Collaborative Bone Study

The aim of the study is to document as fully as possible the extent of bone involvement in as many as possible UK Gaucher disease patients. This involves a clinical history and physical examination of the patients with emphasis on mobility, quality of life and pain. The patients’ clinical experience will then be compared with findings on X-Ray, MRI and bone mineral density investigations.

‘This information will be used to develop severity scoring systems for bone disease and, it is hoped, find factors which predict later bone disease. Such factors might include the age at which a patient first develops symptoms and whether the patient had a splenectomy.  An important component of the project is to search for and then evaluate chemical markers of bone disease that might be of use in prediction and in monitoring severity of bone disease and response to particular treatments.

Recruitment has been achieved mainly through speaking to patients in clinic and going through the ethical consent process with them. They are interviewed for the bone study on the day of their normal clinic visit and a careful physical examination of patients’ joints is carried out. The information received from the patients is then entered on to the  computer database. Blood and urine samples are taken from the  participants and stored. All the patients’ previous X-Rays and MRI studies and, indeed, bone mineral density assessments are reviewed as part of the bone project.

Second Phase
By September 2006, the task of gathering all of the information and all new acquisition of data will be completed. The study will then move on to the analytical phase of the project. Dr Elena Pavlova from the Russian Republic of Yakutia has been recruited to carry out much of the laboratory based analysis of the blood and urine samples.

‘Dr Pavlova is a paediatrician with a longstanding interest in the bone complications of  severely-affected Russian children with Gaucher disease that she looked after. She will examine a number of potentially interesting chemical markers of Gaucher disease with a view to finding ways to predict future bone disease and monitor response of existing bone disease to treatment.

Update report on the Collaborative Bone Study (Gauchers News July 2008)  

Even in the era of effective enzyme, in Europe today, Gaucher disease is principally a disease of bones. These manifestations occur largely in those patients whose condition was established before enzyme therapy could be introduced – either because it was unavailable or because the diagnosis was not made in time.

As part of this project Dr Patrick Deegan in his clinical survey conducted with Jane Tindall and colleagues, surveyed 100 adult and 13 paediatric patients with Gaucher disease nationally at the four original participating centres.

This research was funded generously by the Association and was recently reported in outline at the Association 15th Anniversary Conference in January last year. Dr. Deegan set out the burden of disease in the UK and described the frequency of the modeling deformities, avascular necrosis (osteonecrosis or bone crises), the bone thinning (osteoporosis), and osteolysis, the regional disease in which bone is destroyed focally at sites of Gaucher tissue within the marrow cavity.

Bone Crises
Of all these conditions, avascular necrosis appears to be the most disabling and, once established, the most difficult to treat. It frequently leads to destruction of big joints particularly the hips but also the knees and shoulders; it may also cause diffuse pain by affecting the difficultto-visualise bones of the pelvis.

Avascular necrosis is associated with severe episodes of pain at the time with no findings either on clinical examination by the doctors -or by conventional X-rays- in the first instance; only MRI and more sophisticated methods of diagnostic imaging can identify the affected bone during the crises. Careful doctors know that this is a diagnosis based on experience - and on simply believing the patient.

We know that avascular necrosis (‘bone crises’) occur particularly at critical points of growth and are common at puberty but they also occur at other times. Dr Deegan in an analysis of the survey data as part of this project has shown that they occur more frequently close to the time that a spleen was removed in the old days as a treatment for Gaucher disease.

The manifestations of disease in the bone reflect both the release of powerful enzymes from the lysosomes (as shown in part by Dr. Mary Teresa Moran – whose research was also funded by the Association) and cytokines studied by us and numerous other groups. These factors influence local events in bone but also have widespread effects on the skeleton; they are believed in some way to impede a critical supply of blood. Some believe that abnormal blood coagulation and abnormal capillary supply in the bone reflect a blocking of the blood supply with accompanying death of living bone tissue.

The Need for Research into Biomarkers
The frequency of bone crises appears to be markedly reduced by enzyme therapy. At the same time, existing biomarkers measured in the blood at visits to hospital (plasma chitotriosidase and  Cl-18/PARC – discovered jointly by Dr. Mary Teresa Moran with Prof. Aerts’s group in Amsterdam) reflect the activity and bulk of Gaucher cells in other organs such as the spleen. We thus propose that these and other components that change in the blood might be able to predict the risk for occurrence of these complications in the bone. Certainly there is a longstanding association between severe bone disease in patients with Gaucher disease and the prior removal of the spleen before enzyme therapy was introduced.

Predictive biomarkers
At present specialised biological “markers” are used in the clinic to follow the patients’ responses to treatments. In fact currently licensed treatments for Gaucher disease (Cerezyme and Miglustat) reduce the amounts of the markers chitotriosidase and CCL18 in the plasma and are used by many centres to follow the course of the condition during treatment. However, at thepresent time clear evidence that abnormalities in these biomarkers, predict a particular complication for Gaucher disease is lacking.

Other candidate biomarkers which can be used to follow the course of the condition and possibly may have a role in predicting complications have been under consideration from the research carried out mostly by European investigators particularly those in the Netherlands and elsewhere. These cytokines or chemokines mediate inflammation by blood cells at sites where Gaucher disease is active. They can be measured and some of them have been shown to be elevated in untreated patients with Gaucher disease.

We have then a problem in determining the clinical value of serum biomarkers:-

• Does measurement of these biomarkers play any role in helping doctors distinguish between patients with and without the bone manifestations of Gaucher disease?
 • We also ask whether or not of certain biomarkers be used to treat the treatment response?
 • Most importantly, does the extent to which the biomarkers are elevated in the blood predict the risk of developing of bone complications?

These questions, though simple to ask are difficult to answer – particularly in the era in which most patients are receiving appropriate treatment. Thus any attempt to determine the value of biomarkers is hampered by the effects of preexisting intervention. Given that this is not an ideal world and that we do not have a large bank of serum from patients before treatment, enabling us to measure the biomarkers in advance and then measure the outcome of the patients’ illness over a long period of time subsequently, we are forced then to interpret and analyse changes in samples we already have.

Details of the Project
To get a handle on this problem, Dr Elena Pavlova catalogued the independent data collected by Dr Deegan and Sister Tindall from the UK patients at the various centres. She also used a technique to measure numerous candidate and known biological markers of Gaucher disease in the patients’ serum.

Our clinical database has been established on more than 100 patients  – 100 adults and 13 children. This database has unbiased clinical information including the results of physical examination, radiological assessment of the bones and clinical laboratory data that were routinely recorded. At the same time, the research team collected samples of serum, plasma and urine from the 100 adult participants and Dr Pavlova analysed the potential biomarkers in these samples.

Here we report on the characteristics of 100 adult patients
 • 60 female, 40 male
 • Average age: 49 years
 • Average age at presentation: 17 years
 • Average age at diagnosis: 21 years.
 • History of avascular necrosis: 43
 • Spleen removed: 44
 • Those with type 3 disease: 4
 • Enzyme replacement therapy: 92
 • Genotype: 13 out of 89 in whom genotype known were homozygous N370S.
 • Average Zimran severity score: 10.5

Of this group of 100 adult patients, 84 were receiving enzyme replacement therapy without ongoing avascular necrosis; a minority receiving enzyme replacement therapy (8 patients) had ongoing episodes of avascular necrosis.

Of the 8 patients who were not receiving enzme replacement therapy, none of these fortunately had developed avascular necrosis.

Laboratory Studies
The analytical methods used multiplex flow cytometric bead array assays to measure 15 cytokines simultaneously on a very small sample of serum; in addition Dr Pavlova developed a separate flow cytometric bead assay for CCL18/PARC which gave very good operating characteristics – and required some development.

Several serum were found to be elevated significantly in the patients with Gaucher disease compared with healthy control subjects serum biomarkers of several cytokines that were stable in stored serum and plasma. This included macrophage chemotactic protein 1 (MCP-1); macrophage inhibitory protein 1a and 1b (MIP-1b and MIP1-a); interleukin 8.

Dr Pavlova found that those patients who gave a history of avasclar necrosis that had been determined in an unbiased way before the analysis had a significantly elevated concentrations of both MIP1-b and IL8 as well as CCL18/PARC.

The study of the receiver operating characteristics for these cytokines showed indeed that they had some considerable specificity and sensitivity in detecting those patients with avascular necrosis. The best results were found with Interleukin 8 which at best had a highly significant performance with 78% specificity and 60% sensitivity for detecting avascular necrosis where a cut off value of greater than 25 picogram/ml was chosen. However, as they stand, these values would mean that about 30% of the patients would be  mischaracterised.

We then asked the question: Are the biomarkers associated with the achievement of therapeutic goals – in this case avoidance of avascular necrosis?

Indeed Interleukin 8, CCL18/PARC, MIP1a and MIP1b were all significantly elevated in patients who had continued to develop  avascular necrosis after receiving ERT compared with those who did not have avascular necrosis while receiving enzyme therapy. We then further asked the question – could we distinguish between patients who had had avascular necrosis after enzyme replacement therapy, and those who had no avascular necrosis after enzyme therapy- compared with those who had never had avascular necrosis. In this case, the significance was seen strongly in favour of those who developed avascular necrosis, whose measured cytokine and chemokine concentrations were significantly higher.

We thus concluded that avascular necrosis of bone with a failure to meet a key therapeutic goal was associated with elevated serum cytokines including the one discovered by Dr Mary Teresa Moran in conjunction with Professor Aerts and colleagues in Amsterdam and now in current use as CCL18/PARC. MIP1a, MIP1b and Interleukin 8 were also correlated with failure to meet this therapeutic goal.

Reservations and plans for the Development of this work.
 We are aware that there may be some flaws in the design of this study, it is a cross-sectional study and makes assumptions, however is the best available method to give a proper survey of bone disease in the UK as seen in our Gaucher patients.

What the study shows to our great surprise is a strong association between biomarkers, whose abundance correlates with Gaucher disease activity (and decrease with specific enzyme treatment), and ocurence of the disabling complication, avascular necrosis. In all the cases studied, the bone events occurred many months or years before the serum samples were collected.

Future Extension of the Study
We are now planning to set up prospective studies to determine whether the serum proteins can truly serve as predictive biomarkers for this complication. This would allow us to establish target values of the biomarkers so that enzyme replacement therapy and other treatments can have standard limits which should be attained in order to reduce the risk of avascular necrosis.

The study then is very important in setting the ground rules for an important piece of work which would add further lustre to the development of therapeutic goals and, if we may say so here, to the work supported by the Association through the specialist centres where treatment is given.

Members of the Association will be pleased to know that a grant has been obtained in collaboration with investigators in Germany, Italy and Sweden further to investigate aspects of the burden of Gaucher disease and other lysosomal disorders in the European population under the Framework Package 7 funded by the European Union. The very recent award of this grant should enable us to do prospective studies as demanded by the findings here in several large populations of Gaucher patients  including the UK, in an attempt to answer these key questions definitively.

We feel that such studies relate very much to the daily experience of patients in their own lives. We have been very fortunate in having the support of the Association to launch this study and when the further work has been done will undertake not only to report back but to implement any new standards of care that will be implicit in the findings.