Dynamics of Sequence and Structural Cell-Free DNA Landscapes in Small-Cell Lung Cancer

Abstract

Purpose: Patients with small-cell lung cancer (SCLC) have an exceptionally poor prognosis, calling for improved real-time noninvasive biomarkers of therapeutic response.

Experimental design: We performed targeted error-correction sequencing on 171 serial plasmas and matched white blood cell (WBC) DNA from 33 patients with metastatic SCLC who received treatment with chemotherapy (n = 16) or immunotherapy-containing (n = 17) regimens. Tumor-derived sequence alterations and plasma aneuploidy were evaluated serially and combined to assess changes in total cell-free tumor load (cfTL). Longitudinal dynamic changes in cfTL were monitored to determine circulating cell-free tumor DNA (ctDNA) molecular response during therapy.

Results: Combined tiered analyses of tumor-derived sequence alterations and plasma aneuploidy allowed for the assessment of ctDNA molecular response in all patients. Patients classified as molecular responders (n = 9) displayed sustained elimination of cfTL to undetectable levels. For 14 patients, we observed initial molecular responses, followed by ctDNA recrudescence. A subset of patients (n = 10) displayed a clear pattern of molecular progression, with persistence of cfTL across all time points. Molecular responses captured the therapeutic effect and long-term clinical outcomes in a more accurate and rapid manner compared with radiographic imaging. Patients with sustained molecular responses had longer overall (log-rank P = 0.0006) and progression-free (log-rank P < 0.0001) survival, with molecular responses detected on average 4 weeks earlier than imaging.

Conclusions: ctDNA analyses provide a precise approach for the assessment of early on-therapy molecular responses and have important implications for the management of patients with SCLC, including the development of improved strategies for real-time tumor burden monitoring. See related commentary by Pellini and Chaudhuri, p. 2176.

Figure 1.

Study methodology and cohort analyzed. Overall, 33 patients with SCLC who received systemic treatment with either chemotherapy or immunotherapy-containing regimens were analyzed for this study. A, High-depth targeted error-correction sequencing (TEC-seq) was performed on cfDNA extracted from serial plasma samples collected at baseline and longitudinally throughout the course of treatment, alongside matched WBC DNA. Sequence and structural alterations were directly detected in cfDNA for each patient at each time point analyzed and used to evaluate longitudinal changes in cfTL using a combined tiered approach. WES was further performed on pretreatment tumor and matched WBC DNA derived from buffy coat from a subset of 5 patients and used to evaluate the concordance between copy-number profiles computed from tumor next-generation sequence data and PA. Finally, molecular response classifications were assigned to each patient based on dynamic changes in cfTL and used to predict clinical outcomes. B, Swimmer plot showing disease course in each patient, treatment, and samples collected. The mean time to first blood sample collection after initiation of treatment for patients included in the study was 9 weeks (median 3 weeks). The mean time to first imaging assessment in this cohort was 8 weeks (median 6 weeks), and the mean time to BOR assessment was 11 weeks (median 7 weeks).

Figure 2.

Landscape of sequence alterations in plasma. The type and origin of variants detected across plasma samples analyzed for this study are shown. Alteration frequencies for each gene across plasma samples analyzed are shown on the right and per-sample mutation counts are displayed in the bar plot on top. Panels indicating sampling time point, treatment type, PA scores, combined molecular response, OS, and PFS are shown below. Germline variants (filled circles) were observed in a total of 8 genes (FLT3, FGFR1, CDK6, ALK, POLE, BRAF, PTCH1, and BRCA2) on a patient-specific basis. Variants attributed to CH (crosses) were identified in canonical CH genes (DNMT3A, TP53, and ATM) in >50% (18/33) of patients analyzed for the study and across 14 additional genes at a lower prevalence. Overall, tumor-derived sequence alterations (solid border) were detected in plasma from 30 of the 33 patients analyzed and were most frequently detected in the TP53 gene, which was mutated in 26 patients. Of these 26 patients, 3 shared a mutation in RB1.

Figure 3.

Chromosomal arm-level somatic copy-number profiles and PA. Targeted sequencing data were used to assess genome-wide arm-level SCNAs across serial plasma samples. Representative examples of genome-wide SCNA profiles are shown for 2 patients. A, Patient 10 displayed multiple copy-number gains (chromosomes 1, 3q, 5p, 7, 12p, 15, 17, and 20) and losses (chromosomes 2, 3p, 4, 5q, 8, 9q, 10, 13, and 16) in plasma at baseline sampling. Analysis of these regions across follow-up (week 3–73) plasma samples collected after cisplatin/etoposide treatment indicated a return to normal ploidy, which was detected prior to the radiographic assessment of partial or complete (PR/CR) response at week 11. B, Patient 5 displayed widespread genome-wide copy-number aberrations across tumor (WES; top) and matched serial plasma samples (bottom). This included gains across chromosomes 1, 8, 12, 13, 17, 18, 19, and 20 and losses across chromosomes 2, 3p, 4, 5q, 7, 10q, 11, 14, 16, 21, and 22. All SCNAs detected in the tumor were captured in cfDNA at baseline and persistently across all follow-up time points analyzed in plasma after initiation of second-line ipilimumab/nivolumab combination therapy. This was consistent with a radiographic assessment of PD 5 weeks after initiation of ipilimumab/nivolumab combination therapy. C–E, The most aberrant alterations in individual chromosome arm-level copy number were used to calculate a genome-wide composite measure of PA (termed PA score) for each sample. Examples of longitudinal trends in PA scores are shown for 3 patients who had no detectable tumor-derived sequence alterations across plasma time points analyzed. C, For patient 3, aneuploidy was detected in plasma 4 weeks after baseline sampling, followed by a reduction to normal ploidy by the week 8 timepoint, consistent with an OS time exceeding 21 months from baseline sampling. In contrast, radiographic assessment at week 6 indicated PD for this patient. D–E, Similarly, in patients 19 and 29, aneuploidy was detected at baseline followed by a reduction to normal ploidy by week 3–4 and week 5–7 time points, respectively. This preceded radiographic assessments of PR at weeks 6 and 15, respectively.

Figure 4.

Dynamic changes in cfTL during therapy. Longitudinal changes in cfTL across plasma time points analyzed for each patient were used to assign a combined molecular response classification. Representative examples are shown of patients who were assigned to each of the 3 classifications. A, Patient 10 was classified as a molecular responder based on the complete elimination of cfTL, assessed using tumor-derived sequence alterations, between baseline and week 5 sampling during cisplatin/etoposide chemotherapy treatment (indicated by the green shaded area). A reduction in PA scores to undetectable levels from baseline was also observed in this patient. B and C, Patients 26 and 21 were assigned a classification of molecular response followed by recrudescence based on the elimination of cfTL between baseline and intermediate time points [during atezolizumab/etoposide/carboplatin (purple) and carboplatin/etoposide (green) treatment, respectively], after which an increase in cfTL was observed at the final time points analyzed. C, In patient 21, a shift in mutation profiles defined by the presence of tumor-derived RET (p.Y314F) and TP53 (p.V173E) mutations at recrudescence, which were not present at the baseline timepoint, was observed. D, Patient 1 was classified as a molecular progressor based on the persistence of cfTL, defined by a tumor-derived TP53 (p.C135S) sequence alteration, across all time points analyzed during treatment with nivolumab (blue). E, Combined molecular responses were significantly associated with clinical evaluations of best radiographic response (P = 0.003, Fisher exact test). F, A broader comparison between the elimination of cfTL at any timepoint analyzed for the study and radiographic assessments further revealed concordance (P = 0.001, Fisher exact test) between each variable. G, Molecular responses were determined on average 4 weeks prior to best radiographic response assessments in 28 patients with comparable ctDNA and imaging assessments in this cohort (mean 5.61 weeks vs. 10.21 weeks; P = 0.01 Mann–Whitney U test). Patients without baseline plasma samples available (n = 2) and cases with discordant molecular and radiographic responses (n = 3) were excluded from analyses. Mean times to response assessment are shown alongside standard error for each modality.

Figure 5.

Combined tiered ctDNA molecular responses and the prediction of survival outcomes. Of the 33 patients included in the study cohort, patients who were assigned a classification of molecular response (green) based on longitudinal dynamic changes in cfTL displayed superior (A) overall survival (OS; median survival not reached vs. 12.35 vs. 6.48 months; log-rank P 0.0006) and (B) progression-free survival (PFS; median survival not reached vs. 6.18 vs. 1.74 months; log-rank P < 0.0001) compared with patients with an initial molecular response followed by recrudescence (orange) or molecular progression (red). C and D, Molecular responses derived from ctDNA (dark purple) were a stronger predictor of OS at 12 (AUC 78.1 vs. 73.3) and 36 (AUC 80.0 vs. 71.8) months, and (E and F) early post-therapy PFS at 3 (AUC 91.7 vs. 82.0) and 12 (AUC 83.5 vs. 78.8) months, compared with radiographic assessment (PR/CR vs. SD/PD/MR; light purple). f/b, followed by.