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Technical Specifications
The results of this test should be interpreted and utilized after review of the following specifications:
Product Indication
The test genotypes a single nucleotide polymorphism (SNP), rs396991, in the FCGR3A gene (encoding the Fc gamma IIIa receptor to which rituximab binds)
that results in an amino acid-changing polymorphism, V158F. This SNP has been shown to be associated with response to rituximab in the treatment of
Follicular, CD20-positive NHL. (G Cartron et al. Blood, 99:754-758. 2002; WK Wang & R Levy. Journal of Clinical Oncology, 21(21):
3940-3947. 2003.).
Description Of Genetic Assays
A DNA sample is genotyped by restriction fragment length polymorphism (RFLP) analysis, which includes amplification of the gene region containing
the specific SNP by polymerase chain reaction (PCR) followed by restriction enzyme digestion and resolution of the restriction enzyme digestion
fragments using gel electrophoresis and visualization.
Description Of Methods
- Sample acquisition: Whole blood is collected via finger stick on to an FTA Classic Card (Whatman , Middlesex, UK) from which genomic DNA is isolated.
Bar code identifiers are used to identify and track samples through a laboratory information management system.
- PCR amplification: Amplification of the DNA by PCR is used to generate templates for restriction enzyme digestion. In addition to the patient DNA sample,
a positive, negative, and blank control are included in the analysis.
- Genotyping: After the PCR reaction, an aliquot from the amplified DNA is used for restriction enzyme digestion. The restriction enzyme digestion fragments
are resolved using gel electrophoresis and visualized in the presence of ethidium bromide.
FCGR3A Analysis
The FCGR3A receptors are located at the surface of Natural Killer (NK) cells where they bind to the Fc region of Immunoglobulin G1 (IgG1), thus promoting
the NK cell immune response. The FCGR3A receptor 158V allele displays a higher affinity for human IgG1 than the 158F allele, as well as increased
antibody-dependent cellular cytotoxicity. (S Dall’Ozzo et al. Cancer Research, 64:4664-4669. 2004).
In the treatment of Follicular, CD20-positive Non-Hodgkin’s Lymphoma, patients homozygous for the FCGR3A-158V allele have a 75% to 90% chance of
experiencing a clinical response to rituximab monotherapy, while patients carrying one or two copies of the 158F allele have only a 25% to 51% chance of
responding to the treatment. (G Cartron et al. Blood, 99:754-758. 2002; WK Wang & R Levy. Journal of Clinical Oncology, 21(21):3940-3947. 2003.).
The receptor amino acids valine (V) and phenylalanine (F) are encoded respectively by the G and T nucleotides of the SNP (rs396991), which is located at
position 4985 in exon 4 of the FCGR3A gene. The restriction enzyme HincII is used to genotype this SNP in the RFLP analysis.
- Quality Control
Two trained technicians independently score all the fragments and a trained supervisor reconciles discrepancies.
Report Generation: The patient’s V158F genotype will be reported. The final report is reviewed and signed by a CLIA-licensed Laboratory Director.
Performance Characteristics
- Accuracy: The RFLP assay used to determine genotypes for this SNP has been validated. Genotype results obtained from this method have been
analyzed for accuracy by comparing them with genotypes obtained using bidirectional sequencing. In all the accuracy tests, the genotype calls obtained
by the RFLP method were identical to the genotype calls obtained by bidirectional sequencing.
- Limitations: Failure to follow sample collection and shipping instructions might reduce DNA quality and affect the results.
- The PGxPredict™:RITUXIMAB Test will identify the 20% of patients who are more likely to respond to rituximab with 94-100% specificity.
The PGxPredict:RITUXIMAB Test is 20-35% sensitive. (G Cartron et al. Blood, 99:754-758. 2002; WK Wang & R Levy. Journal of Clinical Oncology,
21(21):3940-3947. 2003.).
This test was developed and its performance characteristics determined by Clinical Data, Inc. FDA approval is not currently required for clinical use of this test.
Validation was done as required by the Clinical Laboratory Improvement Act of 1988 (CLIA).
Test Interpretation
The genotypes will be interpreted according to the following table:
| G4985T Genotype |
V158F Genotype |
Rituximab Efficacy |
| GG |
V/V |
More likely to respond |
| TG |
V/F |
Less likely to respond |
| TT |
F/F |
Less likely to respond |
1-877-2-PGXHEALTH (877-274-9432)
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