Determining the Mutational Landscape of Waldenström’s Macroglobulinemia By Liquid Biopsy: Results of the Prospective Ecwm-2 Trial of the European Consortium for Waldenström’s Macroglobulinemia

Background: In Waldenström Macroglobulinemia (WM) genetic alterations such as MYD88, CXCR4 and TP53 mutations can affect treatment outcome in the era of Bruton’s tyrosine kinase inhibitors, highlighting the importance of understanding the molecular landscape in this disease. Liquid biopsy and the analysis of cell-free (cf)DNA allows for a minimally invasive approach for the diagnostic assessment and genotyping in lymphomas. Here, we aim to characterize the recurrent mutations in a treatment naïve cohort of patients with WM to determine the suitability of cfDNA for the initial diagnosis and genotyping of WM in the setting of a multicenter prospective clinical trial.

Methods: Diagnostic plasma samples from 37 treatment naïve WM patients treated within the ECWM-2 study (NCT03620903) were sequenced using a targeted capture assay (Euroclonality-NGS DNA Capture assay (EC-NDC, Univ8® Genomics, UK)) for the identification of single nucleotide variants (SNVs), structural variants and immunoglobulin (IG) receptor rearrangements. Data were analyzed by ARResT/Interrogate (Bystry, Bioinformatics, 2017) with an adapted pipeline for targeted capture in cfDNA. MYD88 mutations and CXCR4 frameshift (FS) or nonsense (NS) mutations from amino acid position 308 to 352 were called with ≥3 unique reads at ≥0.1% variant allele frequency (VAF). Other SNVs were called with ≥3 unique reads at ≥1 % VAF. CHIP mutations were called with ≥3 unique reads at ≥2 % VAF. Detectable MRD was called with ≥2 unique MYD88 reads. DNA from peripheral blood was analyzed in parallel for germline control in 32/37 patients. Patients in the ECWM-2 trial received 6 cycles (C) (d=28) of Bortezomib (1.6 mg/ m² s.c. d1,8,15), Rituximab (375 mg/m² i.v (C1d1), 1400 mg absolute s.c (C2-6 d1) and Ibrutinib (420 mg p.o. daily) followed by maintenance with Rituximab (1400 mg absolute s.c; D1 every 2nd month) combined with Ibrutinib for 24 months and subsequent ibrutinib treatment until progression or non-tolerated toxicity. Plasma samples obtained after 3 cycles of treatment were sequenced to track cell dynamics and MRD response (35/37 patients).

Results: Before start of treatment all patients had detectable cfDNA (median 1813 hGE/ml, range 268- 3668 hGE/ml), however cfDNA levels were lower compared to other lymphomas such as diffuse large B-cell lymphoma. No correlation of IgM level or bone marrow (BM) infiltration with the amount of cfDNA and ctDNA was observed. Clonal IG rearrangements were detected in 29/37 patients (76 %).

MYD88 ^L265P was detected in 35/37 samples (95%), with a median MYD88 ^L265P VAF of 5.1% (range 0.1% to 44.2%). No other non-L265P MYD88 mutations was observed. CXCR4 mutations were detected in 13/37 samples (35%) with a median VAF of 1.3% (range 0.1% to 8.2%). CXCR4^S338X was detected in 7/13 samples, while FS mutations were found in 6/13 cases. Interestingly, CXCR4 was mostly mutated on a subclonal level in relation to MYD88, ranging from 2.8% to 100% of MYD88 VAF. No MYD88 wildtype samples were CXCR4 mutated.

Targeted capture mutation data were confirmed by pathology routine sequencing of infiltrated BM in 35/37 (95%) MYD88 and 34/37 (92%) CXCR4 positive cases. In addition, targeted capture data was confirmed by droplet digital (dd)PCR for MYD88 ^L265P in 33/34 (97%) of cases in plasma samples from the same patients.

Additional putative somatic mutations were identified in 15/37 (41%) patients, the most frequent mutations comprising ARID1A (14%), CREBBP (8%) and TP53 (8%). Interestingly, CHIP mutations were found in plasma in 11/37 (30%) of patients, comprising mostly DNMT3A (14%), TET2 (5%) and ASXL1 (3%) and were confirmed in genomic DNA from PB.

In plasma samples obtained after 3 cycles of treatment, MYD88 VAF decreased in all patients and reached MRD negativity as analyzed by capture in 10/35 (29%) samples. Parallel ddPCR performed in 29/35 patients confirmed MRD results in 23 cases while capture showed low-level MRD in 4 ddPCR negative cases and ddPCR was positive in 2 capture negative cases.

Summary: Minimally invasive (liquid biopsy-based) diagnostics by targeted capture sequencing using EC-NDC reliably identifies clinically relevant genetic aberrations in WM and thus might represent a useful tool for genotyping in cases with low-level BM infiltration or lacking circulating lymphoma cells. The role of CHIP variants needs to be deeper explored.