Sequential Analysis of cfDNA Reveals Clonal Evolution in Patients with Neuroblastoma Receiving ALK-Targeted Therapy

Abstract

Purpose: The study of cell-free DNA (cfDNA) enables sequential analysis of tumor cell-specific genetic alterations in patients with neuroblastoma.

Experimental design: Eighteen patients with relapsing neuroblastoma having received lorlatinib, a third-generation ALK inhibitor, were identified (SACHA national registry and/or in the institution). cfDNA was analyzed at relapse for nine patients and sequentially for five patients (blood/bone marrow plasma) by performing whole-genome sequencing library construction followed by ALK-targeted ddPCR of the hotspot mutations [F1174L, R1275Q, and I1170N; variant allele fraction (VAF) detection limit 0.1%] and whole-exome sequencing (WES) to evaluate disease burden and clonal evolution, following comparison with tumor/germline WES.

Results: Overall response rate to lorlatinib was 33% (CI, 13%-59%), with response observed in 6/10 cases without versus 0/8 cases with MYCN amplification (MNA). ALK VAFs correlated with the overall clinical disease status, with a VAF < 0.1% in clinical remission, versus higher VAFs (>30%) at progression. Importantly, sequential ALK ddPCR detected relapse earlier than clinical imaging. cfDNA WES revealed new SNVs, not seen in the primary tumor, in all instances of disease progression after lorlatinib treatment, indicating clonal evolution, including alterations in genes linked to tumor aggressivity (TP53) or novel targets (EGFR). Gene pathway analysis revealed an enrichment for genes targeting cell differentiation in emerging clones, and cell adhesion in persistent clones. Evidence of clonal hematopoiesis could be observed in follow-up samples.

Conclusions: We demonstrate the clinical utility of combining ALK cfDNA ddPCR for disease monitoring and cfDNA WES for the study of clonal evolution and resistance mechanisms in patients with neuroblastoma receiving ALK-targeted therapy.

Figure 1.

Clinical efficacy of lorlatinib. A, Clinical evolution in a 10-year-old boy with neuroblastoma, INRGSS M, without MYCN amplification (patient 17). Successive ¹²³I-MIBG scans indicate widespread osteomedullary disease at diagnosis. Initial therapy consisted of rapid COJEc (PD), CAV (PD), BEACON irinotecan-temozolomide (SD), pazopanib (PD), etoposide (PD), and cyclophosphamide (PD). Lorlatinib (single agent) resulted in an important reduction of the SIOPEN score (PR according to INRC). Following progression, trametinib was empirically associated with lorlatinib following (PD), based on a possibility of sensitivity to MEK inhibition in combination, while awaiting cfDNA analysis. Palliative irradiation to symptomatic metastatic sites followed by etoposide was given (PD). The patient passed away 67 months after diagnosis. The cfDNA concentrations (ng/mL, in blue) and ALK VAF (%, in orange) are indicated. PD, progressive disease; SD, stable disease; PR, partial response. B, Efficacy of lorlatinib in 18 patients with high-risk neuroblastoma treated with lorlatinib, with or without chemotherapy, achieving SD or PR. The bar indicates the duration of lorlatinib treatment. Triangles indicate the start of response to treatment. Discontinuation of treatment by lorlatinib occurred at relapse/progression.

Figure 2.

Droplet digital PCR maintains linearity for ALK F1174L and ALK R1275Q with or without WGS libraries process. A, Comparison analysis of measured ALK R1275Q VAFs’ (%) average (n = 4) in ddPCR between two conditions: with WGS libraries process or no WGS libraries process for each group of dilutions. B, Comparison analysis of measured ALK F1174L VAFs’ (%) average (n = 4) in ddPCR between two conditions, with WGS libraries process or no WGS libraries process for each group of dilutions. Comparison of the VAF means of the quadruplicates of each dilution demonstrated that no statistically significant difference was observed between the two conditions (Mann–Whitney test; P < 0.05). C, Correlation analysis of measured ALK R1275Q VAFs (%) without or with WGS libraries process obtained by calculating an average on four replicates (n = 4) represented respectively in the x-axis and the y-axis (as mean ± SD; n ≥ 3). Pearson’s correlation coefficient (r) and P-value are indicated. D, Correlation analysis of measured ALK F1174L VAFs (%) without or with WGS libraries process obtained by calculating an average on four replicates (n = 4) represented respectively in the x-axis and the y-axis (as mean ± SD; n ≥ 3). Pearson’s correlation coefficient (r) and P-value are indicated.

Figure 3.

ALK hotspot mutations in cfDNA extracted from plasma samples of patients with neuroblastoma and comparison with disease status measured by ddPCR and WES. A, Patient 6 (5 years, INRG M, no MNA): ALK R1275Q VAFs (%; y-axis) by ddPCR analysis on WGS libraries of cfDNA extracted from sequential blood or bone marrow plasma. The number of days after diagnosis is represented in the x-axis. Treatments are indicated by letter: (a) HR-NBL 1.7, (b) Temodal, and (c) lorlatinib. Treatment according to HR-NBL1.7 was administered (a) with a decrease of ALK R1275Q VAF between D6, to 0% at D 41, D88, and D479, correlating with clinical complete remission. Upon relapse (D603) an increase of ALK R1275Q VAF to 17% was observed, corresponding to PD. Treatment with Temodal (b) resulted in further PD, with a further increase of ALK R1275Q VAF to 24%. After the start of lorlatinib (c), a small decrease of ALK R1275Q VAF to 22% at D739 was observed. The patient passed away due to PD. B, Patient 7 (14 years, INRG M, MNA): ALK F1174L VAFs (%; y-axis) by ddPCR analysis on WGS libraries of cfDNA extracted from sequential blood or bone marrow plasma. The number of days after diagnosis is represented in the x-axis. Treatments are indicated by letter: (a) HR-NBL 1.7, (b) lorlatinib, and (c) TOTEM. Initial treatment HR-NBL 1.7 (a) resulted in a decrease of ALK F1174L VAF from 13% at D5 to almost 0% at D101, correlating with clinical very good PR. On D179, ALK F1174L VAF increased to 4.5%, whereas imaging (CT scan, MIBG) concluded persistent VGPR. Relapse was confirmed at D222 (by local imaging and MIBG), indicating that ddPCR detected relapse 43 earlier than imaging. Lorlatinib (b) treatment was commenced D236 with a further increase of ALK F1174L VAF to 24%, with new PD on D349. TOTEM (c) commenced on D354 resulted in ALK F1174L VAF 10%, indicating clinically stable disease. The patient passed away due to PD. C, Patient 17 (10 years, INRG M, no MNA): ALK F1174L VAFs (%; y-axis) by ddPCR analysis on WGS libraries of cfDNA extracted from sequential blood or bone marrow plasma. The number of days after diagnosis is represented in the x-axis. Treatments are indicated by letter: (a) HR-NBL 1.7, (b) Beacon, (c) Pazopanib, (d) RT + Etoposide, (e) cyclophosphamide, (f) lorlatinib. ALK F1174L VAFs at D12 was 26% (blood) versus 7% (bone marrow). Induction of HR-NBL1 resulted in stable disease, with an ALK F1174L VAF persisting at 12% at D96. Beacon (b) commenced on D102 and resulted in SD, with ALK F1174L VAF 7% on D229. Pazopanib (c) treatment began on D253 with a temporary decrease of ALK F1174L VAF to 1.5% at D364 followed by a renewed increase to 7.755% at (D) lorlatinib (f) was commenced on D834, with ALK F1174L VAF subsequently attaining nearly 0%. A reemergence of ALK F1174L to 2% concomitant with a single MIBG lesion, preceding multimetastatic relapse. The patient passed away due to PD. D, Patient 16 (3 years, INRG M with no identified primary, no MNA) ALK I1170N VAFs (%; y-axis) by ddPCR analysis on WGS libraries of cfDNA extracted from sequential blood or bone marrow plasma. The number of days after diagnosis is represented in the x-axis. Treatments are indicated by letter: (a) HR-NBL 1.7, (b) Beacon, (c) CAdO, (d) lorlatinib, and (e) etoposide. ALK I1170N VAFs, at 19% at diagnosis decreased to 0% at D196 and D387 following HR-NBL 1.7 (a) treatment. At D506, ALK I1170N VAF increased to 2% with confirmation of PD by imaging on D514. ALK I1170N VAF was 0.4% at the beginning of lorlatinib treatment on D665 with a decrease to 0.169% at D716 and 0.260 at D770 but a new increase to ALK I1170N 10% on D840 coinciding with new PD. Salvage treatment proved inefficient, with ALK I1170N VAF at 45%. The patient passed away due to PD.

Figure 4.

Venn diagrams and clonal evolution based on cfDNA analysis via WES in sequential plasma samples of patients with neuroblastoma treated with lorlatinib. For each patient, the graph on the (left) indicates the number of somatic SNVs detected by cfDNA WES in each analyzed sample. CCFs were calculated to determine clonal composition and clonal evolution. The graph on the (right) indicates the clones according to the cancer cell fraction of all somatic SNVs identified in sequential plasma samples. The clone of origin is depicted in (blue), shrinking clones are indicated in (green, gray, and purple), and emerging clones are indicated in (red) and (pink).

Figure 5.

Top enriched gene sets after Gene Set Enrichment Analysis (GSEA) (http://www.gsea-msigdb.org/gsea/index.jsp, ontology gene sets C5—biological processes) for emerging (top) and persisting (bottom) SNVs after lorlatinib treatment. Depicted is the number of genes in the ontology overlap (x-axis), colored by patient contribution, and the P-value significance indicated (right).