoptosis (Baba et al., 2012). They also correspond well with recent data showing that loss of FLCN leads to metabolic transformation in cells. However, it is currently unclear why loss of FNIP1 leads to such specific phenotypes in the immune system, only affecting a subset of lineages. It would be interesting to determine whether FLCN and its other binding proteins – FNIP2, PKP4, RPT4 and the Rag proteins – control the development of other immune cell lineages. If so, this would provide compelling evidence to support previous observations that FLCN function changes in different cell types (Hudon et al., 2010).

 

  • Baba M, Keller JR, Sun HW, Resch W, Kuchen S, Suh HC, Hasumi H, Hasumi Y, Kieffer-Kwon KR, Gonzalez CG, Hughes RM, Klein ME, Oh HF, Bible P, Southon E, Tessarollo L, Schmidt LS, Linehan WM, & Casellas R (2012). The folliculin-FNIP1 pathway deleted in human Birt-Hogg-Dubé syndrome is required for murine B-cell development. Blood, 120 (6), 1254-61 PMID: 22709692
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www.bhdsyndrome.org – the primary online resource for anyone interested in BHD Syndrome.

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Topical Rapamycin might not be an effective treatment for fibrofolliculomas

Last week, the results of a clinical trial testing the effectiveness of topical rapamycin as a treatment for BHD were published in PLOS ONE (Gijezen et al., 2014). The study was performed by Professor Dr Maurice van Steensel’s team at the Maastricht University Medical Centre.

19 patients were enrolled in the trial, and were given two topical treatments – a 0.1% Sirolimus oral solution, and a placebo treatment containing just the solvent – and asked to use each treatment on different sides of their face every day for six months.  The study was double-blind, randomised, facial left-right controlled, so patients and doctors did not know which side of their face was being treated with the drug. The cosmetic appearance, size and number of patients’ fibrofolliculomas were then assessed at 3 months and 6 months.

Cosmetic appearance was reported by both doctors and patients. Doctors reported no improvement in 17 patients, and improvements on both facial sides in 2 patients. 9 patients reported improvement with rapamycin treatment, 7 reported no improvement at all, and 5 patients reported improvement with placebo. Thus, rapamycin does not appear to improve the cosmetic appearance of fibrofolliculomas.

A reduction in the number of fibrofolliculomas was observed in 6 rapamycin treated facial sides, and 7 placebo sides. Additionally, at three months, difference in size of measured fibrofolliculomas was not statistically significant.  Thus rapamycin did not significantly reduce the number of fibrofolliculomas or halt their growth in this trial.

13 patients reported one or more side effects on the rapamycin treated facial side, including burning, redness (erythema), dryness and itching, and one patient had to leave the trial after 3 months due to a tearing eye. However, 11 patients reported similar side effects with the placebo treatment, suggesting that many of the side effects were caused by the solvent the drug was dissolved in – which included ethanol – rather than the drug itself.

These results suggest that topical Rapamycin is not an effective treatment for fibrofolliculomas. Since this trial was started, two papers have been published showing that under certain conditions FLCN activates mTOR signalling. If this is the case in skin cells, rapamycin would not be predicted to treat fibrofolliculomas, which may explain the results of the trial.

On the other hand, FLCN has been seen to inhibit mTOR signalling in other studies, making rapamycin an appropriate treatment to test. This is a very small study, with only 19 patients completing the trial, meaning that any effects of rapamycin treatment would have to be quite large to conclusively prove any effect, and subtle or stratified effects would not be found. It is also possible that the dose of Sirolimus was not high enough to affect fibrofolliculomas.

Studies in TSC have shown that Sirolimus is better tolerated when dissolved in an emollient (Foster et al., 2012, Koenig et al., 2012). Thus it is possible that if the drug were delivered in this way, higher doses could be used, or treatment could be tolerated for longer due to fewer side effects. If taken at a higher dose, or for longer, it is possible that rapamycin may still prove to be an effective treatment to prevent fibrofolliculoma growth, or to improve the appearance of existing lesions.

  • Foster RS, Bint LJ, & Halbert AR (2012). Topical 0.1% rapamycin for angiofibromas in paediatric patients with tuberous sclerosis: a pilot study of four patients. The Australasian journal of dermatology, 53 (1), 52-6 PMID: 22309333
  • 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
  • Koenig MK, Hebert AA, Roberson J, Samuels J, Slopis J, Woerner A, & Northrup H (2012). Topical rapamycin therapy to alleviate the cutaneous manifestations of tuberous sclerosis complex: a double-blind, randomized, controlled trial to evaluate the safety and efficacy of topically applied rapamycin. Drugs in R&D, 12 (3), 121-6 PMID: 22934754

www.bhdsyndrome.org – the primary online resource for anyone interested in BHD Syndrome.

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The three Rs of animal research: replacement, refinement, reduction

Following on from last week’s blog discussing animal research, this week’s blog discusses the work of the National Centre for the Replacement, Refinement & Reduction of Animals in Research (NC3Rs). But before discussing how NC3Rs are reducing the numbers of animals in research, how many animals are currently used in research?

The Home Office statistics for 2011 show that, in the UK,  just over 3.7 million animals were used in research, 93% of which were mice, rats or fish. While this sounds a lot, to put this number into perspective, this means that for each member of the 63 million strong UK population, 0.06 of an animal was used in research in 2011. Furthermore, this means that, assuming an average life expectancy of 80 years, for each person in the UK, 4.7 animals will be used in research in their lifetime, 3.4 of which will be mice, and 1 of which will be a rat or a fish.

NC3Rs was set up in 2004 by the UK government to investigate the role of animals in research, and to provide a scientific lead in discussions surrounding this issue. Their mission is to use the principles of replacement, refinement and reduction to support scientific discovery, and address societal concerns about animal research. In 2013, NC3Rs awarded over £8.2 million to 3Rs projects investigating ways to reduce the numbers of animals used in research, or to improve the welfare of those currently used.

Replacement describes the use of an alternative method that avoids the use of protected animals. Exciting technological advancements in this area have yielded a number of alternatives to using animals in research: a lung-on-a-chip to replicate pulmonary oedema (Huh et al., 2012); growing mouse mini-livers from stem cells to use for drug screens (Huch et al., 2013); and the use of amoeba to determine the biological mechanism behind seizures and to screen for better epilepsy drugs (Chang et al., 2014). Together, these advances are estimated to avoid the use of tens of thousands of mice.

Refinement refers to improving scientific procedures in order to improve the welfare of the animals used in the experiment. For example, it takes less than 40 minutes to train a macaque to voluntarily give blood without being restrained. Not only is the procedure less stressful for the animal, but the experiment is not confounded by increased levels of stress hormones in the animal’s blood, thus simultaneously improving the animal’s wellbeing, and the accuracy of the experiment.

Reduction refers to the use of methods that allow researchers to collect a similar quantity and quality of data using fewer animals. This can be achieved by improved experimental designs and statistical analysis, or more sophisticated analysis techniques. For example, improved non-invasive scanning techniques mean a single animal can be analysed multiple times. However, using too few animals to obtain a conclusive result is as wasteful and unethical as using too many animals. Therefore experiments must be designed carefully beforehand to ensure that they do not need to be repeated.

By engaging with the public about animal research, NC3Rs, Understanding Animal Research and researchers hope to communicate the messages that animal research is necessary for medical progress, it is highly regulated, it is being constantly monitored to identify where improvements can be made, and that animal use will inevitably decrease as technology improves. This will hopefully increase public support for the use of animals in research where there is no viable alternative.

Animal research is, quite rightly, a complex and emotive issue. However, it is worth bearing the following in mind: as described above, fewer than 5 animals, most of which will be mice, rats or fish, are used in research during the lifetime of each person in the UK. While not everyone will get seriously ill, more than 1 in 3 people in the UK get cancer and will need treatment, most people will have surgery in their lifetime, and nearly all will take paracetamol for a headache. None of these interventions would be possible, or as effective, without the use of animals in research.

 

Useful resources:

Understanding Animal Research Website

Government guidance on research and testing using animals

The National Centre for the Replacement, Refinement and Reduction of Animals in Research

 

  • Chang P, Walker MC, & Williams RS (2014). Seizure-induced reduction in PIP3 levels contributes to seizure-activity and is rescued by valproic acid. Neurobiology of disease, 62, 296-306 PMID: 24148856
  • Huch M, Dorrell C, Boj SF, van Es JH, Li VS, van de Wetering M, Sato T, Hamer K, Sasaki N, Finegold MJ, Haft A, Vries RG, Grompe M, & Clevers H (2013). In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration. Nature, 494 (7436), 247-50 PMID: 23354049
  • Huh D, Leslie DC, Matthews BD, Fraser JP, Jurek S, Hamilton GA, Thorneloe KS, McAlexander MA, & Ingber DE (2012). A human disease model of drug toxicity-induced pulmonary edema in a lung-on-a-chip microdevice. Science translational medicine, 4 (159) PMID: 23136042

www.bhdsyndrome.org – the primary online resource for anyone interested in BHD Syndrome.

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