WO2014057263A1

WO2014057263A1 - Chronic kidney disease assay

Info

Publication number: WO2014057263A1
Application number: PCT/GB2013/052628
Authority: WO
Inventors: Stephen Harding; Paul Cockwell
Original assignee: The Binding Site Group Limited
Priority date: 2012-10-10
Filing date: 2013-10-09
Publication date: 2014-04-17
Also published as: GB201218137D0; EP2906955A1

Abstract

A method of identifying the risk of a patient with chronic kidney disease (CKD) requiring renal replacement therapy (RRT) within a predetermined period of time comprising determining a value of free light chains (FLC), preferably combined FLC, in a sample from a patient, comparing the value with a predetermined value and assigning a risk score to that value of FLC in the sample to indicate the risk of the patient requiring RRT over a predetermined length of time.

Description

Chronic Kidney Disease Assay

The invention relates to methods of identifying patients with reduced risk of requiring renal replacement therapy and assays for use in such methods.

Antibodies comprise heavy chains and light chains. They usually have a two-fold symmetry and are composed of two identical heavy chains and two identical light chains, each containing variable and constant region domains. The variable domains of each light- chain/heavy-chain pair combine to form an antigen-binding site, so that both chains contribute to the antigen-binding specificity of the antibody molecule. Light chains are of two types, κ and λ. There are approximately twice as many κ as λ molecules produced in humans, but this is different in some mammals. Usually the light chains are attached to heavy chains. However, some unattached "free light chains" are detectable in the serum or urine of individuals. FLC may be specifically identified by raising antibodies against the surface of the free light chain that is normally hidden by the binding of the light chain to the heavy chain. In FLC this surface is exposed, allowing it to be detected immunologically. Commercially available kits for the detection of κ or λ FLC include, for example, "Freelite™", manufactured by The Binding Site Group Limited, Birmingham, United Kingdom. The Applicants have previously identified that measuring the amount of free κ, free λ and/or free κ/free λ ratios, allows the detection of monoclonal gammopathies in patients. It has been used, for example, as an aid in the diagnosis of intact immunoglobulin multiple myeloma (MM), light chain MM, non-secretory MM, AL amyloidosis, light chain deposition disease, smouldering MM, plasmacytoma and MGUS (monoclonal gammopathies of undetermined significance). Detection of FLC has also been used, for example, as an aid to the diagnosis of other B-cell dyscrasia and indeed as an alternative to urinary Bence Jones protein analysis for the diagnosis of monoclonal gammopathies in general.

Conventionally, an increase in one of the λ or κ light chains is looked for. For example, multiple myelomas result from the monoclonal multiplication of a malignant plasma cell, resulting in an increase in a single type of cell producing a single type of immunoglobulin. This results in an increase in the amount of FLC, either λ or κ, observed within an individual. This increase in concentration may be determined, and usually the ratio of the free κ to free λ is determined and compared with the normal range. This aids in the diagnosis of monoclonal disease. Moreover, the FLC assays may also be used for the following of treatment of the disease in patients. Prognosis of, for example, patients after treatment for AL amyloidosis may be carried out.

Katzmann et al (Clin. Chem. (2002); 48(9): 1437-1944) discuss serum reference intervals and diagnostic ranges for free κ and free λ immunoglobulins in the diagnosis of monoclonal gammopathies. Individuals from 21-90 years of age were studied by immunoassay and compared to results obtained by immunofixation to optimise the immunoassay for the detection of monoclonal FLC in individuals with B-cell dyscrasia.

The amount of κ and λ FLC and the κ/λ ratios were recorded allowing a reference interval to be determined for the detection of B-cell dyscrasias.

The Applicants had identified that assaying for FLC and especially total or combined FLC (cFLC) can be used to predict long-term survival of individuals over a period of a number of years, even when the individual is an apparently healthy subject. They have found that FLC concentration is statistically, significantly linked to long-term survival. Moreover, this link appears to be similar or better than the link for existing long-term survival prognostic markers such as cholesterol, creatinine, cystatin C and C-reactive protein.

More recently cFLC have been shown to be prognostic for a number of clinical scenarios, including chronic kidney disease (CKD) (Stringer S. Haematology Reports (2010) 2(S2) page 6). Elevated cFLC in samples of serum in patients referred to a haematology unit have been shown to correlate to increased frequency of death in patients after 100 days (Basu S et al J Clin Pathol (2012) doi: 10.1136/jclinpath-2012-200910).

Not all patients with CKD will progress to needing renal replacement therapy (RRT). RRT is, for example, dialysis or a kidney transplant. Most patients in CKD stages 3b-5 are monitored in secondary care by follow-up appointments often in the specialist renal units at hospitals. For example, patients with CKD stage 4, showing estimated glomerular filtration rates (eGFR) of 15-30ml/min/1.73m , are typically followed-up at between 6 weekly and 3 monthly appointments. This is very time consuming and expensive, not only for the patient, but also for the specialist renal unit to which they are often referred. Tangri et al (JAMA, (2011) E1-E7) discloses a predictive model for progression of CKD to kidney failure. That model looked at a variety of factors including age, sex, eGFR, albuminuria, serum calcium, serum phosphate, serum bicarbonate and serum albumin concentrations to produce a model which produces a probability of renal failure expressed as a percentage over time. This can be used as a guide to indicate those at more or less risk of renal failure over 5 years. It does not provide specific guidance on interpretation of the data in regards to individual patient management.

The Applicant has now assessed the risk of RRT by looking at free light chains and other markers or risk factors to produce an improved, easier to use, scoring system.

The invention provides a method of identifying the risk of a patient with chronic kidney disease (CKD) requiring renal replacement therapy (RRT) within a predetermined period of time comprising determining a value of free light chains (FLC) in a sample from the patient, comparing the value to a predetermined value and assigning a risk score to that value of FLC in the sample to indicate the risk of the patient requiring RRT. The time period of time may be at least 6, 8 or 10 or approximately 12 months or 361 days, or 2, 3, 4 or 5 years.

The risk stage of CKD may be any risk stage. It may be, for example, risk stage 3b-5 or 4.

One or more additional risk factors may be determined selected from. The presence of diabetes in a patient; the eGFR value, serum phosphate value, serum calcium value, serum cystatin C value, ACR value; ethnicity of the patient, underlying renal diagnosis of the patient, sodium value, potassium value, bicarbonate value, creatinine value, C reactive protein (CRP) value, an albumin value and a parathyroid hormone (PTH) value. These values may be measured by techniques generally known in the art.

Table 1 shows regression values associated with risk of progression to RRT.

The method may typically determine one or more additional risk factors in the patient by determining whether the patient has diabetes by measuring the eGFR value for the patient, measuring a serum phosphate value for the patient, and/or measuring an albumin/creatinine ratio (ACR) value for the patient. Typically all of these risk factors are determined, though alternatively only one or two may be determined. Typically, at least FLC, eGFR, serum phosphate, and ACR, and optionally diabetes levels, values and scores are determined.

A risk score may be assigned for each risk factor. For example, the or each value determined for the patient may be compared to a predetermined value to identify a risk score associated with the value. That risk may be expressed as a positive or negative or zero risk, or for example if the value is above a particular level and shows a risk then a score of +1 is given, or below that level a score of zero is given. Similarly if the patient has diabetes then a positive risk, or +1 score is given, or if the patient does not have diabetes a negative or zero score is given.

Table 1. Cox regression analysis of complete CKD cohort (stages 1-5) and risk of progression to RRT (n=867), of which 169 progress to RRT

HR (95% CI) P- value

Age 1.003 (0.994-1.012) 0.461

Gender

Male 1

0.940 Female 0.988 (0.730-1.337)

Ethnicity

Caucasian 1

Black 1.793 (1.048-3.069) 0.033

Asian 1.687 (1.162-2.451) 0.006

Renal Diagnosis (%)

Glomerulonephritis 1

Pyelonephritis 1.940 (0.989-3.802) 0.054 Polycystic kidney 3.938 (2.356-6.584) <0.001 Renal vascular disease 1.584 (0.995-2.524) 0.053 Interstitial nephritis 1.312 (0.468-3.680) 0.606 Diabetes 5.692 (3.559-9.106) <0.001 Unknown/other 2.376 (1.439-3.924) 0.001

Diabetes (as co-morbidity) 1.794 (1.302-2.473) <0.001

Cardiovascular disease 0.975 (0.666-1.426) 0.894

ACEi 1.122 (0.815-1.546) 0.480

Albumin 0.948 (0.925-0.971) <0.001

Calcium 0.204 (0.104-0.402) <0.001

Phosphate 20.070 (12.358-32.597) <0.001

Creatinine * 15.706 (12.011-20.537) <0.001 eGFR (MDRD)* 0.100 (0.079-0.126) <0.001

CKD group

1 1

2 N/A N/A

3a 0.781 (0.081-7.507) 0.830 3b I .406 (0.176-11.239) 0.748 4 I I .519 (1.599-82.960) 0.015 5 61.714 (8.583-443.752) <0.001

ACR* 1.611 (1.483-1.751) <0.001 hsCRP* 1.128 (1.001-1.271) 0.048

Cystatin C 2.127 (1.951-2.318) <0.001 Serum kappa* 5.275 (4.247-6.551) <0.001

Serum lambda* 5.479 (4.418-6.795) <0.001

Serum cFLC* 5.855 (4.694-7.302) <0.001

Urinary kappa* 1.806 (1.527-2.135) <0.001

Urinary lambda* 1.804 (1.555-2.093) <0.001

* Data analysed on a logarithmic scale

A risk score is typically determined for FLC, eGFR, serum phosphate and ACR and optionally diabetes for the patient.

If a patient has values for all of the risk factors determined and none of the values show a risk, then the patient is unlikely to require RRT within 12 months. A score of one positive risk factor suggests that the patient is unlikely to need RRT within 6 months.

The FLC may be kappa or lambda FLC. However, preferably the total FLC concentration is measured as detecting kappa FLC or lambda FLC alone may miss, for example abnormally high levels of one or other FLC produced for example monoclonally in the patient.

Combined FLC (cFLC) means the total amount of free kappa plus free lambda light chains in a sample.

The term "total free light chains" means the amount of κ and λ free light chains in the sample from the subject.

The sample is typically a sample of serum from the subject. However, whole blood, plasma or urine may be used. Serum may also be used to determine serum phosphate for example, and the ACR value determined in urine.

Typically the FLC, such as total FLC, is determined by immunoassay, such as ELISA assays or utilising fluorescently labelled beads, such as Luminex™ beads. Alternatively, it may be used in the form of a lateral flow point of care test kit generally known in the art.

ELISA, for example uses antibodies to detect specific antigens. One or more of the antibodies used in the assay may be labelled with an enzyme capable of converting a substrate into a detectable analyte. Such enzymes include horseradish peroxidase, alkaline phosphatase and other enzymes known in the art. Alternatively, other detectable tags or labels may be used instead of, or together with, the enzymes. These include radioisotopes, a wide range of coloured and fluorescent labels known in the art, including fluorescein, Alexa fluor, Oregon Green, BODIPY, rhodamine red, Cascade Blue, Marina Blue, Pacific Blue, Cascade Yellow, gold; and conjugates such as biotin (available from, for example, Invitrogen Ltd, United Kingdom). Dye sols, metallic sols, chemilumine scent labels or coloured latex may also be used. One or more of these labels may be used in the ELISA assays according to the various inventions described herein, or alternatively in the other assays, labelled antibodies or kits described herein.

The construction of ELISA-type assays is itself well known in the art. For example, a "binding antibody" specific for the FLC is immobilised on a substrate. The "binding antibody" may be immobilised onto the substrate by methods which are well known in the art. FLC in the sample are bound by the "binding antibody" which binds the FLC to the substrate via the "binding antibody".

Unbound immunoglobulins may be washed away.

In ELISA assays the presence of bound immunoglobulins may be determined by using a labelled "detecting antibody" specific to a different part of the FLC of interest than the binding antibody.

Flow cytometry may be used to detect the binding of the FLC of interest. This technique is well known in the art for, e.g. cell sorting. However, it can also be used to detect labelled particles, such as beads, and to measure their size. Numerous text books describe flow cytometry, such as Practical Flow Cytometry, 3rd Ed. (1994), H. Shapiro, Alan R. Liss, New York, and Flow Cytometry, First Principles (2nd Ed.) 2001, A.L. Given, Wiley Liss.

One of the binding antibodies, such as the antibody specific for FLC, is bound to a bead, such as a polystyrene or latex bead. The beads are mixed with the sample and the second detecting antibody. The detecting antibody is preferably labelled with a detectable label, which binds the FLC to be detected in the sample. This results in a labelled bead when the FLC to be assayed is present. Other antibodies specific for other analytes described herein may also be used to allow the detection of those analytes.

The Applicant has found that cFLC levels above 50mg/L in serum may identify patients at risk of needing RRT, for example within 12 months more typically above 80, 90, 96.3, 100, 104 mg/L, above 120mg/L or above 150 mg/L may be used as the cut off, above which a positive risk factor is shown and a score may be recorded for the FLC value in the sample.

Diabetes may optionally be used. It increases the risk of RRT. Hence it may produce a positive or +1 risk value eGFR may be calculated using the 4-variable modification of diet in renal disease (MDRD) comprised of gender, age, ethnicity and serum creatinine (Vervoot 2002, Nephrol Dial Transplant. 17(11): 1909- 13).

Urine albumin creatine ratio (ACR) may be measured on Roche Modular analysers. Urine Albumin is measured by automated immunoturbidimetry using antibody against human albumin. Urine creatinine is measured by a compensated version of the Jaffe reaction using alkaline picrate. eGFR levels of below 25, typically below 20 or below 15 ml/min/1.73m indicates a risk of progression to RRT. Hence below such a value may be given a positive or +1 risk value. Similarly serum phosphate levels above 1.3, typically above 1.36 or above 1.4mmol/L; and/or the presence of albuminuria, either micro, typically ACR ratios above 2.5, 3 or 3.5, or macro, typically ACR ratios above 25, 28 typically above 30 or 35mg/mmol indicate risks of factors for requiring RRT and may then be given a positive or +1 risk value.

Calcium values of below 2.1 mmol/L serum indicates increased risk, as does serum albumin levels below 34 g/L serum.

Assay kits for determining the combination of cFLC value and one or more additional values are also provided. In practice the Applicant has found that no score of the values described above indicates that the patient is unlikely to require RRT within 12 months.

This means that the patient can be monitored by their local doctor at this time, rather than having to attend specialist renal clinic, or else may attend specialist renal clinics less frequently, thus reducing costs for those clinics and freeing valuable time for members of those clinics

Those patients identified at high risk are then typically monitored more closely and/or frequently in specialist renal units They then may receive treatment by drug or dialysis, for example.

The invention will now be described by way of example only with reference to the following figures:

Figure 1 shows Kaplan-Meier patient progression to RRT based on zero, one, two or three (top to bottom lines) of positive risk factors.

Data: Using a cohort of CKD stage 4 patients from Birmingham (UHB), we assessed the risk of progression to RRT.

Figure 2 shows further analysis of data discussed below. Kaplan-Meier analysis of patients from the development population who progressed to RRT over (A) the duration of patient follow-up and (B) within the first year of follow-up. Patients were scored by the number of risk factors for which they were positive (- 0, - 1, - 2+ - upper, middle, lowest line, in (B) the upper and middle lines overlap). Risk factors included eGFR<20ml/min/1.73ni2, ACR>30mg/mmol, phosphate>1.4mmol/L and cFLC>120mg/L

All available biochemical parameters were associated with an increased risk of progression (using clinically relevant categories below) Table 2. Factors associated with progression to RRT in patients with CKD stage 4, using categorical variables

cFLC cut-off was ROC determined. eGFR cut-off is the median for CKD stage 4

The optimal cut-off for risk assessment of progression using combined FLC (cFLC) was 96.3mg/L. We have also shown (in a different cohort) that cut-offs as low as 50 can split the population into progressors and non-progressors. Using the above variables an optimal model was developed which classified patients by the number of risk factors that they were positive for. These risk factors included: Diabetes (Yes/No), eGFR<20ml/min/1.73m , serum phosphate>1.36mmol/L, albumin/creatinine ratio (ACR) >30 (i.e. marco-albuminuria), and serum cFLC >104mg/L.

Table 3. Optimal risk model for 5 year risk of progression/non-progression to RRT in CKD stage 4 patients

Using this model there were no patients with 0 risk factors that progressed during the 5 year follow-up period. Patients with 3+ positive variables were grouped, and compared to patients with 2, 1, and 0 positive variables.

When this risk of progression was capped to 1 year, there were no patients with 0 risk factors who progressed, and 1 patient with 1 risk factor who progressed at 361 days. The patients who died before progression did not die from chronic renal complications. Table 4. Risk of progression/non-progression to RRT at 12 months in patients with CKD sta

Summary: Using this model we could identify patients with 0 risk factors that are unlikely to progress within the next 12 months, and patients with 1 risk factor who are also unlikely to progress within the next 6 months.

These patients could potentially be followed-up less stringently within hospital outpatient clinics, or in some cases, may be discharged to follow-up under primary care (GP) until their relative risk of progression changes.

Further Data

This model was further improved by using the following risk factors: cFLC > 120 mg/L, ACR > 30, eGFR <20 mL/min/1.73m" and serum phosphate > 1.4 mmol/L. This has been found to classify patients without the need to determine whether diabetes is present in the patient, though this may still optionally be determined.

This was based on an analysis of 201 patients with GFR, ACR, FLC and phosphate. Patients with missing results were excluded. The data also includes bicarbonate which was only available on 172 patients. The cFLC shown here is the median, not ROC optimised cut off. Table 5

Variable Hazard Ratio P

Age 0.970 (0.954-0.985) <0.001

Sex (Female) 1.102 (0.662-1.836) 0.709

Ethnicity

White 1

Black 2.919 (1.300-6.555) 0.009

Asian 1.954 (1.074-1.836) 0.028

Renal Diagnosis

GN 1

Pyelonephritis 3.560 (1.230-10.307) 0.019

Polycystic 3.231 (1.420-7.354) 0.005

RVD 0.811 (0.351-1.871) 0.623

Interstitial Nephritis 1.780 (0.495-6.397) 0.377

Diabetes 2.239 (0.985-5.088) 0.054

Unknown/Other 1.293 (0.478-3.500) 0.613

Diabetes Co-Morbidity 1.100 (0.653-1.855) 0.720

History of CVD 0.639 (0.332-1.231) 0.181

Albumin<34g/L 3.849 (1.200-13.342) 0.023

Calcium<2.1mmol/L 2.528 (1.273-5.023) 0.008

Phosphate>1.4mmol/L 1.944 (1.165-3.243) 0.011

Bicarbonate<21mmol/L 1.373 (0.582-3.238) 0.469

eGFR<22.5ml/min/1.73m² 4.339 (2.302-8.177) <0.001

ACR>30mg/mmol 3.732 (2.165-6.434) <0.001

cFLC>83.8mg/L 2.447 (1.436-4.171) <0.001

Table 6 shows cross-tabulation of patients from the development population who progressed to RRT over the duration of patient follow-up and within the first year of follow-up. Patients were scored by the number of risk factors for which they were positive; risk factors included eGFR≤20ml/min/1.73m², ACR>30mg/mmol, phosphate>1.4mmol/L and cFLC>120mg/L. Kaplan Meier analysis of this data is shown in Figures 2(a) and (b).

Total Follow-Up First Year Follow-up

Positive Non- Progressors Non- Progressors Total

Risk Progressors Progressors

Factors

0 51 0 51 0 51

1 48 13 60 1 61

2 21 27 46 2 48

3+ 21 20 37 4 41

This shows that two or more risk factors improve the stratification of the patients.

Claims

1. A method of identifying the risk of a patient with chronic kidney disease (CKD) requiring renal replacement therapy (RRT) within a predetermined period of time comprising determining a value of free light chains (FLC), preferably combined FLC, in a sample from a patient, comparing the value with a predetermined value and assigning a risk score to that value of FLC in the sample to indicate the risk of the patient requiring RRT over a predetermined length of time.

2. A method according to claim 1, additionally determining one or more of the following risk factors in a patient selected from : the presence of diabetes, the eGFR value, serum phosphate value, serum calcium value, serum cystatin C value, ACR value, ethnicity of the patient, underlying renal diagnosis of the patient, sodium value, potassium value, bicarbonate value, creatinine value, C-reactive protein value, albumin value and PTH value.

3. A method according to claim 2 additionally comprising determining one or more, preferably two or more, of the following risk factors in the patient by determining the eGFR value for the patient, the serum phosphate value for the patient and/or the albumin/creatinine (ACR) value in the patient

4. A method according to claim 3, additionally comprising determining whether the patients has diabetes.

5. A method according to claim 1 to 4, wherein a risk score is assigned to the or each risk factor in the patient.

6. A method according to claims 3 to 5, wherein all of the risk factors as measured.

7. A method according to any preceding claim, wherein the or each value determined for the patient is compared to a predetermined value and if the value shows it is a risk value, that value is given a positive score or if the patient has diabetes the patient is given a positive risk score.

8. A method according to claim 3 to 7, wherein the risk score for FLC, diabetes eGFR, serum phosphate and ACR is determined.

9. A method according to claim 8, wherein the risk score for diabetes is additionally determined.

10. A method according to claims 6 to 9, wherein if the patient does not have any positive scores, then the risk of progression to RRT within 1 year is zero.

11. A method according to any preceding claim, wherein the patient has a positive risk factor if the value for the or each risk factor is;

FLC above 50 mg/L or above 104 mg/L in serum;

eGFR less than 20 ml/min/1.73m²;

serum phosphate at least 1.36 mmol/L;

ACR at least 30mg/mmol;

or if the patient has diabetes a positive risk factor is given

12. A method according to claim 11, wherein the patient has a positive risk factor if the value for the or each risk factor is: cFLC above 120 mg/L

eGF≤20ml/min/1.73m²

serum phosphate above 1.4 mmol/L;

ACR above 30mg/mmol.

13. An assay kit for carrying out a method according to any preceding claim comprising anti-FLC antibodies or fragments thereof and instructions to use the antibodies in a method according to any preceding claim.

14. An assay kit for carrying out a method according to claims 1 to 12, comprising of multiple analytes, such as FLC, creatinine and albumin, by multiplex method on a single or dual analyser in a sample from the patient.