A practical strategy for the routine use of BIOMED‐2 PCR assays for detection of B‐ and T‐cell clonality in diagnostic haematopathology

Abstract

SummaryBIOMED-2 polymerase chain reaction (PCR) assays for clonality analysis of immunoglobulin (IG) and T-cell receptor (TCR) gene rearrangements were evaluated in routine haematopathological practice where paraffin-embedded tissues constitute the majority of specimens. One hundred and twenty-five fresh/frozen and 316 paraffin specimens were analysed for DNA quality and clonality. Seventy-nine per cent of paraffin specimens yielded PCR products of over 300 bp. These specimens and all fresh/frozen specimens were analysed with the complete set of BIOMED-2 reactions for IG (8 reactions) and/or TCR (6 reactions) gene rearrangements. The rate of detection of clonality was 96% in mature B-cell neoplasms and 98% in mature T-cell neoplasms and there were no significant differences in these rates between paraffin and fresh/ frozen specimens. As the value of sole use of any individual BIOMED-2 reaction in clonality detection was limited, we assessed combinations of reactions that gave the greatest sensitivity with fewest reactions and were applicable for both fresh/frozen and paraffin specimens. For IG gene rearrangements, three reactions combining one targeting the IG heavy chain framework-2 region and two targeting the IG kappa locus achieved a 91% detection rate. For TCR gene rearrangements, the two TCR gamma reactions gave a 94% detection rate. We therefore recommend this strategy as the firstline assays for routine B-and T-cell clonality analysis in diagnostic haematopathology.

Nasal-type NK/T-cell lymphomas are more frequently T rather than NK lineage based on T-cell receptor gene, RNA, and protein studies: lineage does not predict clinical behavior

“…22 For FFPE samples, the sensitivity has been reported to range from 80 to 98% for TCR. [23][24][25][26] ENKTLs are frequently accompanied by necrosis and apoptosis of the tumor cells, leading to degradation of DNA, ineffective PCR reactions, and false-negative results. 27 In a study by Pongpruttipan et al, 8 clonal TCR gene rearrangements were documented in only three out of six TCR protein-positive cases.…”

“…Cell types of extranodal NK/T-cell lymphoma M Hong et al 17,26 recent studies using immunohistochemistry showed variable degrees of TCR expression, ranging from 0 to 23% of these tumors. 7,8,10,20 Rodriguez-Pinilla et al 10 showed that ENKTLs primarily affecting the skin or subcutaneous tissue (n = 11) were all negative for TCR-γ.…”

Somatic Mutation Screening Using Archival Formalin-Fixed, Paraffin-Embedded Tissues by Fluidigm Multiplex PCR and Illumina Sequencing

“…This was performed by PCR of variably sized genomic fragments (Table 1), 14 using a 2-ng template DNA in a 10-mL reaction mixture. PCR conditions were 95 C for 10 minutes, 40 cycles of 95 C for 20 seconds, 60 C for 20 seconds, 72 C for 1 minute, and a final extension at 72 C for 5 minutes.…”

A20, ABIN-1/2, and CARD11 Mutations and Their Prognostic Value in Gastrointestinal Diffuse Large B-Cell Lymphoma

“…DNA was extracted by using standard proteinase K digestion, followed by phenol/chloroform/ isoamyl-alcohol extraction or by using the QIAamp DNA Micro Kit (QIAGEN). The quality of the DNA samples was assessed by the PCR amplification of variably sized gene fragments (33), and those with successful amplifications of genomic fragments in excess of 300 bp, in addition to the DNA samples from the frozen tissues, were used for mutational screening.…”