Longitudinal analysis of plasma DNA and RNA for therapy monitoring of ALK-positive non-small cell lung cancer

Steffen Dietz ^1,2 Petros Christopoulos ^2,3 Lisa Gu ^1,2 Volker Endris ⁴ Zhao Yuan ⁵ Simon Ogrodnik ^1,2 Tomasz Zemojtel ⁶ Marc A. Schneider ^2,7 Anna-Lena Volckmar ⁴ Michael Meister ^2,7 Thomas Muley ^2,7 Martin Reck ⁸ Matthias Schlesner ^2,5 Michael Thomas ^2,3 Albrecht Stenzinger ^2,4,9 Holger Sültmann ^1,2

¹Division of Cancer Genome Research, German Cancer Research Center (DKFZ), German Cancer Consortium (DKTK), and National Center for Tumor Diseases (NCT), Heidelberg, Germany
²German Center for Lung Research (DZL), TLRC Heidelberg, Heidelberg, Germany
³Department of Oncology, Thoraxklinik at University Hospital Heidelberg, Heidelberg, Germany
⁴Institute of Pathology, Heidelberg University, Heidelberg, Germany
⁵Bioinformatics and Omics Data Analytics, German Cancer Research Center (DKFZ), Heidelberg, Germany
⁶Genomics Core Facility, Berlin Institute of Health (BIH), Charité University Medical Center, Berlin, Germany
⁷Translational Research Unit, Thoraxklinik at University Hospital Heidelberg, Heidelberg, Germany
⁸Lung Clinic Grosshansdorf, Airway Research Center North, German Center for Lung Research, Grosshansdorf, Germany
⁹German Cancer Consortium, DKTK, Heidelberg, Germany

Non-small cell lung cancer (NSCLC) patients with ALK rearrangements are routinely treated with tyrosine kinase inhibitors (TKIs), leading to improved survival. However, clinical courses vary widely and the disease remains incurable. Therefore, early detection and molecular characterization of treatment failure is important for patient outcome.

To identify indicators of therapy response and tumor progression, we performed an analysis of circulating tumor DNA (ctDNA) from serial plasma samples of 46 NSCLC patients with ALK rearrangements. Using hybrid-capture-based targeted sequencing and shallow whole genome sequencing (sWGS), we achieved mean unique target coverages of 5000x and 0.5x, respectively. Variable mutation levels were marked in all patients and correlated with clinical features. For example, mutant ctDNA levels were low in cases of stable disease, but increased at the time of TKI failure. Targeted sequencing identified known and novel mutations indicating TKI resistance. We also found mutated TP53 at the time of progression in patients with initially TP53 wildtype tumors. The progression-free survival of patients with acquired TP53 mutations was comparable to that of primarily TP53 mutated and shorter than that of persistently TP53 wildtype cases. sWGS of ctDNA identified copy number variations, some of which might contribute to disease progression. We also measured miRNA abundances in corresponding serum samples and noted fluctuating miRNA levels during therapy that reflected the remission status and correlated withthe clinical course in several cases.

Our data suggest that liquid biopsies can improve ALK⁺ NSCLC patient care through early detection of disease progression and tailored treatment of resistant tumors. ctDNA and circulating miRNA can indicate the need to switch treatment and provide information to guide the selection of next-line therapy. Detection of acquired TP53 mutations in liquid rebiopsies at the time of disease progression identifies additional high-risk cases and suggests potential clinical utility of ctDNA monitoring for this disease beyond profiling of ALK resistance mutations.