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Vectra® Measures 12 Biomarkers Implicated in Key Pathways of Rheumatoid Arthritis

The 12 Biomarkers included in the Vectra score provide a molecular measure of inflammation

VCAM-1 Vascular Cell Adhesion Molecule-1 VCAM-1

(Vascular cell adhesion molecule-1) is an adhesion molecule expressed by endothelial and synovial cells. VCAM-1 may contribute to cellular recruitment to synovial tissue, as well as to cartilage invasion and destruction by fibroblasts. Vectra measures the soluble form of VCAM-1.

IL-6 Interleukin 6

IL-6 (Interleukin 6) is a cytokine produced by multiple cells, including leukocytes, fibroblasts, and skeletal cells in RA. IL-6 is a major driver of RA inflammation, cartilage degradation, bone erosion, and the hepatic acute phase response.

MMP-3 Matrix Metalloproteinase-3 or Stromelysin-1

MMP-3 (Matrix metalloproteinase-3 or stromelysin-1) is an enzyme that degrades glycosaminoglycan components to cartilage and also activates the collagenase MMP-1.

Resistin

Resistin is also a hormone associated with the adipose tissue. Like leptin, it can be secreted by synovial tissue and bone and promotes inflammation and bone remodeling. Resistin has been associated with obesity and diabetes.

EGF Epidermal Growth Factor

EGF (Epidermal growth factor) is a growth factor secreted by macrophages, fibroblasts, and endothelial cells in RA joint tissue. EGF is associated with proliferation and differentiation of stromal cells (fibroblasts, chondrocytes, endothelial cells) and can induce production of inflammatory mediators and proteinases in these cells. EGF may also modulate the hepatic acute phase response.

TNF-RI Tumor Necrosis Factor Receptor, Type 1

TNF-RI (Tumor necrosis factor receptor, type 1) is a receptor for TNF-a that is expressed on the membranes of numerous cell types. TNF-R1 mediated signaling contributes to multiple effects of TNF-a including induction of cell death. Vectra measures the soluble form, TNF-R1, which binds to and neutralizes TNF-a.

YKL-40 Human Cartilage Glycoprotein 39

YKL-40, or human cartilage glycoprotein 39, is secreted primarily by chondrocytes and differentiated macrophages. YKL-40 may promote chondrocyte and fibroblast proliferation and antagonize cartilage destruction. YKL-40 reactive T cells have been found in RA patients, suggesting that it may contribute to RA autoimmunity.

SAA Serum Amyloid

SAA (Serum amyloid) is another major acute phase protein secreted by the liver in response to inflammation. Like CRP, elevated SSA may be associated with cardiovascular risk. SAA may also be produced by synovial fibroblasts and chondrocytes, and may induce proinflammatory activation of fibrobasts, macrophages, and T cells.

VEGF-A Vascular Endothelial Growth Factor A

VEGF-A (Vascular endothelial growth factor A) is a potent angiogenic growth factor and vascular permeability factor expressed by various synovial cells including fibroblasts and macrophages. VEGF-A promotes inflammation, fluid accumulation, and bone erosion.

MMP-1 Matrix Metalloproteinase-1 or Collagenase-1

MMP-1 (Matrix metalloproteinase-1 or collagenase-1) is a collagen-degrading enzyme that contributes to cartilage destruction in RA and to leukocyte invasion and angiogenesis in the synovial tissue.

Leptin

Leptin is a hormone secreted by adipose tissue, synovial tissue and bone. A satiety factor associated with obesity, leptin can also activate leukocytes and regulate bone remodeling.

CrP C-Reactive Protein

CRP (C-reactive protein) is a major acute phase protein secreted by the liver in response to inflammation. Elevated CRP levels are associated with an elevated risk of cardiovascular disease.

Vectra® Technical Specifications
Crescendo Biosciences, Inc. Effective Date: July, 2020

TEST RESULTS SHOULD BE USED ONLY AFTER REVIEW OF THE FOLLOWING SPECIFICATIONS:

Indications and Use

Intended Use
Vectra® is a multi-biomarker disease activity (MBDA) test for measuring rheumatoid arthritis (RA) inflammatory disease activity. It is validated for use in adults diagnosed with RA. Vectra measures the serum concentrations of 12 protein biomarkers reflecting the pathobiology of RA and combines them with demographic and clinical information (age, gender and adiposity) to generate an integer score on a scale of 1 to 100 that represents the level of RA disease activity in that patient.

Description of Method

Vectra can only be tested on serum.  The serum must be prepared from a blood sample collected in a Serum Separator tube, processed following the applicable instructions of the manufacturer and received by the laboratory within 7 days of the sample draw date. Samples should be refrigerated until they are shipped, at which time they should be packaged according to the provided instructions. Alternatively, serum obtained as described above can be frozen at -80ºC and shipped on dry ice. At -80ºC samples are stable for several years and can be processed for testing.

Upon receipt in the laboratory, patient samples are assigned a unique bar-code for robotic specimen tracking, and the serum is aliquoted and diluted in preparation for the assay.

The Vectra test uses multi-spot 96-well immunoassay plates (MSD) to measure biomarker concentrations in wells that have been spotted with biomarker-specific capture antibodies. These antibodies are grouped in 3 multiplex panels, with one panel multi-spotted per well: Panel A (EGF, IL-6, leptin, and VEGF-A), Panel B (CRP, SAA, and VCAM-1) and Panel C (MMP-1, MMP-3, resistin, TNF-RI, and YKL-40). Patient samples and serum controls are diluted in panel-specific assay diluents and loaded by robot onto the plates for testing alongside pre-diluted biomarker standards. After plates are incubated and washed, detecting antibodies conjugated to SULFO-TAG are added. After another wash, a Read Buffer containing a co-reactant for light generation is loaded into each well. Plates are read by electrochemiluminescence on the Sector Imager 6000 (MSD). Automated robotic devices perform these steps. Data acquisition utilizes the Discovery Workbench software (MSD) for:

1) Simultaneous quantification of analytes in multiplex formats using electrochemiluminescence detection,

2) Generation of biomarker standard curves, and

3) Interpolation of concentrations for each sample and control. Biomarker concentration data are uploaded into a laboratory information management system for quality control assessment, Vectra score generation and reporting of results.

4) Results include an adjusted Vectra score, an estimated 1-year risk of radiographic progression visualized in a risk curve determined from the validation cohort, assessment of change in score in relation to minimally important difference for the Vectra score for patients with a score in the high or moderate disease activity categories, and interpretation of the Vectra score.

Performance Characteristics/Limitations

Accuracy of the Vectra Test
To evaluate the accuracy of each biomarker assay, biomarker concentrations were measured in 16 sample mixtures and the percent recovery, relative to the expected concentration, was calculated. Test samples were prepared by mixing equal parts of two samples with known high and low biomarker concentrations. For the 12 biomarkers in the Vectra test, the average recovery ranged from 92% to 102%.

Precision of the Vectra Test
Inter-assay and intra-assay precision of the 12 individual biomarker assays was determined. Inter-assay precision (between plates) was evaluated by testing 6 samples on approximately 40 plates per panel over 10 days with multiple operators and instruments. The average inter-assay coefficient of variation (CV)  ranged from 7% to 18%. Intra-assay precision (within plate) was tested by calculating the average CV for each biomarker using 14 replicates of 4 serum pools analyzed on 4 plates. For the 12 biomarkers, the average intra-assay CV ranged from 5% to 19%. Inter-run and intra-run precision of the Vectra score were determined by analyzing multiple replicates of Vectra samples in a separate experiment. Three inter-batch replicates were run, each with two intra-batch replicates of 101 samples. Inter-run standard deviation of the Vectra score was 0.86. Intra-run standard deviation, calculated using the same dataset, was 0.78. The overall reproducibility was found to be 1.17 Vectra score units.

Sensitivity of the Vectra Test
Upper and lower limits of quantitation (LOQ) were established for each of the 12 biomarkers in the Vectra test. The LOQ is defined as the concentration of analyte that can be reliably detected in a sample, and at which the total analytical error meets the laboratory requirements for accuracy and precision.  The acceptable accuracy requirement at the LOQ was 80% to 120% recovery, and the acceptable precision requirement at the LOQ was 20% CV. The upper limit (ULOQ) and the lower limit (LLOQ) of quantitation define the analytical measurable range. They are then dilution-adjusted to define the clinically reportable range for each biomarker. LLOQ and ULOQ were determined by performing multiple measurements of biomarker concentrations using a panel of 8 serum samples (with targeted concentrations at the upper and lower limits of the standard curves) for approximately 40 plates. LOQs were initially established in the analytical validation studies and might be subsequently adjusted when new reagent lots are manufactured. In a sample for which any biomarker concentration is measured above the ULOQ or below the LLOQ, the Vectra score is calculated using the ULOQ/LLOQ of that biomarker as its clinically reportable value.

Interference
The level of interference caused by each of the following potential sources has been evaluated for the Vectra test: Common sample abnormalities (hemolysis, icterus and lipemia), therapeutic drugs commonly administered to patients with RA (abatacept, acetaminophen, adalimumab, certolizumab pegol, etanercept, golimumab, ibuprofen, infliximab, methotrexate, methylprednisone, rituximab, and tocilizumab), heterophilic antibodies (rheumatoid factor [RF]) and human anti-mouse antibodies (HAMA). To evaluate common sample abnormalities and therapeutic drug interference, serum samples that contained varying endogenous biomarker concentrations were spiked with selected levels of each interferant and biomarker measurements were compared to a corresponding spike control sample (e.g., a sample not containing the interferant). Spiking concentrations for sample abnormalities and therapeutic drugs were determined from either CLSI standard EP07-A2 (Interference Testing in Clinical Chemistry) or published pharmacokinetic data. Interference from antibodies was evaluated by mixing serum samples with known high concentrations of RF or HAMA with samples that had low/no RF or HAMA in equal amounts and comparing the measured biomarker concentrations to the expected results based on dilution alone. The acceptance criterion to demonstrate that a substance did not interfere with an individual assay at the biomarker concentration level was defined as either a mean or median percentage recovery between 80% and 120% across all of the samples tested. Vectra scores were calculated using the individual biomarker concentrations for each sample containing the various interferants and were compared to the Vectra scores of the corresponding spike control samples or to the Vectra scores of the unmixed samples, as appropriate. For each of the 12 biomarkers in the Vectra test, the mean or median percentage recoveries were within the range of 80% to 120% for all interferants tested. The absolute difference between the Vectra score of a sample containing an interferant and its corresponding spike control was ≤ 3 SD for each sample tested, indicating the results were within the assay variability of the Vectra test.

Quality Control Measures
Manufactured standard solutions with known biomarker concentrations and a set of control serum samples with high and low Vectra scores (prepared from pooled human serum samples) are run on each sample plate. These controls are analyzed to verify that the expected results have been obtained.

Cross-reactivity
Cross-reactivity with biologically relevant analogs was assessed for each of the 12 Vectra analytes by the vendors who supply the capture and detection antibodies for the immunoassays or by additional testing performed at Crescendo Bioscience. Cross-reactivity was observed to be absent or at levels considered to be analytically and clinically insignificant for each biomarker.

Limitations
The Vectra test is not intended or validated to:
–        Diagnose RA or guide therapy selection.
–        Diagnose other inflammatory or non-inflammatory joint diseases.
–        Assess disease activity in joint diseases other than RA.
–        Be used in patients younger than 18 years of age.
–        Be used for interpretation of individual biomarker results.
Patient serostatus may affect the risk of radiographic progression. Thus, the actual risk of radiographic progression may be higher if a patient is seropositive and lower if this patient is seronegative.

Sample Rejection Criteria
Inappropriate sample types can cause cancelation of the test. Inappropriate sample types include: Samples from patients not diagnosed with RA, unspun and partially spun samples, samples not collected in Serum Separator tubes or collected in expired Serum Separator tubes, and heavily hemolyzed samples. Samples for which insufficient clinical information has been provided may be canceled. Samples of insufficient serum quantity (<1 ml) or quality may also be canceled. Insufficient quality may be due to damage occurring during shipping, an extended period of time between sample collection and receipt by the laboratory, or sample exposure to temperature >22ºC (72ºF). A test may also be canceled if the time from a previous Vectra test is <14 days.

Summary and Interpretation − Vectra Score
The Vectra test measures the following 12 biomarkers: adhesion molecules (vascular cell adhesion molecule 1 [VCAM-1]); growth factors (epidermal growth factor [EGF] and vascular endothelial growth factor A [VEGFA]); cytokine-related proteins (interleukin 6 [IL-6] and tumor necrosis factor receptor type I [TNFRI]); matrix metalloproteinases (matrix metalloproteinase 1 and 3 [MMP-1]; [MMP-3]); a skeletal-related protein (cartilage glycoprotein 39 [YKL-40]); hormones (leptin and resistin); and acute-phase proteins (serum amyloid A [SAA] and C-reactive protein [CRP]). The concentrations of these 12 biomarkers are measured in serum and entered in a proprietary formula to generate a score on a scale of 1 to 100 that represents the level of RA disease activity. By design, the formula is the sum of terms that are biomarker-based predictions of the 28-joint tender joint count (TJC28), 28-joint swollen joint count (SJC28) and patient global assessment, each of which uses many of the 11 non-CRP Vectra biomarkers; and a CRP term. This biomarker-based equation is analogous to the formula of the Disease Activity Score in 28 joints with CRP (DAS28-CRP). DAS28 (with ESR or CRP) is the most widely accepted tool for assessing disease activity in adults with RA in clinical trials.

In the Vectra equation, linear scaling is applied in combination with min, max, and round operations to produce an integer score between 1 and 100.

This original Vectra score, described above, was validated in adults diagnosed with RA. It was shown to be significantly associated with the DAS28-CRP in seropositive (AUROC 0.77, P < 0.001) and seronegative (AUROC 0.70, P < 0.001) patients.

Since December 4, 2017, the original Vectra score has been adjusted for age, gender and adiposity (based on leptin concentration), with this adjustment being applied to all commercial Vectra tests performed after this date. As before, the adjusted Vectra score is an integer on a scale of 1 to 100 with Vectra disease activity categories of Low (1-29), Moderate (30-44) and High (45-100). The adjusted Vectra score has been validated against DAS28-CRP and as a predictor of risk for radiographic progression over the following year.

Summary and Interpretation − Minimally Important Difference (MID)
The change between two Vectra scores can be interpreted in terms of the Minimally Important Difference (MID) for Vectra. The MID is the smallest absolute change in Vectra score that is likely to reflect change in disease activity and not be due only to random variation. The MID for patients with a moderate or high Vectra score is 8. The MID has not been established for patients with a low Vectra score. Treatment management decisions should be made in the context of all available clinical information.

Summary and Interpretation – 1 year risk of Radiographic Progression (RP)
The Sharp scoring system provides separate scores for erosions and for joint space narrowing seen on X-rays. Their sum, the total Sharp score, is an objective measurement of bone and cartilage damage in joints. Disease progression can be measured by comparing Sharp scores over time and subtracting the first total Sharp score value from the second.

The Vectra Score correlates with the risk of RP. As the Vectra Score increases the risk of RP increases. The risk of RP can be reported as a function of the patient’s Vectra Score. The definition of rapid RP is a change in total Sharp score over 1 year of >5 units. For Vectra scores in the Low (1-30) category the predicted risk of rapid RP is 1% to 3% and for Vecta scores in High (45-100) category the predicted risk of rapid RP is 7% to 47%. Patient serostatus may affect the risk of radiographic progression independently. Thus, the actual risk of radiographic progression for a patient may be higher if the patient is seropositive and lower if the patient is seronegative.

An increase of five Sharp units per year has been reported to be the minimal clinically important difference needed for rheumatologists to change therapy. A change >5 units has been used to define rapid radiographic progression in clinical trials.

Vectra is a prognostic test for predicting the risk of rapid radiographic progression., thus providing a convenient means of assessing the risk of new joint damage without using x-rays or advanced imaging.

A continuous risk curve has been generated that provides a risk estimate for any adjusted Vectra score. This curve demonstrates that the risk of radiographic progression approaches zero when the Vectra score is low and continuously increases with an increasing Vectra score.

References

  1. Brahe CH, Østergaard M, Johansen J, et al. Predictive value of a multi-biomarker disease activity (MBDA) score for clinical remission and radiographic progression in patients with early rheumatoid arthritis (RA) – a posthoc study of the OPERA trial. Scandinavian journal of rheumatology. 2018;46:353-358.
  2. Hambardzumyan K, Bolce R, Saevarsdottir S, et al. Pretreatment multi-biomarker disease activity score and radiographic progression in early RA: results from the SWEFOT trial. Annals of the rheumatic diseases. 2015;74:1102-9.
  3. Hambardzumyan K, Bolce RJ, Saevarsdottir S, et al. Association of a multibiomarker disease activity score at multiple time-points with radiographic progression in rheumatoid arthritis: results from the SWEFOT trial. RMD open. 2016;2(1):e000197. doi: 10.1136/rmdopen-2015-000197.
  4. Li W, Sasso EH, van der Helm-van Mil AH, et al. Relationship of multi-biomarker disease activity score and other risk factors with radiographic progression in an observational study of patients with rheumatoid arthritis. Rheumatology (Oxford). 2016;55:357-66.
  5. Markusse IM, Dirven L, van den Broek M, et al. A multibiomarker disease activity score for rheumatoid arthritis predicts radiographic joint damage in the BeSt study. Journal of rheumatology. 2014;41:2114-9.
  6. Van Der Helm-Van Mil AH, Knevel R, Cavet G, et al. An evaluation of molecular and clinical remission in rheumatoid arthritis by assessing radiographic progression. Rheumatology (Oxford). 2013;52:839-46.
  7. Curtis JR, et al. Uptake and Clinical Utility of the Multi-Biomarker Disease Activity Testing in the U.S. J Rheumatol. 2018; Nov 15.
  8. Chernoff P, et al. Determination of the minimally important difference (MID) in multi-biomarker disease activity (MBDA) test scores: impact of diurnal and daily biomarker variation patterns on MBDA scores. Clin Rheumatol. 2018; Aug 29.
  9. Curtis JR, et al. Validation of a novel multibiomarker test to assess rheumatoid arthritis disease activity. Arthritis Care Res. 2012; 64 (12): 1794-1803.
  10. Inoue E, et al. Comparison of Disease Activity Score (DAS)28- erythrocyte sedimentation rate and DAS28- C-reactive protein threshold values. Ann Rheum Dis 2007; 66: 407-409.
  11. Curtis J, et al. Validation of the Adjusted Multi-biomarker Disease Activity Score as a Prognostic Test for Radiographic Progression in Rheumatoid Arthritis: A Combined Analysis of Multiple Studies. EULAR 2020; Jun 02
  12. Huizinga T, et al. Predicting Risk of Radiographic Progression for Patients with Rheumatoid Arthritis. American College of Rheumatology, Annual meeting 2019; Abstract # 466
  13. van der Heijde DM. How to read radiographs according to the Sharp/van der Heijde method. The Journal of Rheumatology. 2000;27:261-263.
  14. Bruynesteyn K, van der Heijde D, Boers M, et al. Determination of the minimal clinically important difference in rheumatoid arthritis joint damage of the Sharp/van der Heijde and Larsen/Scott scoring methods by clinical experts and comparison with the smallest detectable difference. Arthritis & Rheumatism. 2002;46(4):913-920.
  15. Vastesaeger N, Xu S, Aletaha D, St Clair EW, Smolen JS. A pilot risk model for the prediction of rapid radiographic progression in rheumatoid arthritis. Rheumatology. 2009;48(9):1114-1121.
Validations

Vectra® was validated to assess RA disease activity through a rigorous development process.

The 12 protein biomarkers were selected from a pool of 396 candidate biomarkers
  • Validated against the DAS28-CRP in both seropositive (AUROC = 0.77; P<0.001) and seronegative patients (AUROC = 0.70; P<0.001)
  • Significantly correlated with change in disease activity based on DAS28-CRP and DAS28-ESR
  • Precise and reproducible — 28 samples were each assayed 10 times over multiple days with multiple operators (median coefficient of variation = 1.6%)
Crescendo Bioscience Laboratory Certifications

Federal CLIA Certificate
The Centers for Medicare & Medicaid Services (CMS) regulate all laboratory testing performed on humans in the U.S. through the Clinical Laboratory Improvement Amendments (CLIA). The objective of the CLIA program is to ensure quality laboratory testing.

CAP Laboratory Accreditation Program
The commission on Laboratory Accreditation of the College of American Pathologists examined Crescendo Bioscience’s laboratory records and quality control procedures for the preceding 2 years. In addition, staff qualifications, laboratory equipment, and the facility’s safety program and record, as well as the overall lab management were examined.

New York State Clinical Laboratory Permit
The Wadsworth Center at the New York Department of Health through its Clinical Laboratory Evaluation Program (CLEP) issued a laboratory permit to Crescendo Bioscience to provide services to physicians/patients in New York. The excellence of the CLEP program has been acknowledged by CMS through their granting of exempt status from the federal CLIA for laboratories located in and holding NYS clinical laboratory permits.

CMS coverage policy issued by Palmetto GBA for Vectra
Palmetto GBA posted a favorable coverage article for Vectra on May 10, 2013 under the Medicare Part B program, which also drives coverage policies for the large Medicare Advantage Plans.

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