Clinical trials for rare diseases – finding and keeping patients

International Clinical Trial day (May 20th) celebrates the medical advances as a result of clinical trials. Clinical trials are essential to ensure drug safety and efficacy, and the recent increase in the development of orphan drugs has led to an increase in rare disease clinical trials. The nature of rare diseases creates specific challenges for clinical trial design and patient recruitment.

A major challenge in any clinical trial is the recruitment of enough suitable patients – something made more difficult with a rare disease because there are so few patients meaning recruitment has to occur from a wide geographical area. Patient support groups are key in identifying and contacting these patients, and often know of equivalent groups in other countries. Increasingly they have patient registers that can contain information on patient location, genetics, health and sometimes biological sample data. Early contact with a patient support groups can also help identify clinical or research experts and current treatment centres (and their patients) that could assist with a trial. Additionally a dedicated trial website, suitably advertised, enables patients to learn more about the trial independently and enquire if interested.

A good trial design ensures valid and useful results however the “gold-standard” of a randomised, parallel group controlled trial may not be feasible with rare diseases due to low patient numbers and a lack of comparative interventions. Instead rare disease trials are more likely to have under 50 patients and be single arm, non-randomised, open label trials (Bell & Tudar Smith 2014). Details of the different approaches for designing an effective clinical trial design for rare disease can be found in a methodical review from Gagne et al., (2014).

Rare disease clinical trial logistics also require additional thought and a more patient-centric approach can ensure adequate recruitment and retention. Early consideration of acceptable physical burden, treatment durations and travel requirements, which support groups can often advise on, can speed up patient recruitment.

Many rare diseases are debilitating and/or affect children so patients will require an accompanying carer for visits – which increases travel and accommodation costs. Multiple trial centres, potentially across several countries, can help reduce the required travel and be favourable to patients. An example is the AKU Society developAKUre trial which has trial centres in the UK, France and Slovakia enabling more local participation. These countries were chosen based on patient support groups and registries, clinical and research expertise and in Slovakia a higher than average patient density. AKU patients from throughout Europe are also travelling to the UK to participate which requires additional planning and patient support such as translation services.

An alternative clinical trial strategy, which takes patient preference and potential difficulty travelling into account, is the provision of an in-home clinical service; the majority of simple clinical procedures (treatment administration, blood draws, health monitoring) are administered by a clinical nurse at home with travel to the designated clinical trial site only required for certain appointments. This significantly reduces the disruption to patients and their families in terms of time and can reduce travel and accommodation costs for the sponsor. In-home supported clinical trials have been found to have reduced withdrawal rates and the reduced burden on patients can encourage greater enrolment and completion of trials ahead of estimates (Norris et al., 2012).

Choosing to take part in a clinical trial is a personal decision and a patient has the right to withdraw at any time. By supporting rare disease patients from the start and throughout, making taking part in clinical trials as easy as possible, more effective treatments for a range of rare diseases can hopefully be approved for use. Information about ongoing trials can be found at clinicaltrials.gov. Participation in a clinical trials is not entirely risk free and should always be discussed with your doctor. You can find more information about the process of clinical trials here.

  • Bell SA, Tudur Smith C. A comparison of interventional clinical trials in rare versus non-rare diseases: an analysis of ClinicalTrials.gov. Orphanet J Rare Dis. 2014 Nov 26;9:170. PubMed PMID: 25427578.
  • Gagne JJ, Thompson L, O’Keefe K, Kesselheim AS. Innovative research methods for studying treatments for rare diseases: methodological review. BMJ. 2014 Nov 24;349:g6802. Review. PubMed PMID: 25422272.
  • Norris N, Pascale W, Tulipano D. In-home Clinical Services: Reducing patient burden and improving patient participation in studies. ACRP Clinical Research Articles. April 2012. http://www.symphonyclinicalresearch.com/Final%20ACRP-Publication.pdf

Thank you to Oliver Timmis at the AKU Society for providing insight into the developAKUre trial.

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A role for Matrix Metalloproteinases in BHD?

The BHD protein folliculin (FLCN) plays a role in numerous signalling pathways and cellular processes. Although mutations in FLCN are only firmly linked to the development of fibrofolliculomas, pulmonary cysts and renal tumours it is possible that disruption of these pathways also plays a role in other phenotypes. Recently Kapoor et al., (2015) reported three cases studies of women with BHD who presented with intracranial vascular pathologies. There are few other reports of vascular pathologies in BHD and further studies would be required to determine any causative link. Kapoor et al. proposed two hypotheses to link BHD to aneurysms and vascular malformations: aberrant HIF-1α signalling and increased matrix metalloproteinase 9 (MMP-9) activity.

MMP-9 is a proteolytic enzyme that cleaves several connective tissue proteins and has multiple roles in tissue extra-cellular matrix (ECM) remodelling, angiogenesis and cell migration. Regulation of MMP-9 is predominantly transcriptional; low background levels result from inhibition by SMAD7 (Kim et al., 2012, Yu et al., 2013). A significant reduction in SMAD7 expression, alongside other TGF-β signalling targets, was reported in FLCN-null cells (Hong et al., 2010), supporting a link between reduced FLCN and increased MMP-9.

MMPs are important for vascular wall matrix remodelling and increased activity is associated with potentially-pathogenic increased degradation of structural proteins in vascular walls (Maradni et al., 2013). Increased levels of MMP-9 have been seen in extra- and intra-cerebral, abdominal aortic and intracranial aneurysm walls (Pannu et al., 2006, Maradni et al., 2013). The expression of MMP9 is locally, rather than systemically, perturbed in such patients as healthy vascular tissue from aneurysm patients shows no increase in MMP-9 compared to healthy controls (Kim et al., 1997). Although an increase in MMP-9 has not been investigated in aneurysm wall samples from BHD patients, a role for FLCN in the control of MMP9 expression could support the potential for increased risk of intracranial vascular pathologies in BHD.

Aberrant MMP-9 activity has also been associated with a number of pulmonary disorders and previously been discussed in the context of BHD. MMP9 is expressed at low levels in healthy adult lung tissue but marked increases have been reported in cystic fibrosis (Sagel et al., 2005), asthma, IPF, and COPD (Atkinson & Senior, 2003) associated with airway and vascular remodelling. Increased MMP-9 expression is also seen in Lymphangioleiomyomatosis (LAM) patients associated with ECM breakdown and cystic lesions (McCormack, 2008).

In BHD patients increased MMP9 expression has been reported in alveolar epithelial cells, macrophages and neutrophils (Hayashi et al., 2010, Pimenta et al., 2012). However, these reports are not conclusive as there was a lack of comparison to healthy tissue (Hayashi et al., 2010) or no published genetic confirmation of BHD (Pimenta et al., 2012). Additionally increased production of MMP-9 as a result of increased pulmonary inflammation has been reported in mouse models of BHD (Goncharova et al., 2014). Contrary to these reports Nishii et al., (2013) found that MMP-9 levels were unchanged in their patient’s lung samples suggesting that further, large-scale analysis of expression is required in BHD patients.

The patient reported by Pimenta et al. (2012) was initially misdiagnosed with LAM and received standard treatment with the MMP-inhibitor doxycycline. Although this patient was subsequently suspected to have BHD she did show an improvement in pulmonary function during the treatment. Further research is required to understand the role, if any, that MMP-9 or other MMPs play in the pathology of BHD. If disruption of TGF-β signalling is resulting in a pathogenic activation of MMP-9 then it may be possible to utilise the well-established inhibitors such as doxycycline for the treatment of BHD pathologies.

 

  • Atkinson JJ, Senior RM (2003). Matrix metalloproteinase-9 in lung remodeling. Am J Respir Cell Mol Biol, Jan;28(1):12-24. Review. PubMed PMID: 12495928.
  • 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 Rep, Apr 24;7(2):412-23. PubMed PMID: 24726356.
  • Hayashi M, Takayanagi N, Ishiguro T, Sugita Y, Kawabata Y, Fukuda Y (2010). Birt-Hogg-Dubé syndrome with multiple cysts and recurrent pneumothorax: pathological findings. Intern Med, 49(19):2137-42. PubMed PMID: 20930443.
  • Hong SB, Oh H, Valera VA, Stull J, Ngo DT, Baba M, Merino MJ, Linehan WM, Schmidt LS. Tumor suppressor FLCN inhibits tumorigenesis of a FLCN-null renal cancer cell line and regulates expression of key molecules in TGF-beta signalling (2010). Mol Cancer, Jun 23;9:160. PubMed PMID: 20573232.
  • Kapoor R, Evins AI, Steitieh D, Bernardo A, Stieg PE (2015). Birt-Hogg-Dubé syndrome and intracranial vascular pathologies. Fam Cancer, May 8. [Epub ahead of print] PubMed PMID: 25952757.
  • Kim SC, Singh M, Huang J, Prestigiacomo CJ, Winfree CJ, Solomon RA, Connolly ES Jr (1997). Matrix metalloproteinase-9 in cerebral aneurysms. Neurosurgery, Sep;41(3):642-66. PubMed PMID: 9310982.
  • Kim S, Han J, Lee SK, Koo M, Cho DH, Bae SY, Choi MY, Kim JS, Kim JH, Choe JH, Yang JH, Nam SJ, Lee JE (2012). Smad7 acts as a negative regulator of the epidermal growth factor (EGF) signaling pathway in breast cancer cells. Cancer Lett, Jan 28;314(2):147-54. PubMed PMID: 22033246.
  • Maradni A, Khoshnevisan A, Mousavi SH, Emamirazavi SH, Noruzijavidan A (2013). Role of matrix metalloproteinases (MMPs) and MMP inhibitors on intracranial aneurysms: a review article. Med J Islam Repub Iran, Nov;27(4):249-54. Review. PubMed PMID: 24926188.
  • McCormack FX (2008). Lymphangioleiomyomatosis: a clinical update. Chest, Feb;133(2):507-16. Review. PubMed PMID: 18252917.
  • Nishii T, Tanabe M, Tanaka R, Matsuzawa T, Okudela K, Nozawa A, Nakatani Y, Furuya M (2013). Unique mutation, accelerated mTOR signaling and angiogenesis in the pulmonary cysts of Birt-Hogg-Dubé syndrome. Pathol Int, Jan;63(1):45-55. PubMed PMID: 23356225.
  • Pannu H, Kim DH, Guo D, King TM, Van Ginhoven G, Chin T, Chang K, Qi Y, Shete S, Milewicz DM (2006). The role of MMP-2 and MMP-9 polymorphisms in sporadic intracranial aneurysms. J Neurosurg, Sep;105(3):418-23. PubMed PMID: 16961137.
  • Pimenta SP, Baldi BG, Nascimento EC, Mauad T, Kairalla RA, Carvalho CR (2012). Birt-Hogg-Dubé syndrome: metalloproteinase activity and response to doxycycline. Clinics (Sao Paulo), Dec;67(12):1501-4. PubMed PMID: 23295609.
  • Sagel SD, Kapsner RK, Osberg I (2005). Induced sputum matrix metalloproteinase-9 correlates with lung function and airway inflammation in children with cystic fibrosis. Pediatr Pulmonol, Mar;39(3):224-32. PubMed PMID: 15635615.
  • Yu H, Zhao G, Li H, Liu X, Wang S (2012). Candesartan antagonizes pressure overload-evoked cardiac remodeling through Smad7 gene-dependent MMP-9 suppression. Gene, Apr 15;497(2):301-6. PubMed PMID: 22326534.
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Advances in immunotherapy for renal cell carcinoma

In recent years there has been renewed interest in immunotherapies to treat of metastatic renal cell carcinoma (mRCC). Immunotherapies increase the body’s natural finely regulated multiple-step anti-tumour immune response (Figure 1). The balance of stimulatory and inhibitory signals (reviewed in Chen & Mellman, 2013) are important in maintaining self-tolerance and modulating normal immune responses to minimising health tissue damage. However, tumours can develop mechanisms to evade this immune response.Immuno cycle

Figure 1: Immune Response Cycle. The major steps in the immune response cycle to cancer cells. Immunotherapies target a number of different steps (red). Modified from Chen & Mellman (2013) Immunity 39, 1-10.

The approved immunotherapies for RCC are the cytokines Interferon-alpha (IFN-α) and Interleukin-2 (IL-2) to stimulate T-cell activity. Unfortunately this can result in a variety of treatment related toxicities. The response rate to these drugs is relatively low but the responses are more likely to be durable. An alternative strategy is to inhibit specific immune checkpoints that normally act to limit excessive immune activity. The numerous stimulatory and inhibitory checkpoints in the immune system minimise the death of healthy, “self” cells in an immune response. The presence of inhibitory signals on tumour cells can therefore stop an immune response but the blockade of these inhibitory signals can increase the immune response and tumour cell death. A list of ongoing trials is available here.

One such inhibitor is Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) which when bound to B7-1 or B7-2 disrupts early T-cell activation and blocks cytokine production. Ipilumumab binds to CTLA-4 and blocks this inhibition resulting in increased production of auto-reactive T-cells and cytokines. While some patients have responded well to ipilumumab there are some severe adverse events (AE) associated with off-target effects of this drug (Yang et al., 2007).

A different checkpoint target is the Programmed cell Death-1 (PD-1)/PD-L1 inhibitory signalling pathway which reduces T-cell signalling and activation helping to maintain peripheral tolerance. Anti-PD-1 antibodies – nivolumab, pembrolizumab and pidilizumab – and the anti-PD-L1 antibody MPDL3280A are effective at blocking this signalling. PD-1 is expressed on a range of immune cells and PD-L1 can be expressed by tumour cells minimising the immune response. Nivolumab is the most frequently used anti-PD-1 in RCC and early phase trials have shown enhanced long-term survival (Lipson et al., 2013), with varied doses of nivolumab inducing comparable response rates with manageable toxicity profiles (Motzer et al., 2015).  There are also numerous ongoing trials investigating pembrolizumab which showed some response in melanoma patients but can have severe AE (Martin-Liberal et al., 2015).

An alternative, and potentially complimentary, approach would be the use of cancer vaccines to prime the immune system for response to cancer cells. The introduction of antigen presenting cells (APCs) loaded with tumour-derived RNA, such as AGS-003, induces a tumour-specific immune response (Amin et al., 2015). A synthetic vaccine, IMA901, consists of 10 Tumour Associated Peptides (TUMAPs) bound to HLA ligands for presentation to the immune system and as in early trials (Bedke & Stenzl, 2013).

The immune related AEs (irAE) seen in immunotherapy drug trials can differ from AE from other drug types and can be severe (Weber et al., 2012). Early diagnosis and effective toxicity management is required to ensure ongoing treatment is tolerable. Early trials suggest a correlation between response rate and frequency of irAEs and perhaps such irAEs could be worth enhanced survival. Further trials will also determine any risk of sequential toxicities that could impact on treatment regimens.

In addition to the development of therapies there is also a great need for research into relevant biomarkers; currently there are only indicative and not predictive biomarkers to determine which patients are most likely to response. Patient selection could also reduce the use of ineffective treatments which have a high irAE risk but no or minimal health benefit. Patient fitness and toxic burden also needs to be considered as frailer patients may not be able to tolerate AEs or to survive an initial decline before response begins.

As immunotherapies develop they may also be beneficial as adjuvant treatments following surgical resection. The responses to immunotherapies can be durable, continuing long after treatment has completed indicating the formation of tumour specific immune memory; they could therefore also be useful in reducing recurrence risk in high risk patients such as those with hereditary forms of RCC.

 

  • Amin A, Dudek AZ, Logan TF, Lance RS, Holzbeierlein JM, Knox JJ, Master VA, Pal SK, Miller WH Jr, Karsh LI, Tcherepanova IY, DeBenedette MA, Williams WL, Plessinger DC, Nicolette CA, Figlin RA (2015). Survival with AGS-003, an autologous dendritic cell-based immunotherapy, in combination with sunitinib in unfavorable risk patients with advanced renal cell carcinoma (RCC): Phase 2 study results. J Immunother Cancer Apr 21;3:14. PMID: 25901286.
  • Bedke J, Stenzl A (2013) IMA901: a peptide vaccine in renal cell carcinoma. Expert Opin Investig Drugs Oct;22(10):1329-36. Review. PMID: 23899354.
  • Chen DS, & Mellman I (2013). Oncology meets immunology: the cancer-immunity cycle. Immunity, 39 (1), 1-10 PMID: 23890059.
  • Lipson EJ, Sharfman WH, Drake CG, Wollner I, Taube JM, Anders RA, Xu H, Yao S, Pons A, Chen L, Pardoll DM, Brahmer JR, Topalian SL (2013). Durable cancer regression off-treatment and effective reinduction therapy with an anti-PD-1 antibody. Clin Cancer Res Jan 15;19(2):462-8. PMID: 23169436.
  • Martin-Liberal J, Kordbacheh T, Larkin J (2015). Safety of pembrolizumab for the treatment of melanoma. Expert Opin Drug Saf Apr 30:1-8. [Epub ahead of print] PMID: 25927979.
  • Motzer RJ, Rini BI, McDermott DF, Redman BG, Kuzel TM, Harrison MR, Vaishampayan UN, Drabkin HA, George S, Logan TF, Margolin KA, Plimack ER, Lambert AM, Waxman IM, Hammers HJ (2015). Nivolumab for Metastatic Renal Cell Carcinoma: Results of a Randomized Phase II Trial. J Clin Oncol May 1;33(13):1430-7. PMID: 25452452.
  • Weber JS, Kähler KC, Hauschild A (2012). Management of immune-related adverse events and kinetics of response with ipilimumab. J Clin Oncol Jul 20;30(21):2691-7. PMID: 22614989
  • Yang JC, Hughes M, Kammula U, Royal R, Sherry RM, Topalian SL, Suri KB, Levy C, Allen T, Mavroukakis S, Lowy I, White DE, Rosenberg SA (2007). Ipilimumab (anti-CTLA4 antibody) causes regression of metastatic renal cell cancer associated with enteritis and hypophysitis. J Immunother Nov-Dec;30(8):825-30. PMID: 18049334.
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Highlights from the 10th European International Kidney Cancer Symposium

Last weekend, the Kidney Cancer Association (KCA) held its 10th European International Kidney Cancer Symposium in Lyon. The meeting brought together kidney cancer specialists and researchers from around the world to discuss current and future treatments for Renal Cell Carcinoma (RCC), and other considerations relevant to treatment. Videos of the presentations are available here.

Professor Hein Van Poppel opened the conference with the de Mulder memorial lecture discussing total and partial nephrectomies. Partial nephrectomies are associated with increased kidney function (Scosyrev et al., 2014) but not increased survival (Van Poppel et al., 2011). Although partial nephrectomies require more surgical skill it is possible to remove tumours <7cm, with minimal additional healthy tissue removed, with a low local recurrence risk.

Dr James Brugarolas spoke about the relevance of cancer genes in developing and selecting targeted therapies. A new HIF2α targeting drug is on the horizon and could be useful in targeting multiple tumourigenic pathways. Dr Brugarolas also discussed the subclassification of RCC patients based on mutations in the VHL, BAP1, and PBRM1 genes to screen for therapies effective in specific cohorts.

Dr Nizar Tannir discussed the development of new the immunotherapies – cancer vaccines that aim to stimulate the innate cancer immune response, and checkpoint inhibitors that blockade the immunosuppressant nature of tumours enabling an effective immune response. Further optimisation is required, including patient selection techniques, but immunotherapy could offer long term survival to suitable patients.

In the interdisciplinary discussions Dr Alessandro Volpe and Professor Bernard Escudier commented on how biopsies can be useful in diagnosis and play a role in selecting targeted treatments, but are invasive and unnecessary if irrelevant to proposed treatment. Professor Antoni Alcaraz and Professor Manuela Schmidinger discussed the increased use of minimalistic approaches to kidney cancer following advances in technology but warned against using potentially less effective techniques based on socio-economic pressure or trends. Dr Axel Bex and Dr Viktor Grünwald agreed that the choice to do a cytoreductive nephrectomy was patient dependent based on potential impact on tumour burden and quality of life. Dr Julien Garnon and Professor Jean-Jacques Patard commented on the high success rate of cryoablation as an alternative to surgery for tumours under 4cm.

There are several challenges presented to clinicians as a result of RCC treatments. Dr Christiane Thallinger discussed the high incidence of dermatological adverse effects in patients and the potential effects on dose reduction or treatment withdrawal. Dr Stephane Ederhy stated that hypertension was a common adverse effect with Tyrosine Kinases Inhibitor (TKI) treatments and that patients should be assessed and treated in advance, and be monitored carefully throughout. Professor Romano Danesi warned of the effects of variable TKI blood concentration in patients and the effect of other drug interactions that can impact on toxicity and efficacy. Dr James Larkin discussed the toxicity risks of checkpoint inhibitors and the need to educate patients and carers to these adverse events to enable early diagnosis and treatment.

The first day ended with a session concerning novel treatment targets. Dr Laurence Albiges discussed treatments that target FGFR signalling and MET signalling in clear cell (cc) RCC and non-ccRCC. Dr Primo Lara further discussed T-cell checkpoints with a focus on the PD-1/PD-L1 pathway and commented on the unanswered immunotherapy questions in RCC. Dr Eric Raymond discussed a regain of aberrant mTOR/AKT activity in tumours that evade sunitinib supporting the use of everolimus as a second line treatment. Dr Hans Hammers commented on the development and ongoing trials for cancer vaccines noting that it may necessary to use such treatments in combination with checkpoint inhibitors.

The second day of the conference began with a look back on recent trials and their impact on treatment plans. Professor Martin Gore commented that first line treatments have not changed and that the “smarter” drugs in development have not been as effective as hoped. Dr Cora Sternburg discussed optimal sequential treatment with a TKI being more efficacious as a first line treatment than a mTOR inhibitor, but both TKIs and mTOR inhibitors are recommended as a second line treatment. Professor Bernard Escudier spoke about the use mTOR inhibitors as a first line treatment in poor risk and non-ccRCC patients and the need for further trials to assess mTOR inhibitors in the less common papillary and chromophobe RCC cases. Dr Camillo Porta discussed the need for more third line treatment guidance and the need for more treatments in the future as patient survival increases.

This year’s Schonfeld Lecture was given by Professor Sylvie Négrier and was focused on the role of immunotherapy in metastatic RCC. Although immunotherapies have a relatively low response rate they are more likely to be durable. Future treatments might be made more effective by using knowledge of tumour specific antigens or neo-antigens. Immunotherapy treatments require optimisation especially with regards to combination therapies and adverse events.

The final session of the conference covered open questions in RCC treatment. Professor Alain Ravaud discussed the use of surgery to treat lung and liver metastases and ablation techniques when surgery was not an option. Dr Ronan Tanguy then discussed the use of radiotherapy alongside standard to treatments for liver, lung, brain and spinal metastases that are not suitable for surgery. Professor Tim Eisen gave his predictions for immunotherapy treatments by 2020 based on current trials, and discussed the need to consider patient fitness and symptoms in deciding on treatments with higher risk factors. Professor Tom Powles ended the conference by discussing the use of different imaging techniques to detect early metastases and monitor treatment responses.

The conference was a wonderful opportunity to hear from world leaders in kidney cancer treatment and hear about on ongoing trials and upcoming therapies. The treatment of hereditary renal cancers such as BHD can vary from sporadic renal cancer as the risk of further tumour development makes it more important to retain optimum kidney function through partial nephrectomies. Further biomarker classification may also identify treatments that are more effective with a specific histology such as the chromophobe or hybrid tumours most often seen in BHD.

 

  • Scosyrev E, Messing EM, Sylvester R, Campbell S, Van Poppel H. Renal function after nephron-sparing surgery versus radical nephrectomy: results from EORTC randomized trial 30904. Eur Urol. 2014 Feb;65(2):372-7. PubMed PMID: 23850254.
  • Van Poppel H, Da Pozzo L, Albrecht W, Matveev V, Bono A, Borkowski A, Colombel M, Klotz L, Skinner E, Keane T, Marreaud S, Collette S, Sylvester R. A prospective, randomised EORTC intergroup phase 3 study comparing the oncologic outcome of elective nephron-sparing surgery and radical nephrectomy for low-stage renal cell carcinoma. Eur Urol. 2011 Apr;59(4):543-52. PubMed PMID: 21186077.

 

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FindZebra: a specialised search engine for rare diseases

When you hear hoof beats think of horses, not zebras

Dr Theodore Woodward

 

Every doctor is taught to think of the common causes of a problem before contemplating the rare ones. This works for the majority of patients but occasionally the cause is something unusual – a “zebra” – and isn’t so easy to find. For rare disease patients a specific diagnosis can be elusive with 46% receiving at least one incorrect diagnosis and 20% waiting over five years for a final diagnosis (Rare Disease UK, 2010).

Medical experts, and non-experts, use the internet for health related searches. The ability to mine the increasing quantity of available information can help clinicians to diagnose difficult cases. It is also common for patients to research their symptoms to try and find a diagnosis. However the standard search engines are not optimised to find results relevant to rare diseases. Search engines generally rank results based on popularity and can return irrelevant and untrustworthy results.

FindZebra is a specialist search engine designed specifically to help doctors diagnose rare diseases (Dragusin et al., 2013). FindZebra only mines for relevant information from reputable rare disease sources – OMIM, Orphanet, GARD, NORD and other smaller rare disease databases – to limit irrelevant results and reduce the time required for assessment. A search can be based on multiple phenotypes and reference points, and FindZebra will return near match results as well as exact matches to account for missing or non-specific phenotypes. A recent addition to FindZebra is a separate list of genes associated with the search term. This provides rapid access to relevant genes for future genetic testing or alternatively allows for the identification of diseases associated with mutation in a particular gene.

Dragusin et al. (2013) compared the ability of FindZebra and Google to find disease-relevant information based on the symptoms of 56 difficult diagnostic cases. Within the top 20 results FindZebra produced a relevant result in 68% of cases, compared to only 32% when using Google. Limiting Google to the same datasets used by FindZebra produced relevant information in only 11% of cases demonstrating the unsuitability of the Google ranking system for this this kind of query. FindZebra is also significantly better with long search queries, such as complex medical histories, than Google which is optimised for short queries.

A Google search for individual BHD symptoms does not produce any immediately relevant results on the first page. The exception is a search for “fibrofolliculomas” but as a highly BHD-specific term this is unlikely to be a common differential diagnostic search term. Combinations of BHD phenotypes yield more prominant relevant results. In comparison a search on FindZebra for “spontaneous pneumothorax” listed BHD in the top results, and has FLCN as the most relevant gene. On FindZebra, as with Google, a search for “renal cell carcinoma” is not specific enough to identify BHD from other hereditary RCC diseases such as VHL or TSC. However the addition of “hybrid” or “multifocal” to the search puts BHD and FLCN in the top results. Again combinations of phenotypes result in BHD being a top result.

One of the biggest differences between the results produced by Google and FindZebra is the ease of reading and assessment. A Google search result involves visiting new pages and sometimes finding the relevant information can be difficult. The FindZebra system shows the detailed result, with the disease in the title, alongside the initial search results and highlights relevant information making it easier to quickly scan for relevance and compatibility.

FindZebra was launched in 2013 and produced over 1,000,000 diagnostic hypotheses for 30,000 unique visitors in the first five weeks (Dragusin et al., 2013b) although no data has been published yet regarding the accuracy of these hypotheses. As more clinicians become aware of the benefits of using FindZebra undoubtedly its user group will grow. Such a tailored search engine has great potential to aid in the rapid diagnosis of numerous rare disease patients.

FindZebra is undoubtedly one of the fastest and most reliable ways to link crucial symptoms to causal genes… [and] has rapidly become an integrated part of our diagnostic set up for rare diseases.

Finn Cilius Nielsen, Center for Genomic Medicine, University of Copenhagen

 

  • Dragusin R, Petcu P, Lioma C, Larsen B, Jørgensen HL, Cox IJ, Hansen LK, Ingwersen P, Winther O (2013a). FindZebra: a search engine for rare diseases. Int J Med Inform. Jun;82(6):528-38 PubMed PMID: 23462700.
  • Dragusin R, Petcu P, Lioma C, Larsen B, Jørgensen HL, Cox IJ, Hansen LK, Ingwersen P, Winther O (2013b). Specialized tools are needed when searching the web for rare disease diagnoses. Rare Diseases, May 16;1:e25001. PubMed PMID: 25002998.
  • Rare Disease UK (2010). Experiences of Rare Disease: An Insight from Patients and Families. http://www.raredisease.org.uk/experiences-of-rare-diseases.htm
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Rhabdomyomas: an additional BHD hamartoma phenotype?

Hamartomas are benign, focal malformations formed by an excess of normal tissue growing in a disorganised fashion. Several hamartoma syndromes have been linked to aberrant mTOR signalling including BHD and Tuberous Sclerosis Complex (TSC). In addition to the predisposition of BHD patients to develop hair follicle hamartomas or fibrofolliculomas (Birt et al., 1977), Fuyura et al., (2012) propose that the pulmonary cysts in BHD patients are hamartoma-like cystic alveolar formations. The benign nature of these BHD growth phenotypes, in comparison to the potentially malignant growth of BHD renal cell carcinomas, shows that folliculin (FLCN) haploinsufficiency gives a less severe pathology than FLCN loss-of-function.

A recently published study from Bondavalli et al., (2015) was the first report of a cardiac rhabdomyoma (hamartoma) in an infant carrying a FLCN mutation. Cardiac rhabdoyomas are the most common cardiac tumour in children and can be sporadic or syndromic. Syndromic cardiac rhabdomyomas are associated with TSC and mutations in the TSC1 and TSC2 genes, however no such mutations were found in the infant.

A diagnosis of BHD in the extended family subsequently led to identification of a three base pair deletion in FLCN (c.469_471delTTC) in the infant, his mother and grandmother. Although genetic testing for BHD, typically an adult onset disease, is not always recommended for children it was deemed necessary in this case to determine if the child require further monitoring for TSC-associated phenotypes. Examination of the extended family identified a history of fibrofolliculomas but only one case of pneumothorax and one case of RCC.

Two other rhabdomyomas have been reported in BHD patients to date. A laryngeal rhabdomyoma was reported among the cohort assessed in Toro et al (2008). The second patient was diagnosed with BHD and multiple endocrinopathoes and found to have an adult rhabdomyoma in a presumed parathyroid adenoma (Mikesell et al., 2014). In addition cardiac rhabdomyomas have been reported in the Nihon rat model of BHD (n=15/125, Kouchi et al., 2009).

Cardiac rhabdomyomas in TSC patients have been linked to aberrant mTOR signalling (Kotulska et al., 2009) through the loss of regulation by the hamartin (TSC1) and tuberin (TSC2) complex. The mechanism by which FLCN regulates mTOR signalling is not fully understood and appears to be context dependent, but its dysregulation has been linked to the acknowledged BHD pathologies (Baba et al., 2006, Fuyura et al., 2012). Further research is required to determine if FLCN haploinsufficiency in muscle tissue also affects mTOR signalling and can subsequently be linked to rhabdomyoma formation in BHD patients and models.

Bondavalli et al. raise the possibility that mutations in FLCN could be the cause of cardiac rhabdomyomas and argue for BHD to be included in the differential diagnosis of these hamartomas. However they also believe that routine cardiac monitoring of children from BHD is not required. It is possible that the presence of FLCN mutations in patients with these hamartomas is more common but a limited knowledge of BHD, especially among paediatric cardiologists, is resulting in cases going unrecognised. These few reports, whilst insufficient to causatively link BHD and rhabdomyomas, could suggest an additional source of hamartomas associated with a haploinsufficiency of FLCN.

 

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Intragenic deletions in folliculin identified and mapped

The phenotypes associated with BHD – fibrofolliculomas, pulmonary cysts with an increased risk of pneumothorax, and an increased risk of renal cell carcinoma (RCC) – show variable penetrance between and within families, making diagnosis difficult when only one symptom is present. Studies have found that up to 10% of patient cohorts diagnosed with Primary Spontaneous Pneumothorax (PSP) have folliculin (FLCN) mutations (Ren et al., 2008, Johannesma et al., 2014).

Patients suspected of having BHD undergo genetic testing via DNA sequencing to detect FLCN mutations. However, for a minority patients with clinical BHD no mutation can be detected using these techniques. Large intragenic deletions and duplications have been identified in such patients (Kunogi et al., 2010, Sempau et al., 2010, Benhammou et al., 2011). New work from Ding et al., (2015) has identified and mapped three large intragenic deletions in the FLCN genes of 40 patients from nine Chinese families with a family history of PSP and lung cysts.

Ding et al., (2015) analysed 12 families in total including five families identified in Ren et al., (2008) that were found not to have small mutations detectable by DNA sequencing. The families showed reduced fibrofolliculoma penetrance and no RCC. The families were identified through a proband admitted after a spontaneous pneumothorax, so this selection bias might make the reduction in fibrofolliculomas and RCC less significant. Larger comparative studies are required to determine if there are significant difference in phenotype penetrance in different ethnic groups.‎

Ding et al., used Multiplex Ligation-Dependent Probe Amplification (MLPA) and breakpoint analysis to identify a large deletion across exons 9-14 (c.872-492_1740+1763del) in five families, a deletion across exon 14 (c.1539-536_1740+1701del) in two families and a large deletion across exons 1-3 (c.-504-1303_-25+845del) in two families. The FLCN start codon is in exon 4 therefore deletion of exons 1-3 would disrupt the promotor reducing expression (Benhammou et al., 2011). The other deletions would prematurely truncate FLCN and as several identified functions rely on the C-terminal (Baba et al., 2006) these mutations would be pathogenic.

A 5.5Mb disease haplotype was identified around the exon 9-14 deletion suggesting a founder mutation. In addition all five families shared a point mutation in exon 5. This deletion was estimated to have occurred approximately 16 generations ago. Haplotypes around the exon 14 and exon 1-3 deletions were also found, however estimating the age was not possible from two families.

To date one large intragenic duplication and 11 large intragenic deletions (Figure 1) have been identified in 20 BHD families and 2 sporadic patients. Two of the deletions identified by Ding et al., have previously been reported (Kunogi et al., 2010) but not mapped in detail. The three deletions mapped in this work and several of the other deletions have Alu repeat sequences (black arrows in Figure 1) on either side. Interestingly the FLCN gene has a high density of Alu repeats compared to the genome in general and this increases the risk of homologous recombination resulting in deletions and duplications.

This work supports the conclusion that a percentage of PSP patients are actually undiagnosed BHD patients, and that the 10% previously suggested could be an underestimation if only DNA sequencing is used to detect FLCN mutations. MLPA analysis would therefore be an important and useful additional DNA testing tool for detecting mutations in suspected BHD patients.

FLCN mutations

 

Figure 1: Intragenic deletions and duplications identified in FLCN 
Previously identified deletions and duplications are shown in blue and the three mutations found in Ding et al., (2015) are shown in red. Alu repeats are represented by black arrows. Taken from Ding et al., 2015, American Journal of Medical Genetics Part A 21 MAR 2015 DOI: 10.1002/ajmg.a.36979

 

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