Longitudinal Assessment of Circulating Tumor Mutational Burden Using a Next-Generation Sequencing Cancer Gene Panel: A Potential Biomarker of Response to Programmed Cell Death 1 (PD-1) Blockade in Patients with Relapsed/Refractory Classical Hodgkin Lymphoma

Introduction: The programmed cell death 1 (PD-1) monoclonal antibodies (MoAbs) nivolumab and pembrolizumab induce response rates exceeding 70% in relapsed/refractory (R/R) classical Hodgkin lymphoma (cHL). The lack of response to PD-1 MoAbs, and the relapse occurring in most patients who had responded to PD-1 blockade suggest that tools to identify the determinants of response/resistance to PD-1 MoAbs are urgently required. We hypothesized that the characterization of the mutational profile of circulating tumor DNA (ctDNA) could represent a valuable tool to track clonal evolution-driven resistance to checkpoint inhibitors.

Patients and Methods: 21 R/R cHL (median age, 32 years; range, 19-51) who had received a median of 5 (range, 3-7) chemotherapy lines, including autologous stem cell transplantation (77%) and brentuximab vedotin (100%), were treated with PD-1 MoAbs. Blood samples were profiled by CAPP-Seq strategy. We analyzed ctDNA and paired DNA from peripheral blood mononuclear cells (PBMCs), as source of germline DNA to filter out polymorphism and sequencing errors. A targeted resequencing panel optimized to include the coding exons and splice sites of 133 genes (320 Kb) that are recurrently mutated in B-cell lymphomas was used. Libraries were prepared from ctDNA and germline gDNA according to the CAPP-seq targeted enrichment strategy (Nimblegen-Roche) and subjected to ultra-deep-next generation sequencing (NGS) using the Nextseq 500 platform (Illumina). The sequencing was performed to obtain a depth of coverage >2000x in >80% of the target region in all samples, which allowed a sensitivity of 3x10^-3. A stringent and completely automated bioinformatic pipeline was applied to call non-synonymous somatic mutations, using the somatic function of VarScan2.

Results: After a median of 26 (range, 9-63) cycles of PD-1 inhibitors best response was complete remission (CR) for 9 patients (42%), partial remission (PR) for 6 (29%) and progressive disease (PD) for 6 (29%). Patients achieving PR experienced a disease control lasting for 4.5 to 24 months and subsequently underwent PD. Plasma and PBMC samples were collected at baseline, every five cycles of therapy, and end-of-therapy (EOT). At baseline, 18 of 21 patients could be successfully genotyped, whereas three were not. Evaluable patients showed a mean (±SD) number of mutated genes and mutations per patient of 7.3±5.1 (range, 2-22) and 9.9±8.4 (range, 2-37), respectively. Genes recurrently affected by non-synonymous somatic mutations in >20% of R/R cHL included STAT6 (45%), SOCS1 (40%), ITPKB (35%), GNA13 (35%), TP53 (20%), TNFAIP3 (15%). At baseline, no association of distinct DNA mutations with resistance to PD-1 inhibitors could be demonstrated. Signaling pathways targeted by DNA mutations included JAK-STAT, NF-κB, PI3K-AKT, cytokine, NOTCH, immune evasion. The concentration of ctDNA reported as haploid genome equivalent per ml (hGE/ml) was 592.2 (range, 2-2,746), with values of hGE/ml detected in PD patients being significantly higher as compared to CR patients (P=.0437). As compared to cycle 0, the hGE/ml of ctDNA at cycle 5 showed a significant reduction (592.2 vs. 67, P<.0008) which was followed by further hGE/ml decline in CR patients (to 14 P=.05) and further hGE/ml increase in PD patients (to 1,300 P=.1). At cycle 5, all CR/PR patients showed complete disappearance of baseline mutations, which were replaced by completely novel clones. In all CR/PR patients, this pattern of "clonal reshaping" was repeatedly detected over time. In striking contrast, at cycle 5, PD patients showed the persistence of baseline mutations. In all PD patients, this pattern of "clonal persistence", was repeatedly detected over time. In 4 patients, resistance to PD-1 inhibitors was associated with the appearance of a TP53 mutated clone. Although, a formal correlation of circulating DNA mutations with standard FDG-PET imaging was outside the objective of this study, both the "clonal reshaping" and "clonal persistence" patterns could be demonstrated to correlate with the results of FDG-PET.

Conclusions: Analysis of ctDNA allows detecting tumor-specific mutations in R/R cHL. The longitudinal tracking of circulating DNA mutations in these patients identifies two different patterns of clonal evolution associated with sensitivity (clonal reshaping) or resistance (clonal persistence) to checkpoint blockade.