Rapid direct determination of HLA-DQB1 ∗ 0301 in the whole blood of normal individuals and cancer patients by specific polymerase chain reaction amplification

Abstract

The HLA class II DQB1 ∗ 0301 allele is present at a higher frequency in patients with malignant melanoma than in Caucasian controls. Furthermore, HLA-DQB1 ∗ 0301 identifies a group of melanoma patients presenting with relatively advanced disease, and independently identifies a group of melanoma patients more likely to have disease recurrence. A rapid screening test for HLA-DQB1 ∗ 0301 may be useful in clinical research involving melanoma patients. Standard molecular oligotyping for HLA class II alleles using the polymerase chain reaction (PCR)-sequence specific oligonucleotide (SSO) method is relatively expensive, labor-intensive, and involves the use of radioisotope. We therefore developed an inexpensive, rapid, non-radioactive method using sequence-specific primers, peripheral whole blood as the substrate, and strictly defined reaction conditions in a single-step PCR to allow determination of the presence or absence of genomic HLA-DQB1 ∗ 0301. Comparison of the single-step PCR method with standard PCR-SSO oligotyping on 63 blinded samples from Caucasian melanoma patients demonstrated complete agreement between the two methods in the detection of HLA-DQB1 ∗ 0301. Confirmatory testing in 456 additional cancer patients and healthy controls showed a sensitivity of 98.0% and a specificity of 99.4%. Single-step PCR is accurate, rapid, inexpensive, and does not require radioisotope. These advantages make it the procedure of choice for screening melanoma patients and others for the presence of the HLA-DQB1 ∗ 0301 allele.