PD-L1 and PD-L2 Genetic Alterations Define Classical Hodgkin Lymphoma and Predict Outcome

Abstract

Purpose: Classical Hodgkin lymphomas (cHLs) include small numbers of malignant Reed-Sternberg cells within an extensive but ineffective inflammatory/immune cell infiltrate. In cHL, chromosome 9p24.1/PD-L1/PD-L2 alterations increase the abundance of the PD-1 ligands, PD-L1 and PD-L2, and their further induction through Janus kinase 2-signal transducers and activators of transcription signaling. The unique composition of cHL limits its analysis with high-throughput genomic assays. Therefore, the precise incidence, nature, and prognostic significance of PD-L1/PD-L2 alterations in cHL remain undefined.

Methods: We used a fluorescent in situ hybridization assay to evaluate CD274/PD-L1 and PDCD1LG2/PD-L2 alterations in 108 biopsy specimens from patients with newly diagnosed cHL who were treated with the Stanford V regimen and had long-term follow-up. In each case, the frequency and magnitude of 9p24.1 alterations-polysomy, copy gain, and amplification-were determined, and the expression of PD-L1 and PD-L2 was evaluated by immunohistochemistry. We also assessed the association of 9p24.1 alterations with clinical parameters, which included stage (early stage I/II favorable risk, early stage unfavorable risk, advanced stage [AS] III/IV) and progression-free survival (PFS).

Results: Ninety-seven percent of all evaluated cHLs had concordant alterations of the PD-L1 and PD-L2 loci (polysomy, 5% [five of 108]; copy gain, 56% [61 of 108]; amplification, 36% [39 of 108]). There was an association between PD-L1 protein expression and relative genetic alterations in this series. PFS was significantly shorter for patients with 9p24.1 amplification, and the incidence of 9p24.1 amplification was increased in patients with AS cHL.

Conclusion: PD-L1/PD-L2 alterations are a defining feature of cHL. Amplification of 9p24.1 is more common in patients with AS disease and associated with shorter PFS in this series. Further analyses of 9p24.1 alterations in patients treated with standard cHL induction regimens or checkpoint blockade are warranted.

Fig 1.

Genetic and immunohistochemical analyses of the PD-L1 and PD-L2 loci and PD-1 ligand expression. (A) Location and color labeling of the bacterial artificial chromosome (chr) clones on 9p24.1 used for fluorescent in situ hybridization (FISH). RP11-599H20 including PD-L1, labeled red. RP11-635N21 including PD-L2, labeled green. (B) Representative images of FISH results for the various categories. PD-L1 in red, PD-L2 in green, fused (F) signals in yellow, and centromeric probe (CEP9) in aqua (A). In these images, disomy reflects 2A:2F; polysomy, 3A:3F; copy gain, 3A:6F; and amplification, 15+F. (C) The top panel shows PD-L1 (brown)/PAX5 (red) immunohistochemistry (IHC) in the classical Hodgkin lymphoma (cHL) cases with 9p24.1 disomy, polysomy, copy gain, and amplification from (B). The bottom panel shows PD-L2 (brown)/pSTAT3 (red) IHC in the same cHL cases. Scale bar = 50 μm.

Fig 2.

The spectrum of 9p24.1 alterations in classical Hodgkin lymphoma (cHL). (A) 9p24.1 alterations in evaluated cHLs. The cHLs are classified by the highest observed level of 9p24.1 alteration in Reed-Sternberg (RS) cells: polysomy, copy gain, or amplification (top). Individual tumors are depicted as columns on the x-axis. In each cHL, the percentage of RS cells with 9p24.1 disomy (black), polysomy (light pink), copy gain (pink), and/or amplification (red) is shown on the y-axis. (B) Percentage of RS cells with residual 9p24.1 disomy in cHLs classified by 9p24.1 alterations, as represented as box-and-whisker plots, showing minimum, first quartile, median, third quartile, and maximum. cHLs with 9p24.1 polysomy, copy gain, and amplification have significantly different percentages of residual RS cells with normal (disomic) 9p24.1 copy numbers. P < .001, Kruskal-Wallis test. (C) Association of PD-L1 protein expression and 9p24.1 copy number alterations. Residual 9p24.1 disomy is depicted on the y-axis; PD-L1 immunohistochemistry (IHC) H-score (in quartiles) is shown on the x-axis. Quartiles are indicated by dashed lines. A locally weighted polynomial regression line is shown in black. A highly significant decrease in percentage of residual 9p24.1 disomic cells in cHLs with a higher PD-L1 IHC H-score is shown. P = .005, Kruskal-Wallis test.

Fig 3.

Clinical and genetic predictors of progression-free survival (PFS). (A) PFS by clinical stage in patients with classical Hodgkin lymphoma (cHL), early stage favorable (ES-F; n = 33), early stage unfavorable (ES-U; n = 41), and advanced stage (AS; n = 34). P = .002, log-rank test. (B) PFS by 9p24.1 alterations in patients with cHL (disomy, n = 1; polysomy, n = 5; copy gain, n = 61; amplification, n = 39; translocation, n = 2; P < .001, log-rank test). (C) Percentage of patients with 9p24.1 disomy (1%), polysomy (5%), copy gain (56%), amplification (36%), and translocation (1%) in the current series. (D) Frequency of 9p24.1 alterations (polysomy, copy gain, amplification, translocation, or disomy) by clinical stage (ES-F, ES-U, and AS) in this series. The incidence of 9p24.1 amplification is significantly different in clinically staged patients (ES-F, 24%; ES-U, 34%; AS, 50%; P = .024, Kruskal-Wallis test).

Fig A1.

Chromosomal rearrangements in classical Hodgkin lymphoma (cHL). (A) Location and color labeling of the bacterial artificial chromosome (chr) clones on 9p24.1 used for fluorescent in situ hybridization (FISH) in translocation 2 (Tx #2). RP11-599H20 including PD-L1, labeled red; RP11-610G2 downstream of PD-L2, labeled green. For labeling of bacterial artificial chromosome clones used for translocation 1 (Tx #1), see Figure 1A. (B) FISH analyses of the cHL cases with chromosomal rearrangements. PD-L1 in red, PD-L2 in green, and centromeric probe (CEP9) in aqua. Arrows indicate the rearranged allele.

Fig A2.

Association of PD-L1/PD-L2 protein expression and 9p24.1 copy number alterations. (A) Percentage of 9p24.1 disomic cells in each of the PD-L1 immunohistochemistry (IHC) H-score quartiles. The y-axis shows the percentage of residual 9p24.1 disomic cells; the x-axis shows PD-L1 IHC H-score in quartiles. A statistically significant decrease in the percentage of normal (disomic) cells in the H-score quartiles was found. P = .005, Kruskal-Wallis test. (B) Percentage of residual 9p24.1 disomic cells in each of the PD-L2 IHC H-score quartiles. The y-axis shows the percentage of residual 9p24.1 disomic cells; the x-axis shows the PD-L2 IHC H-score in quartiles. The percentage of residual 9p24.1 disomic cells is statistically different in the quartiles. P = .006, Kruskal-Wallis test. (C) Percentage of residual 9p24.1 disomic cells (y-axis) and PD-L2 IHC H-score (x-axis) plotted for individual cases. Quartiles are indicated with dashed lines, and a trend line (locally weighted polynomial regression line) is shown in black. q, quartile.

Fig A3.

Distribution of genetic alterations in patients with Epstein-Barr virus (EBV) –negative and EBV-positive classical Hodgkin lymphoma (cHL). (A) The status of PD-L1 and PD-L2—disomy, polysomy, copy gain, amplification, and translocation—in EBV-negative (n = 88) and EBV-positive (n = 20) cHLs is visualized with a pie chart. (B) Distribution of EBV-negative and EBV-positive cases in the various PD-L1 immunohistochemistry (IHC) H-score quartiles. The proportion of EBV-positive cases increases as the H-score quartile category increases. P = .019, Kruskal-Wallis test. (C) and (D) Percentage of 9p24.1 residual disomic cells (y-axis) and PD-L1 IHC H-score (x-axis) plotted for individual EBV-negative and EBV-positive cHLs, respectively. Quartiles are indicated with dashed lines, and a trend line (locally weighted polynomial regression line) is shown in black. (E) and (F) Percentages of residual 9p24.1 disomic cells in EBV-negative and EBV-positive cases in the respective PD-L1 IHC H-score quartiles from (C) and (D) are plotted. The percentage of residual 9p24.1 disomic cells is significantly different in the respective H-score quartiles in EBV-negative cHLs in (E). P = .021, Kruskal-Wallis test. q, quartile.

Fig A4.

Progression-free survival (PFS) curves for the clinical risk groups with (gold) or without (blue) 9p24.1 amplification: early stage favorable (ES-F; n = 8 and n = 25, respectively), early stage unfavorable (ES-U; n = 14 and n = 27, respectively), and advanced stage (AS; n = 17 and n = 17, respectively).