Frequency of PDL locus alterations and correlation with transcript expression levels. (A) Representative FISH signal patterns from 2 clinical samples; top: break-apart (primary PMBCL), bottom: amplification (primary PMBCL). (B) The proportion of PDL locus (9p24.1) aberration across 7 different subtypes of B-cell lymphomas observed using an in-house break-apart FISH assay (N values: DLBCL = 134; PCNSL = 130; primary testicular DLBCL [TDLBCL] = 82; PMBCL = 125; CHL = 20; nodular lymphocyte predominant Hodgkin lymphoma [NLPHL] = 12; follicular lymphoma [FL] = 68). P values were < .05 for the number of break-apart cases in PMBCL in comparison with lymphomas with N > 12; P < .05 for the number of amplified cases in PMBCL in comparison with lymphomas with N > 20. (C) CD274 transcript expression in 17 cell lines and 76 clinical samples as determined via qRT-PCR (N values: PMBCL = 48; DLBCL = 19; TDLBCL = 6; PCNSL = 3). The dotted red line represents basal CD274 expression as determined by reactive tonsil cells. The break-apart to neutral comparison reached statistical significance (P = .03). Amplified to neutral (P = .001) and amplified to gain (P = .01) comparisons also reached statistical significance. (D) PDCD1LG2 transcript expression in the cohort described above. The dotted blue line represents basal PDCD1LG2 expression. Statistical significance was researched between break-apart to neutral (P = .0003), break-apart to gain (P = .001), and break-apart to amplified (P = .005) case comparisons. The amplified to neutral comparison also reached statistical significance (P = .002). (E) Differential expression of both PDL transcripts in break-apart cases. Dots circled in red (2) were Sanger-validated to have a translocation involving the CD274 locus, whereas those in blue (5) were found to harbor rearrangements of PDCD1LG2. Note how break-apart events result in disparate expression levels between transcripts.