Saturday, 30 July 2016

CrowdSpeak: using survival analysis makes Charcot 2 cheaper

An alternative power calculation for the Charcot 2 project.  #MSResearch #CrowdSpeak #Crowdacure #MSBlog

"In response to a comment by 'Stats is my name', (Friday, July 29, 2016 9:36:00 pm): 'But you can't compare shedders at 1 month vs shedders at 3 months. You can only compare like with like so you should use the numbers by time period'. This got me thinking about the study design. Even though EBV salivary shedding is intermittent there is no reason why a study design based on a survival analysis, i.e time to an event or in this case time to EBV shedding, won't work."

An example of a survival curve from Wikipedia

The following is a correction from when the post went live:

"Survival analysis is a branch of statistics for analysing the expected duration of time until one or more events happen, such as death in biological organisms. It is also called reliability theory or reliability analysis in engineering, duration analysis or duration modelling in economics, and event history analysis in sociology. Survival analysis attempts to answer questions such as: what is the proportion of a population which will survive past a certain time? It is necessary to define 'lifetime'. In the case of our study survival can be defined as being free of EBV shedding. I therefore propose randomising pwMS to placebo, or FamV, and then do monthly saliva samples and follow them up until we have enough events (shedding events). In the placebo arm we would expect 45% of subjects to shed EBV by 6 months and we predict that this figure will be reduced by 80%, i.e. to 9.2%, in the group of subjects being treated with FamV. With a power of 80% and an alpha of 5% we will need 44 subjects (22 per arm) to have enough events. This study will be followed until for a variable period after the last subject has been randomised."


Calculated using StatsToDo

"In comparison the proposed power calculations using a proportional analysis: based on the conservative assumption that the antiviral drug will only be 80% effective in suppressing EBV reactivation over 6 months with monthly salivary sampling, a power of 80% and an alpha of 5%, we would need at least 36 subjects or 18 per arm. With a 10% dropout rate this design would need 40 subjects."

"I am now going to armed with the above information and speak to our statistician to find-out which is the best study design. Once again thank you for your advice and support. It is truly very inspiring to have the community help by suggesting ideas about statistics. In this way the Charcot 2 study is going truly be a study designed and funded by the community."



Click here to find out more!

CoI: Team G are the recipients of a grant from Crowdacure that was used to perform this research.

Need for Spasticity Agents

Rønning OM, Tornes KD.Need for symptomatic management in advanced multiple sclerosis. Acta Neurol Scand. 2016. doi: 10.1111/ane.12631. [Epub ahead of print]

OBJECTIVE:A majority of patients with advanced multiple sclerosis (MS) need symptomatic treatment. Many MS-related symptoms may not be recognized and thus are not treated. We conducted a study to estimate the prevalence of inadequate symptomatic treatment of patients with advanced MS.
METHODS:Patients with advanced MS admitted to a specialist MS rehabilitation clinic were included in this study. Severity was assessed using the Expanded Disability Status Scale (EDSS). The information we collected included age of onset, initial course, time to sustained disability, pharmacological treatment, degree of spasticity, pain and bladder dysfunction, and unmet needs of symptomatic treatment.
RESULTS: In total, we assessed demographic and clinical characteristics in 129 patients with a mean age of 56 years and a median EDSS of 7.5. The proportion with inadequate symptom treatment was regarding spasticity 46%, pain 28%, and bladder dysfunction 23%.
DISCUSSION:This study showed that a large proportion of patients with advanced MS had lack of symptomatic treatment. These patients probably under use neurological specialist services. Better symptomatic treatment could contribute to improving quality of life of people with MS

This paper says that nearly 50% of people with disability due to MS get inadequate control of their spasticity. So we need more effective anti-spastic agents. We have been developing one and have an ongoing clinical trial.


CoI We are developing and anti-spastic agent

The Sixth Amendment-Spasticity trial

The Sixth Amendment of the US constitution guarantees the right to a Speedy Trial and Impartial panel of Assessors (Jury).We want to do the same. 

Double blind trials have a impartial set of assessors who are there to monitor the good and bad effects of any test drug but how do we make it more speedy.....simple its the Sixth Amendment. 

Every time you want to change anything in the trial you need to make an amendment that goes through ethical review.

The Sixth Amendment to our trial indeed provides more speed. 


The trial was in two parts. In the first part the drug dose was increased to check that it was safe in people with MS, determine how people with MS handle and respond to the drug; to assess efficacy and to see the doses that are tolerated. Then people get the maximum tolerated dose or placebo twice a day for a few weeks.


This had meant coming into hospital every day to do the first part. and so it helped if you lived locally to be shipped backwards and forwards from home each day or you were put up in a hotel during this time. 


The doctors are happy that the drug appears as safe in people with MS and that everyone tolerates the maximum tested dose and therefore the sixth amendment terminates the need to continue with the first part of the trial.

The study (both part I and II)  is blinded and placebo controlled and the results won't be looked at until everyone is recruited. The trial was always powered on the second part of the study and therefore you now only have to visit the hospital a few times for a couple of hours. 

So now there is less commitment needed in terms of time spent at the hospital making it easier to do the study 


There are now centres in London (Barts and UCL) and Liverpool and Sheffield that are actively recruiting and more are being added so if you can commute (e.g. via a taxi/ train-taxi) to one of these sites or want a hotel when things are inconvenient then contact the sites below or get your neurologist to contact the sites. 


The Neuroscience Research Centre, The Walton Centre NHS Foundation Trust
Liverpool, United Kingdom, L9 7LJ
Contact: Susan Whittam    0151 529 5666    susan.whittam@thewaltoncentre.nhs.uk   
Principal Investigator: Dr Wojciech Pietkiewicz         
The Royal London Hospital
London, United Kingdom, E1 1BB
Contact: Maria Espasandin    02035940637/8    Maria.Espasandin@bartshealth.nhs.uk   
Contact: Kimberley Allen-Philbey    02035941765    Kimberley.Allen-Philbey@bartshealth.nhs.uk   
Principal Investigator: Dr Clarence Liu         
The National Hospital for Neurology and Neurosurgery
London, United Kingdom, WC1N 3BG
Contact: Dr Rachel Farrell         
Contact: Michelle Liddicut       Michelle.Liddicut@uclh.nhs.uk   
Principal Investigator: Dr Rachel Farrell         
Royal Hallamshire Hospital, Sheffield Teaching Hospital NHS Foundation Trust
Sheffield, United Kingdom, S10 2JFS
Contact: Kamel Bouakline    0114 271 3339    kamel.bouakline@sth.nhs.uk   
Principal Investigator: Dr Siva Nair         

To participate in the study you need to have lower limb spasticity

Whilst you can use a wheel chair, you will also need to be able to walk 10m as part of the study and get on the bed for assessment.
This was selected for academic and logistical reasons, 


I am sorry to say this is placebo-controlled trial and not an add-on study, but their are scientific reasons to justify this.

Therefore to be eligible, you will need to be drug-free or be willing to come off your drug to do the trial. I am led to believe that this can be a revelation, as one may appreciate how sedative current medications can be and you will get a tailored symptom management plan once the study is complete.

CoI: I am a founder and consultant to Canbex Therapeutics who are performing this trial.

Friday, 29 July 2016

CrowdSpeak: Why am I a bottleneck?

Thank you we now have our power calculations for the Charcot 2 project? #MSResearch #CrowdSpeak #Crowdacure #MSBlog

"David Holden successfully completed all the lab work on the saliva samples and sent me the spreadsheet several weeks ago. I only managed to analyse them two days ago. We have achieved what we wanted to with the samples and now know how many people with MS shed virus in their saliva. This has allowed me to do two sets of power calculations for the proposed trial of our antiviral drug in pwMS."

"This is the summary, or headline, results. Before reading them I need to explain what some of the jargon or stats speak is. In summary sample size determination is the process of defining the number of observations, or replicates, to include in a clinical trial. The sample size is an important feature of any clinical study in which the goal is to make inferences about the population from which the study subjects are recruited. In practice, the sample size is critical to make sure we have sufficient statistical power to answer the question in hand; for example in our proposed study, do we have enough subjects to confident that our drug works in suppressing lytic EBV infection in the salivary glands of pwMS. Although the sample size calculation uses real data we have to make some basic assumptions, for example how effective we expect the drug to be when tested in pwMS. We also have to set other variables, i.e. the actual power of the study and the so called alpha. Traditionally in medical trials the power of studies is set at 80% or 90%, i.e. we have an 80% or 90% chance based on the number of subjects studied to get a positive result. In comparison, the alpha sets the level of significance we are prepared to accept; we typically set this at 5% using a one- or two-sided comparison. This means that if we get a significant result there is a 5% chance of the result being a false positive result. False positive results are not that uncommon in research, which is why we have to reproduce results in second, or follow-on, studies. Please also note the power calculations, i.e. the size of the study, determines its cost."


CrowdaCure EBV Viral Shedding Trial Power Calculation Output


Proposed Power Calculations for a 6-month study
In the INSPIRE samples, which collected monthly saliva for 6 months, 9 out of 20 subjects with MS (45%) shed EBV, i.e. had a detectable EBV virus in their saliva, on at least one occasion.  Based on this data we are now able to do a power calculation of a trial to assess the ability of an antiviral drug to suppress EBV lytic reactivation in the salivary glands.


Power calculation 1: Based on the conservative assumption that the antiviral drug will only be 75% effective in suppressing EBV reactivation over 6 months with monthly salivary sampling we will need at least 29 trial subjects per arm. This calculation is based on having a power of 90% and an alpha of 5%.


Power calculation 2: Based on the conservative assumption that the antiviral drug will only be 80% effective in suppressing EBV reactivation over 6 months with monthly salivary sampling we will need at least 18 trial subjects per arm. This calculation is based on having a power of 80% and an alpha of 5%.


Proposed Power Calculations for a 3-month study
In the Sheffield cohort, which collected monthly samples for 3 months, 44 out of 119 subjects with MS (37%) shed EBV, i.e. had a detectable EBV virus in their saliva, on at least one occasion.  Based on this data we are now able to do a power calculation of a trial to assess the ability of an antiviral drug to suppress EBV lytic reactivation in the salivary glands.


Power calculation 1: Based on the conservative assumption that the antiviral drug will only be 75% effective in suppressing EBV reactivation over 3 months, with monthly salivary sampling, we will need at least 38 trial subjects per arm. This calculation is based on having a power of 90% and an alpha of 5%.


Power calculation 2: Based on the conservative assumption that the antiviral drug will only be 80% effective in suppressing EBV reactivation over 3 months, with monthly salivary sampling, we will need at least 24 trial subjects per arm. This calculation is based on having a power of 80% and an alpha of 5%.

"In the Charcot Project 2 we are using viral shedding of EBV as a readout for antiviral drugs targeting EBV. If our lead drug Famciclovir suppresses virus in saliva then we will be confident of it also being suitable as a treatment for infectious mononucleosis. This hasn't escaped our attention, nor has the case report below. This is why IM is one of our major focuses going forward and forms part of Charcot 3."

"Some of you may ask why are you behind schedule in getting these results out? It is my fault I have too many balls in the air and not enough bandwidth or time to do everything I am involved in. One of my team has called me the bottleneck in the system. When we have our annual research strategy meeting in a few weeks time I am going to hopefully address this problem. May be I should be called Prof. Bottleneck rather than Prof G?"


"Once more, thank you for your support. The money raised has give us the necessary results that we can now use to develop a protocol for our next study. I sincerely hope we will now be able to leverage these results to get funding for our study."



Click here to find out more!


Goldani. Treatment of severe infectious mononucleosis with famciclovir. J Infect. 2002 Feb;44(2):92-3.

We report a patient with severe acute infectious mononucleosis who was successfully treated with famciclovir. A 15-year-old male was admitted with a 6-week history of fever, malaise, generalized lymphadenopathy, and hepatosplenomegaly, the patient was acutely ill with a temperature of 39.0 degrees C. Oropharyngeal examination revealed enlarged tonsils partially obstructing the airways. EBV serology obtained during admission showed a positive Monospot test, virus capsid antigen IgM, 1:320, Epstein-Barr nuclear and early antigen, negative. After 72 hours of treatment with famciclovir(500 mg t.i.d.), the patient was afebrile with important regression of the lymphadenopathy, enlarged tonsils and hepatosplenomegaly. Because acute infectious mononucleosis may be associated with extensive and prolonged disease, the potential therapeutic role of famciclovir in the treatment of severe forms of the disease deserves further studies.

CoI: Team G are the recipients of a grant from Crowdacure that was used to perform this research.

Learning from other conditions may teach us something-Obrigado

Meyer S, Woodward M, Hertel C, Vlaicu P, Haque Y, Kärner J, Macagno A, Onuoha SC, Fishman D, Peterson H, Metsküla K, Uibo R, Jäntti K, Hokynar K, Wolff AS; APECED patient collaborative, Krohn K, Ranki A, Peterson P, Kisand K, Hayday A.
AIRE-Deficient Patients Harbor Unique High-Affinity Disease-Ameliorating Autoantibodies. Cell. 2016 Jul 14. pii: S0092-8674(16)30792-9.

APS1/APECED patients are defined by defects in the autoimmune regulator (AIRE) that mediates central T cell tolerance to many self-antigens. AIRE deficiency also affects B cell tolerance, but this is incompletely understood. Here we show that most APS1/APECED patients displayed B cell autoreactivity toward unique sets of approximately 100 self-proteins. Thereby, autoantibodies from 81 patients collectively detected many thousands of human proteins. The loss of B cell tolerance seemingly occurred during antibody affinity maturation, an obligatorily T cell-dependent step. Consistent with this, many APS1/APECED patients harbored extremely high-affinity, neutralizing autoantibodies, particularly against specific cytokines. Such antibodies were biologically active in vitro and in vivo, and those neutralizing type I interferons (IFNs) showed a striking inverse correlation with type I diabetes, not shown by other anti-cytokine antibodies. Thus, naturally occurring human autoantibodies may actively limit disease and be of therapeutic utility.


The autoimmune regulator  (AIRE) is mutated in the rare autoimmune syndrome Autoimmune Polyendocrinopathy Syndrome type 1 (APS-1a problem of the endocrine glands that make hormones), also known as Autoimmune Polyendocrinopathy-Candidiasis-Ectodermal Dystrophy (APECED).  These patients are also susceptible to chronic Candidiasis (of skin and nails and mucosal (gut) surfaces caused by a Candidia fungus. This fungus also causes "thrush")



T lymphocyte tolerance is essential for limiting autoimmune disease. Tolerance occurs “centrally” (in the thymus) when developing thymocytes (cells from the thymus) with strongly self-reactive T cell receptors (TCRs) (Antigen recognition receptors are autoimmune targeting) are deleted following engagement of self-antigen-derived peptides presented by major histocompatibility complex (MHC) antigens (Each T cell attacks a foreign substance presented within a MHC molecule which it identifies with its receptor. T cells have receptors which are generated by randomly shuffling gene segments. Each T cell attacks a different substance. T cells that attack the body's own proteins are eliminated in the thymus. Medullary thymic epithelial cells (mTEC) express major proteins from elsewhere in the body (so called "tissue-restricted antigens"), and T cells that respond to those proteins are eliminated through cell death (apoptosis), thus it is thought that AIRE therefore drives negative selection. So the self recognitition element that T cells must see to form a productive immune reaction. This + infection gives protective immunity against infection. This plus self can lead to autoimmunity. This needs to be avoided so in the thymus these cells are programmed to commit suicide with is known as negative selection. The cells that can recognise the MHC but are not reactive to self are selected and this process in part of positive selection) . The expression of thousands of tissue-specific self-antigens (TSAs) by medullary thymic epithelial cells (mTEC) is directly promoted by AIRE, a poorly understood transcriptional regulator.

There are also several mechanisms of peripheral (in the lymph glands and the blood) T cell tolerance, including requirements for co-stimulatory signals for the activation of naive T cells; the expression of molecular “brakes” (e.g., CTLA-4, PD-1) by activated T cells; and the suppression of effector T cells  by FOXP3-expressing T-regulatory (T-reg) cells. 



Central (in the case of B cells in the bone marrow) and peripheral tolerance mechanisms likewise shape the B cell compartment. Thus, self-reactive B cells developing in the bone marrow may be censored by clonal deletion, clonal anergy (non-responsiveness), or B cell receptor (BCR) editing in which secondary gene rearrangements replace the initial BCR with a new specificity Peripheral (in the lymph glands and the blood) B cell tolerance is less well characterized but tolerance is imposed as transitional B cells differentiate into naive B cells via T cell dependent mechanisms. Likewise, T cell help is required in the germinal centres where antibody forming plasma cells are created. The question that then arises is whether major defects in central T cell tolerance provoke wide-ranging losses of B cell tolerance at either or both of these stages. An approach to examine this in people who are AIRE-deficient, whose under-expression of TSAs in the thymus is predicted to lead to increased numbers of peripheral autoreactive T cells. 



Thus, people with APS1/APECED have autoantibodies against limited set of TSAs such as products affecting exocrine glands and they block type I interferons and TH17-related cytoines so they do not get rid of Candida infections. These people harboured autoreactive (neutralising antibodies) and by looking at their sequences it appears  that the autoantibodies particularly develop by dysregulated reactions in the germinal centres, where T cells not tolerized in the thymus promote the competitive outgrowth and affinity maturation of B cells that were initially primed to exogenous antigen(s) but whose mutated IgGs bind to self-proteins. 

What has this got to do with MS? Maybe nothing, but in this study they report how autoimmunity may be blocked. However, it gives us ideas about how autoimmunity can develop. So Obrigado Cinara for bringing this to our attention. There are some testable ideas that come from this. 

I am often told that we give our ideas away too cheaply so will need to explain at a later date, when the paper comes out:-)