Patent Application
20150301041
The present invention relates to a method of determining the likelihood of a patient with joint inflammation developing rheumatoid arthritis (RA). This is useful in stratifying patients presenting with non-specific synovitis, into those patients likely to develop RA and those patients where the disease or condition is likely to resolve itself, e.g. those patients with short-term inflammation that can be successfully managed with rest and/or NSAIDs (non-steroidal anti-inflammatory drugs).
Rheumatoid Arthritis is a chronic inflammatory disease for which we lack a cure and good early diagnostic markers capable of distinguishing acute from persistent joint inflammation (1, 2). A window of opportunity exists during the first 3 months of RA where the disease is still evolving and is more responsive to current treatment strategies (reviewed by [3]). In light of this, there is an urgent need for a biomarker capable of stratifying patients for therapy, i.e. distinguishing those with acute resolving synovitis, from those who will develop chronic persistent joint disease (RA). Indeed, this might be considered the ‘holy grail’ of early arthritis diagnosis.
Surprisingly, we have found that changes in adiponectin receptor expression on peripheral blood leukocytes can be used as a biomarker for early diagnosis of RA, including distinguishing patients likely to develop RA from those with other conditions, for instance those with resolving disease.
In particular, patients that develop or are diagnosed with RA have a lower level of surface protein expression of adiponectin receptors-1 and/or adiponectin receptor-2 (ApidoR1 and/or ApidoR2) on certain leukocyte subpopulations compared to patients with resolving joint inflammation. Looking at gene expression, we have shown that the same patients have elevated levels of AdipoR1 mRNA and reduced AdipoR2 mRNA compared to patients with resolving inflammation.
Thus, in a first aspect, the present invention provides the use of AdipoR1 and/or AdipoR2 in leukocytes as a prognostic marker for RA (Rheumatoid Arthritis).
In a further aspect, the present invention provides a method of determining the likelihood of a patient developing RA (Rheumatoid Arthritis), the method comprising determining the expression of AdipoR1 and/or AdipoR2 in leukocytes in a sample from said patient.
Also provided is a method for indentifying a patient likely to develop RA, comprising:
The invention also provides a screening method for identifying, within a population, one or more patients likely to develop RA, comprising:
In other words, a screening method is provided for identifying, within a population, one or more patients likely to develop RA, comprising repeating the present methods to thereby identify said one or more than one patient likely to develop RA. Optionally, this may further comprise stratifying the patients according to their clinical outcome or likely clinical outcome.
The invention also extends to a prognostic or diagnostic method wherein a reduced cell surface protein expression of AdipoR1 and AdipoR2 is detected by an antibody, such as a fluorescent-labelled antibody. Accordingly, provided is a method of determining the likelihood of a patient developing RA, comprising:
One way to potentially referencing to a standard would be to introduce an external reference measurement into the assay. This could be done for example by putting in latex beads coated with AdipoR1 and/or AdipoR2. These could then be measured using the same reagents for assessing cellular levels. It is envisaged that reference bead could be made which fell between RA and non-RA. i.e. measurements below the reference level would indicate RA, whereas reading above it would indicate no RA. Flow cytometry would be a useful tool here to distinguish such beads from blood cells based either of forward and side light scatter, or incorporation of a fluorochrome that did not interfere with measurements from labelled antibodies.
Optionally, therefore, the method may include obtaining a sample from a patient. The anti-AdipoR1 or the anti-AdipoR2 antibody are optionally those listed herein, for instance in the section herein entitled ‘Examples of Antibodies Useful in the Invention.’ The preferred conditions for these antibodies are listed therein as well. These antibodies are commercially available. The antibodies used can preferably distinguish AdipoR1 and AdipoR2 (i.e. they don't bind some sort of common epitope).
It is also optional to introduce beads for standardisation of AdipoR1 and/or adipoR2 expression. As a result, the comparing the binding of the antibody or antibodies may be to cells and to reference beads. This is optionally by flow cytometry.
Optionally, point c) above, the determining that the patient is likely to develop RA, is based upon the reduced binding of said antibody or antibodies to leukocytes compared to the reference beads. This reduced binding to the reference beads is likely due to reduced surface expression of AdipoR1 and AdipoR2 on the leukocytes of patients with RA.
It may be helpful to indicate the negative prognosis for the determining step, i.e.: determining that the patient is likely not to develop RA based upon the increased binding of said antibody or antibodies. The method, therefore, optionally includes the following step, typically at the expense of other determining steps: determining that the patient does not have RA based on binding levels equivalent to or above those seen on the standard reference bead. This is likely due to the expression AdipoR1 and AdipoR2 remaining at the levels seen in healthy donors in patients who do not have RA.
Optional reference levels can be set up using a healthy cohort of blood donors.
Optionally, reduced binding in step c) was due to a reduction in the cell-surface expression of AdipoR1 or R2 (and preferably both).
The invention also provides an in vitro diagnostic use wherein anti-AdipoR1 or anti-AdipoR2 antibodies are detected. Accordingly, provided is in vitro use of an anti-AdipoR1 antibody and/or an anti-AdipoR2 antibody for identifying a patient likely to develop RA, wherein the identified patient displays in a sample a level of AdipoR1 and/or AdipoR2 protein which is less than a reference level.
Patients not likely to develop RA have other conditions, such as resolving disease. ‘Resolving disease’ is used herein to cover a range of possible conditions, other than RA, which may be causing the pain or inflammation in the joint or joints of the patients. Principal among these conditions is inflammation that can be treated effectively with NSAIDs for instance.
The patient or patients identified may be those that are likely to develop RA or they may be those that are likely to develop resolving disease (which as mentioned above includes other, non-RA conditions causing the symptoms). The present uses and methods therefore allow patients to be stratified according to their clinical outcome or likely clinical outcome, i.e. they include, preferably as a final step, said stratification.
Where reference is made throughout this specification to a patient who is “likely to develop RA,” it will be appreciated that this includes a patient that has RA, will develop RA, or is at least likely to go on to develop RA. The same holds true, mutatis mutandis for resolving disease, so that a patient who is “likely not to develop RA” or “is likely to develop resolving disease,” “will develop resolving disease” or “has resolving disease” can be used interchangeably. Furthermore, it will also be appreciated that term “develop” may include “diagnose” so that a patient that is likely to develop RA may be one that is later diagnosed with RA according to the appropriate criteria. Here, the terms ‘RA’ and ‘early RA’ are also used interchangeably.
Increased or decreased levels of the biomarker(s) may be compared to a reference level. The reference level is an AdipoR1 reference level where AdipoR1 is measured. The reference level is an AdipoR2 reference level where AdipoR2 is measured. When both AdipoR1 and AdipoR2 are measured then each biomarker is compared to the reference level for that same biomarker. The reference levels are described further below.
The patient is preferably one that presents with joint inflammation or joint pain that results, or is likely to result, from joint inflammation.
Accordingly, AdipoR1 and AdipoR2 can be considered to be biomarkers for RA. In particular, they can be considered to be biomarkers for stratifying patients with joint inflammation into patients likely to develop RA and patients that are likely to develop resolving disease.
In particular, the sample is a body tissue or body fluid sample. As the expression of the biomarkers is in (or on) leukocytes, the sample comprises said leukocytes (white blood cells) or fragments thereof. Preferred populations of said sampled leukocyte cells are PBMCs (peripheral blood mononuclear cells), PBL (peripheral blood lymphocytes), B cells and Natural Killer cell populations, e.g. NK-T-Cells.
The level (of the, or each, biomarker) determined is expression of said biomarker. This may be the protein or polynucleotide encoding it.
As the biomarkers are both receptors, determining the level of protein expression is typically achieved by measuring the prevalence of the protein form of the biomarker on the cell surface. This may be achieved by known methods, in particular flow cytometry, but other methods such as ELISA, western blotting, or other analytical protocols which yield a quantitative measurement of protein expression are included.
The polynucleotide encoding the protein biomarkers may be DNA, but is most typically RNA, in particular messenger RNA (mRNA). This may be achieved by known methods, such as qPCR.
The levels of protein and mRNA expression can be determined independently (i.e. AdipoR1 or AdipoR2). When levels of both biomarkers are determined (i.e. AdipoR1 and AdipoR2) then, typically, either protein levels of both or RNA levels are looked at. It is considered to be advantageous to combine the determination of the levels of both biomarkers.
The following applies to any of the uses or methods provided herein. The determining of the expression of the biomarker(s) in leukocytes in a sample from said patient may optionally include identifying a number of different determinants, each of which may be used alone or in any combination thereof.
Changes in cell-surface protein expression one or more of the biomarker(s) may be determined. Optionally, therefore, reduced cell-surface protein expression of AdipoR1 and/or AdipoR2 (being AdipoR1, AdipoR2, or AdipoR1 and AdipoR2) on said leukocytes is indicative of early RA. AdipoR1 and AdipoR2 are highly expressed in healthy patients. However, in patients with early RA, a reduction is seen.
This is, in a preferred example, indicated by a population of B cells (a sub-population of leukocytes) where only 60% of the B-cells show either marker. Thus, the reference level for AdipoR1 is that only 60% of the B-cells show the AdipoR1 marker. The reference level for AdipoR2 is that only 60% of the B-cells show the AdipoR2 marker. Above 60% B-cells AdipoR1 and/or AdipoR2 is therefore indicative of the patient being likely to not develop RA. Below 60% B-cells expressing AdipoR1 and/or AdipoR2 is therefore indicative of the patient is likely to develop RA. A range of +/−10% is envisaged as plausible, so the reference level may be set at a threshold of 50%, 55%, 60%, 65% or 70%. ‘Less than’ here includes ‘less than or equal to’.
Thus, a population of B cells where less than 50-70%, and preferably less than 60%, of the B-cells show either marker is indicative of the patient being likely to develop RA.
Changes in leukocyte sub-populations expressing the protein biomarker(s) may also be determined. In another preferred example, the determination is also indicated by another sub-population leukocytes, NK-T-cells, where only 20% of the NK-T-cells show either marker. Thus, the reference level for AdipoR1 is that only 20% of the NK-T-cells show the AdipoR1 marker. The reference level for AdipoR2 is that only 20% of the NK-T-cells show the AdipoR2 marker. Above 20% NK-T-cells AdipoR1 and/or AdipoR2 is therefore indicative of the patient being likely to not develop RA. Below 20% NK-T-cells expressing AdipoR1 and/or AdipoR2 is therefore indicative of the patient is likely to develop RA. A range of +/−10% is again envisaged as plausible, so the reference level may be set at a threshold of 10%, 15%, 20%, 25% or 30%. ‘Below’ here includes ‘less than or equal to.’
These NK and B-cell determinations are illustrated, for example, in
However, optionally, the levels of both AdipoR1 and AdipoR2 are determined (assessed). In this case, there are at least three different options, which may be used alone or in any combination thereof.
Differences in AdipoR1 and AdipoR2 mRNA Expression Levels
Optionally, one assessment is to determine the differences in AdipoR1 and AdipoR2 mRNA expression levels, i.e. by comparing said mRNA levels. Optionally, a difference between AdipoR1 and AdipoR2 mRNA expression levels is indicative of RA. This difference is preferably a relative expression (REU) above 0.14 (compared to 18S as internal standard gene) for AdipoR1 contrasted with an REU below 0.1 for AdipoR2 (compared to 18S as internal standard gene). This is illustrated, for instance, in
if X−Y≧0.036, 0.038, 0.040, 0.042 or 0.044; then RA is indicated.
where: X=REU of AdipoR1/REU of 18S
A reference for calculating REU is Livak K J & Schmittgen T D (2001). Analysis of relative gene expression data using realtime quantitative PCR and the 2-delta Ct method. Methods 25; 402-408.
Optionally, the expression of AdipoR1 and AdipoR2 may be incorporated into other mathematical algorithms. This may optionally provide standard ranges to stratify patients. In these cases, the level of both AdipoR1 and AdipoR2 are determined and further calculations required, so a reference level in the sense described above is not required. Thus, in these cases, the ‘reference level’ is replaced and the step b) of ‘comparing the determined level of said biomarker(s) with a reference level’ is amended to ‘b) calculating one or more ratios based of expression levels of AdipoR1 and AdipoR2 to provide an output or score to compare against a threshold’.
Optionally, other methods for determining whether a patient is likely to develop RA include:
(i) Ratio of AdiopR2:AdipoR1 mRNA Expression
Optionally, this comprises determining the mRNA expression ratio of AdiopR2:AdipoR1 (i.e. AdipoR2 divided by AdipoR1). This may be measured by qPCR. A score of less than or equal to 1 (100%) is indicative of early RA. This is illustrated, for example, in
if Z≧90%, 95%, 100%, 105% or 110%, then RA is indicated.
where Z=[(mRNA expression of AdipoR2)/(mRNA expression of AdipoR1)]×100
(ii) % Leukocytes Displaying AdiopR1 Protein×(Ratio of AdiopR2:AdipoR1 mRNA Expression)
Optionally, the method comprises determining the percentage of leukocytes displaying cell-surface protein expression of AdiopR1. This is then multiplied by the ratio of mRNA expression of AdiopR2 over AdipoR1 (see (i) above). This can be represented by the formula:
(Surface expression of AdipoR1)×ratio score of (i).
This is illustrated, for example, in
Optionally, a score of less than a threshold of 85 is indicative of early RA, although a score of less than or equal to a threshold of 75, more preferably less than a threshold of 70, 65, 60 or 55 and a score of less than or equal to a threshold of 50 is particularly preferred.
For B-cells very early RA patients have scores of less than a threshold of 50. For PBL very early RA patients have scores of less than a threshold of 15. For NK T-cells very early RA patients have scores of less than a threshold of 10. This is shown in
Thus, the percentage of leukocytes positive for surface protein expression of AdiopR1 is measured (preferably by FACs) and multiplied by the ratio from (i) above.
(iii) % Leukocytes Displaying AdiopR2 Protein×(Ratio of AdiopR2:AdipoR1 mRNA Expression)
Optionally, the method comprises determining the percentage of leukocytes displaying cell-surface protein expression of AdiopR2. This is then multiplied by the ratio of mRNA expression of AdiopR2 over AdipoR1 (see (i) above). This can be represented by the formula:
(Surface expression of AdipoR2)×ratio score of (i).
Optionally, a score of less than or equal to a threshold of 75 is indicative of early RA, more preferably less than or equal to a threshold of 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15 and score of less than or equal to a threshold of 10 is particularly preferred in some instances.
For B-cells very early RA patients have scores of less than or equal to a threshold of 30. For PBL very early RA patients have scores of less than or equal to a threshold of 15. For NK T-cells very early RA patients have scores of less than or equal to a threshold of 8. This is shown in
Thus, the percentage of leukocytes positive for surface protein expression of AdiopR2 is measured (preferably by FACs) and multiplied by the ratio from (i) above.
The methods described herein may also include administration of suitable treatments to a patient. In some embodiments, therefore, they can be considered to be methods of treating said patient, whether for RA or non-RA conditions such as resolving disease, the method including the assessment of the AdipoR1 and/or AdipoR2 biomarkers described herein. Thus, in the case of patients determined to be likely to develop RA, anti-RA drugs may be prescribed and, optionally, administered. Alternatively, in the case of patients determined to be likely not to develop RA, NSAIDs may be prescribed and, optionally, administered for instance. Thus, in a still further aspect, we provide a method of treatment of RA comprising administering an anti-RA drug, for instance those known in the art, to a patient determined to be likely to develop RA by the methods described herein. Said anti-RA drug may be for the treatment and/or prophylaxis of RA. Thus, a course of treatment may be correlated to a particular genotype. Provided is, therefore, a method for the treatment or prophylaxis of RA, comprising administering a drug for said treatment or prophylaxis of RA to a patient identified by the methods described herein. Suitable drugs include Analgesics (e.g. Paracetamol, Co-codamol, Tramadol); Non-steroidal anti-inflammatory drugs (NSAIDs, e.g. Ibuprofen, Naproxen); Corticosteroids (e.g. Prednisolone, Depo-medrone); Disease modifying anti-rheumatic drugs (DMARDs, e.g. Methotrexate, Sulfasalazine, Leflunomide, Hydroxychloroquine); and Biologics (e.g. rituximab and anti TNF drugs, such as etanercept, adalimumab and infliximab). The biologics are particularly preferred, especially rituximab and adalimumab.
The invention will now be described with reference to the Figures where:
Adiponectin is an anti-inflammatory cytokine capable of regulating the recruitment of leukocytes to inflamed tissues (e.g. [4; 5]). Deficiency in adiponectin has previously been reported to augment leukocyte adhesion to TNFα-treated venules in mice, a response attributed to elevated levels of endothelial adhesion molecules (E-selectin and VCAM-1) [4]. Moreover, normal levels of recruitment were restored upon administration of exogenous adiponectin [4]. In vitro, we have shown that adiponectin inhibits human peripheral blood lymphocyte (PBL) migration across inflamed endothelium in a dose dependent manner (
For identification purposes, the sequences of AdipoR1 and AdipoR2 are provided herein as examples of AdipoR1 and AdipoR2. Two variants are listed for AdipoR1. Others may also be known or found but are only considered relevant if they code for a functional protein.
Along with mathematical algorithms which will provide ranges allowing the stratification of patients into different clinical outcome groups based on the measurements detailed. Collectively, they can be used to diagnose, i.e. provide a diagnosis for, RA in patients with very early disease where suitable biomarkers discriminating RA from other forms of joint disease are currently lacking. The invention can also be used to provide a prognosis for the likely clinical outcome for a particular patient, namely to distinguish between RA and non-RA conditions.
Where reference is made to a diagnosis or the provision of a diagnosis, it will be appreciated that this also refers to a prognosis, or the provision of a prognosis unless otherwise apparent. Accordingly, the method may comprise the diagnosis and/or the prognosis of rheumatoid arthritis. As with any method providing a diagnosis or a prognosis, the output (i.e. the diagnosis or prognosis itself) is based on an expected outcome or result. As such, an entirely definitive output cannot necessarily be provided. Instead, such methods, including the present invention, provide for a likelihood of a particular outcome. When a patient is diagnosed with RA, then particular treatments can be prescribed. Similarly, when the prognosis is that RA will develop, the patient may be treated accordingly. However, when it is established that it is unlikely that RA will develop or that the patient does not have RA, then alternative treatments can be provided. Thus, if the patient is likely to have resolving disease, then the output (i.e. the advice) may include a course of antibiotics, anti-inflammatories and/or rest. Other suitable treatments to treat joint inflammation are known, but it will be appreciated that these are not effective, especially long-term, when the underlying condition is RA. Accordingly, rapid and accurate diagnosis of RA is clinically and socially important, both from the point of view of the treating the patient and in ensuring that funds for treatment are spent in the most cost-effective way (i.e. by avoiding funding inappropriate treatments).
Low AdipoR2:AdipoR1 ratio and/or low surface expression×ratio score is indicative of an increased likelihood of development of RA. This is increased with respect to, i.e. greater than, the likelihood of the patient having resolving disease. Of course, the patient may already have RA, especially in its early forms, but has preferably not yet been diagnosed with RA. As mentioned elsewhere, to be officially diagnosed with RA, a current set of criteria must be met, even though the disease is present up until the day the decision is made. In other words, although the criteria and length of time required to meet these or receive a decision may change over time, the invention nevertheless provides a way of assessing whether a patient is in an at risk group (for RA) or not. Accordingly, the present method preferably comprises assessing whether a patient will go on to formally meet the criteria for an RA diagnosis. The method may be also be considered to be a method for (or of) assessing whether a patient is in an at risk group (for RA).
The criteria for developing RA are preferably met at the 18 month stage.
Preferably, only one of AdipoR1 or AdipoR2 is assessed at protein level. This may preferably be AdipoR2, but may equally be only AdipoR1. AdipoR2 is generally preferred as it shows less variability, but bigger diagnostic/prognostic changes. Most preferably, however, expression of both AdipoR1 and AdipoR2 is considered and the ratio of the expression levels used to define patient groups. For mRNA, both AdipoR1 and AdipoR2 will preferably be assessed in order to provide the ratio between the two which again can be used alone or with other measured parameters to provide a ‘score’ or ‘threshold’ below which patients are likely to develop RA and above which they will be diagnosed with synovitis which is not RA. Where both are considered, this is preferably at substantially the same time, as this should help to reduce errors caused by potentially changing expression patterns. A “snap-shot” is thus preferred.
The present method may also preferably include, or be used alongside, existing methods of diagnosing RA, such as but not limited to: imaging e.g. via X-rays, MRI or ultrasound; and/or blood tests e.g. test for rheumatoid factor, tests for the presence of the anti-citrullinated protein antibodies (ACPAs) or anti-CCP (cyclic citrullinated peptide) and the anti-MCV assay (antibodies against mutated citrullinated Vimentin).
RA is known to have secondary links with diffuse inflammation in the lungs, the membrane around the heart (pericardium), the membranes of the lung (pleura), and white of the eye (sclera), and also nodular lesions, most common in subcutaneous tissue. These parameters tend to occur later in pathology in some patients. Accordingly, these are secondary indicia that may be used in conjunction with, but not instead of, the main indicator: joint inflammation. The present biomarker is therefore relevant specifically to patients with very early arthritis (i.e. joint swelling and pain/inflammation of less than 3 months).
The resolving disease may be very early arthritis, but which is not rheumatoid arthritis and, therefore is likely to spontaneously resolve without treatment. However, very early rheumatoid arthritis (RA) may be considered to be a condition which ultimately fulfils the criteria for RA, for instance at an 18 month follow-up. It is preferred that those classified as likely to have or go on to develop RA satisfy the requirements of the American College for Rheumatology 2010 classification criteria.
Synovitis causes joint tenderness or pain, swelling and may go on to present hard lumps called nodules. Symptoms of synovitis can be treated with anti-inflammatory drugs, such as NSAIDs or an injection of steroids, or biological therapies such as Rituximab.
It will be appreciated that terms such as ‘determined,’ ‘measured,’ ‘detected’ or ‘assessed’ may be used interchangeably unless otherwise apparent.
The expression is optionally protein expression, optionally on the cell surface. However, expression at the transcript (RNA and most preferably mRNA) is also envisaged. Indeed, the transcript and protein expression may be measured separately. However, it is preferred that they are measured at approximately the same time. Preferably, they are measured from different aliquots of the same sample. Preferably, any measuring is made approximately contemporaneously. It will be appreciated that reference to the term ‘amount’ may also be taken to refer to the term ‘level’ of, for instance, a transcript or protein being measured.
The patient has joint inflammation, which may present as joint stiffness or pain. Most preferably, the patient has (or presents with) synovitis (although it will be appreciated that they may not have been diagnosed with this yet). The patient may present with joint inflammation or synovitis or may have been diagnosed or suspected of having said conditions by a health practitioner.
The patient is preferably a human, but it is also envisaged that non-human mammals may be used in the present method, in which case the expression of AdipoR1 and/or AdipoR2 should be determined. Indeed, it should be appreciated that reference herein to AdipoR1 and/or AdipoR2 includes non-human homologues unless otherwise apparent.
The present method preferably includes the step of stratifying the patient into one or more groups or cohorts. Preferably, these are “patients with or likely to develop early RA” and “patients with other, non-RA related, conditions.” The latter preferably includes patients with resolving disease, i.e. those patients where treatment of the underlying condition can be successfully, or largely successfully, completed, or at least the symptoms alleviated using anti-inflammatory treatments such as NSAIDs as mentioned above. However, those patients with, or likely to develop, RA, are unlikely to ultimately be successfully treated using such anti-inflammatory treatments. This is because the underlying cause of the disease is not known and is chronic in nature and requires more aggressive anti-inflammatory therapies such as the administration of, for example, methotrexate and/or anti-tumour necrosis factor therapy. Notably, such interventions still only alleviate symptoms in approximately 50% of patients with established RA, although diagnosis of patients earlier will allow more effective treatment as described above.
Indeed, the method itself may be viewed as a method of stratifying patients into (i) those having or likely to develop early RA and (ii) those unlikely to have or develop early RA (and instead having resolving disease). Such methods would include determining the expression of AdipoR1 and/or AdipoR2 in leukocytes in the sample from the patient. Of course, reduced surface expression per se, or a ratio of less than 1 of AdipoR2/AdipoR1 (AdipoR2 divided by AdipoR1), or the use of these data in other algorithms is indicative of the development of RA. In other words, the stratification of the patients may predict clinical outcome.
Patients will tend to present with joint inflammation and this will ultimately be either resolving (i.e. is cured with treatment) or persistent (i.e. where treatment is attempted to keep the symptoms in check). RA is considered to be “persistent” because we currently do not understand the cause of the disease and we do not have adequate treatments to “cure” the disease.
The expression of AdipoR1 and/or AdipoR2 may be assayed at the RNA or the protein level. Accordingly, an assay to determine the quantity or level of mRNA expression may be used, such as PCR, in particular quantitative PCR (qPCR). Other means for determining the quantity or level of mRNA are of course also envisaged. Measurements may be made in crude mixed isolates of leukocytes or on cells after fractionation into subsets.
Alternatively, the level or quantity of protein expression may be determined. This is most easily done by determining the quantities or levels of cell surface expression of the resulting AdipoR1 and/or AdipoR2 receptors, including the proportion of the cells that express the protein. This may be measured in conjunction with other surface proteins which are markers of different leukocyte subsets. Alternatively, the level of protein expressed within the cell, or in different cellular fractions can also be determined. Suitable methods include the use of flow cytometry, western blotting and/or confocal microscopy.
Accordingly, where reference is made to the expression of AdipoR1 and/or AdipoR2 in the particular cells, then it will be appreciated that this can include both detection of the polynucleotide transcript, the peptide and/or the resulting functional protein. The functional protein may be expressed on the cell surface, so in this sense, the “in” a certain cell can also include “on” said cell.
The cells in the sample are leukocytes. Preferably, these are Peripheral Blood Leukocytes. Measurements may be made of sub-populations of the leukocyte mixture, preferably peripheral blood mononuclear cells (PBMC) or peripheral blood lymphocytes (PBL) as the AdipoR1 and AdipoR2 receptors are highly expressed on many subsets of circulating PBL and PBMC. The PBL subpopulations may include T-cells, B-cells and various subpopulations of natural killer cells such as NK T-cells. Analysis may be performed on PBMC or PBL, although B cells and/or NK T-cells are in general preferred, as narrower classes affording greater accuracy.
In patients likely to develop RA or those with very early RA, the expression of AdipoR1 and/or AdipoR2 is reduced. In addition, patients likely to develop RA or those with very early RA, the mRNA expression of AdipoR1 is elevated whilst AdipoR2 mRNA expression is reduced.
It will be appreciated that these changes may be in comparison to a healthy control, preferably once suitable filters, such as age and gender, have been applied. Similarly, preferably the same changes are also seen in comparison between the RA patients and those with resolving disease. Again, it is preferred that said resolving disease patients have been filtered accordingly as mentioned above. In other words, the controls against which the change or changes are measured may be completely healthy patients, or patients with joint inflammation (resolving disease) that are ultimately cured. Mathematical algorithms based on the aforementioned changes will provide standard ranges allowing stratification of patients into different clinical outcomes. The control against which a patient is assessed may also be the same patient, sampled at a different point in time. This allows progression of the condition (or not) to be monitored in a particular individual over time. Such an individual may be one that has had a family history of RA or is otherwise identified as being in an ‘at risk group.’ Thus, the method may preferably comprise two or more assessments (determinations of AdipoR1 and/or AdipoR2 expression) over time, i.e. over pre-determined time intervals, with fluctuation preferably being identified. A reduction in AdipoR1 and/or AdipoR2 expression (or use of this information in appropriate algorithms [as above]) over time may, therefore, be indicative of an increased likelihood that the patient is beginning to develop RA, and suitable interventions may then be made or prescribed.
Unfortunately, certain therapeutic interventions such as methotrexate and/or anti-TNF therapy unbalance the Adiponectin/AdipoR system, so this latter aspect is generally not preferred, unless it is known that no therapeutic intervention has been made in the intervening period or, if it has, then that intervention is known not to unbalance the Adiponectin/AdipoR system. In view of this, internal (within the same individual) comparisons between expression of AdipoR1 and AdipoR2 are preferred, see the algorithms (i), (ii) and (iii) above.
The sample is preferably a blood sample, although other fluid samples are envisaged (for example synovial fluid is also preferred). Most preferably, the sample is peripheral blood.
Accordingly, the use and methods also preferably include any of the following steps, alone or in combination:
The AdipoR1 and AdipoR2 receptors are highly expressed on many subsets of circulating PBLs. By way of example only, and although it will be appreciated that a Ct value can change between qPCR runs, the following ranges should generally apply for healthy controls:
The AdipoR1 and AdipoR2 receptors are highly expressed on many subsets of circulating PBLs. By way of example only, and although it will be appreciated that a Median fluorescent intensity (MFI) value can change between flow cytometers and antibodies used, the following ranges should generally apply for healthy controls following subtraction of the isotype control antibody:
For the expression of AdipoR1 and/or AdipoR2 on the cell surface, thresholds of 60% or less of B cells or 20% or less of NK cells are indicative of early RA (i.e. a population of B cells where only 60% of the B-cells show the marker is indicative of early RA, whilst a population of NK cells where only 20% of the NK-cells show the marker is indicative of early RA). In particular, these thresholds preferably apply to both AdipoR1 and AdipoR2, such that thresholds of 60% or less of B cells expressing both AdipoR1 and AdipoR2 is indicative of early RA, or thresholds of 20% or less of NK cells expressing both AdipoR1 and AdipoR2 is indicative of early RA. Preferably, the B-cell and NK-cell thresholds are both measured to improve accuracy. The threshold (cell %) may be assessed by Flow Cytometry, for example.
It will be appreciated that absolute flow cytometry readings, for instance, may vary depending on the reagents and machine used. However, using ratios according to algorithms set out in (ii) and (iii) will overcome this.
Link: http://www.phoenixpeptide.com/cataloq/product info.php?products id=4686
Antibody from Phoenix Pharmaceuticals, Inc.
Link: http://www.phoenixpeptide.com/cataloq/product info.php?products id=4684
Adipo R2 (374-386)/Adiponectin Receptor Type 2 (374-386) (Human)—Purified IgG Antibody from Phoenix Pharmaceuticals, Inc.
Birmingham Early Arthritis Clinics systematically collected blood samples from patients who are categorised into the following groups based on clinical outcome and disease duration [16; 17]:
Blood samples from age and gender matched healthy controls were also obtained. Peripheral blood lymphocytes were isolated and the expression of the adiponectin receptors on individual subpopulations was assessed by flow cytometry. We observed a significant reduction in the surface expression of the adiponectin receptors on circulating leukocytes isolated from patients with early unclassified RA, compared to healthy controls and patients with resolving disease. This pattern was evident for mixed peripheral blood lymphocytes (
It appears that, in patients with undefined synovitis (joint inflammation), a reading of less than 60% of B-cells or 20% NK-T cells positive for adiponectin receptors would be associated with increased risk of developing RA.
Alternatively, peripheral blood mononuclear cells were isolated and the mRNA expression of the adiponectin receptors were assessed by qPCR. We observed an elevation in AdipoR1 and a reduction in AdipoR2 mRNA expression on circulating PBMCs isolated from patients with early unclassified RA, compared to patients with resolving disease (
It appears that in patients with undefined synovitis, a AdipoR2:AdipoR1 ratio of less than 1 would indicate/be associated with increased risk of developing RA.
The surface and gene expression of adiponectin receptors can also be evaluated together to provide additional power in the data. Here the surface expression of adiponectin receptor 1 and/or 2 on individual lymphocyte subsets is multiplied by the AdipoR2:AdipoR1 mRNA ratio. Using the surface expression of AdipoR1 on B-cells for example, we observed a significant reduction in the score of patients with early unclassified RA, compared to patients with resolving disease (
It appears that in patients with undefined synovitis, a surface expression*ratio score of less than 50 would indicate/be associated with increased risk of developing RA.
Thus, expression of adiponectin receptors on lymphocytes has great potential utility as a biomarker in early arthritis clinics to diagnose those patients with RA earlier than is currently possible.
This has the added advantage of distinguishing them from a wider cohort of patients with uncharacterised resolving disease. Current clinical practice requires patients to fulfil the American College of Rheumatology 2010 classification criteria to be diagnosed with RA. As such, patients can often wait several months before receiving a confirmed diagnosis, by which point it is too late, as the crucial window of opportunity for an early intervention has closed.
Accordingly, early detection and diagnosis of RA by this invention will allow earlier clinical intervention that will improve the long-term outcome for patients. Our data indicates that adiponectin receptors on lymphocytes are the first biomarker capable of stratifying patients within days of them presenting to clinic, allowing improved patient-based therapeutic intervention and disease control.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1221011.8 | Nov 2012 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2013/053070 | 11/21/2013 | WO | 00 |
