Profiling of Circulating Tumor DNA for Noninvasive Disease Detection, Risk Stratification, and MRD Monitoring in Patients with CNS Lymphoma

Abstract

Introduction: Clinical outcomes for patients with central nervous system lymphoma (CNSL) are remarkably heterogeneous, yet identification of patients at high risk for treatment failure remains challenging with existing methods. In addition, diagnosis of CNSL requires invasive neurosurgical biopsies that carry procedural risks and often cannot be performed in frail or elderly patients. Circulating tumor DNA (ctDNA) has shown great potential as a noninvasive biomarker in systemic lymphomas. Yet, previous studies revealed low ctDNA detection rates in blood plasma of CNSL patients. In this study, we utilized ultrasensitive targeted high-throughput sequencing technologies to explore the role of ctDNA for disease classification, MRD detection, and early prediction of clinical outcomes in patients with CNSL. Methods: We applied Cancer Personalized Profiling by Deep Sequencing (CAPP-Seq) and Phased Variant Enrichment and Detection Sequencing (PhasED-Seq, Kurtz et al, Nat Biotech 2021) to 85 tumor biopsies, 131 plasma samples, and 62 CSF specimens from 92 CNSL patients and 44 patients with other brain cancers or inflammatory cerebral diseases, targeting 794 distinct genetic regions. Concentrations of ctDNA were correlated with radiological measures of tumor burden and tested for associations with clinical outcomes at distinct clinical time points. We further developed a novel classifier to noninvasively distinguish CNS lymphomas from other CNS tumors based on their mutational landscapes in plasma and CSF, using supervised training of a machine learning approach from tumor whole genome sequencing data and own genotyping analyses, followed by its independent validation. Results: We identified genetic aberrations in 100% of CNSL tumor biopsies (n=63), with a median of 262 mutations per patient. Pretreatment plasma ctDNA was detectable in 78% of plasma samples and in 100% of CSF specimens (Fig. 1a), with ctDNA concentrations ranging from 0.0004 - 5.94% allele frequency (AF, median: 0.01%) in plasma and 0.0049 - 50.47% AF (median: 0.62%) in CSF (Fig. 1b). Compared to ctDNA concentrations in patients with systemic diffuse large B-cell lymphoma (DLBCL, data from Kurtz et al., J Clin Oncol, 2018), plasma ctDNA levels in CNSL were in median more than 200-fold lower (Fig. 1b). We observed a significant correlation of ctDNA concentrations with total radiographic tumor volumes (TRTV) measured by MRI (Fig. 1c,d), but no association with clinical risk scores (i.e., MSKCC score) or concurrent steroid treatment. Assessment of ctDNA at pretreatment time points predicted progression-free survival (PFS) and overall survival (OS), both as continuous and binary variable (Fig. 1e,f). Notably, patients could be stratified into risk groups with particularly favorable or poor prognoses by combining ctDNA and TRTV as pretreatment biomarkers (Fig. 1g). Furthermore, ctDNA positivity during curative-intent induction therapy was significantly associated with clinical outcomes, both PFS and OS (Fig. 1h). Finally, we applied our novel machine learning classifier to 207 specimens from an independent validation cohort of CNSL and Non-CNSL patients. We observed high specificity (100%) and positive predictive value (100%) for noninvasive diagnosis of CNSL, with a sensitivity of 57% for CSF and 21% for plasma, suggesting that a significant subset of CNSL patients might be able to forego invasive surgical biopsies. Conclusions: We demonstrate robust and ultrasensitive detection of ctDNA at various disease milestones in CNSL. Our findings suggest that ctDNA accurately mirrors tumor burden and serves as a valuable clinical biomarker for risk stratification, outcome prediction, and surgery-free lymphoma classification in CNSL. We foresee an important potential future role of ctDNA as a decision-making tool to guide treatment in patients with CNSL. Figure 1 Figure 1. Disclosures Esfahani: Foresight Diagnostics: Current holder of stock options in a privately-held company. Kurtz: Genentech: Consultancy; Roche: Consultancy; Foresight Diagnostics: Consultancy, Current holder of stock options in a privately-held company. Schorb: Riemser Pharma GmbH: Honoraria, Research Funding; Roche: Research Funding; AbbVie: Research Funding. Diehn: BioNTech: Consultancy; RefleXion: Consultancy; Roche: Consultancy; AstraZeneca: Consultancy; Foresight Diagnostics: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; CiberMed: Current holder of stock options in a privately-held company, Patents & Royalties; Illumina: Research Funding; Varian Medical Systems: Research Funding. Alizadeh: Foresight Diagnostics: Consultancy, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Gilead: Consultancy; Roche: Consultancy, Honoraria; Celgene: Consultancy, Research Funding; Janssen Oncology: Honoraria; CAPP Medical: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Forty Seven: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Cibermed: Consultancy, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Bristol Myers Squibb: Research Funding.

Circulating tumor DNA in B-cell lymphoma: technical advances, clinical applications, and perspectives for translational research

“…Here, the association between ctDNA levels and tumor burden or clinical outcomes is not fully established. For example, while previous studies failed to show any correlation in PCNSL [ 65 , 66 ], Mutter et al recently demonstrated that ctDNA mirrors MRI tumor volumes and predicts PFS and OS, both in log-rank and cox regression analyses, using PhasED-seq for ctDNA quantification [ 68 ]. In FL, three studies targeting either ctDNA clonotypes by IgHTS or assessing cfDNA concentrations by ddPCR revealed significant correlations with baseline TMTV and showed that higher ctDNA/cfDNA levels were associated with shorter PFS [ 38 , 69 , 70 ] (Table 2 ).…”

Circulating tumour DNA — looking beyond the blood

“…A subset of patients with PCNSLs have a good initial radiological response to induction chemotherapy but subsequently have very early disease relapse after consolidative autologous stem cell transplantation. If CSF ctDNA can be used to identify this group before the emergence of radiological evidence of disease relapse, an alternative consolidation therapy, such as whole-brain radiotherapy (for presumed chemoresistant disease), could be offered earlier in the course of disease 84 . Biomarker analyses performed in the context of a phase Ib trial using an NGS panel designed specifically for patients with haematological malignancies were able to demonstrate a positive correlation between a radiological complete response and disappearance of CSF ctDNA, with CSF ctDNA recurrence preceding radiological relapse in one patient 83 .…”

CNS prophylaxis for diffuse large B-cell lymphoma

“…63 Cell-free circulating tumour DNA (ctDNA) has recently emerged as a non-invasive prognostic biomarker in patients with lymphoid malignancies 77 and could have a role in cerebral spinal fluid analysis. A recent study 78 of 67 patients with CNS lymphoma (including 12 with isolated secondary CNS lymphoma) identified ctDNA in all cerebral spinal fluid pretreatment samples (100%) and showed a significant correlation of plasma ctDNA concentration with tumour volume and outcomes.…”