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Testing for genetic alterations in tumor tissue allows clinicians to identify patients who most likely will benefit from molecular targeted treatment. EXLIQUID – exploiting liquid biopsies to advance cancer precision medicine – investigates the potential of additional non-invasive tools for guiding therapy decisions and monitoring of advanced cancer patients. The term “liquid biopsy” (LB) refers to non-invasive analysis of tumor-derived circulating material such as cell-free DNA in blood samples from cancer patients. Although recent technological advances allow sensitive and specific detection of LB biomarkers, only few LB assays have entered clinical routine to date. EXLIQUID is a German Cancer Consortium (DKTK)-wide joint funding project that aims at establishing LBs as a minimally-invasive tool to analyze molecular changes in circulating tumor DNA (ctDNA). Here, we present the structure, clinical aim, and methodical approach of the new DKTK EXLIQUID consortium. Within EXLIQUID, we will set up a multicenter repository of high-quality LB samples from patients participating in DKTK MASTER and local molecular tumor boards, which use molecular profiles of tumor tissues to guide targeted therapies. We will develop LB assays for monitoring of therapy efficacy by the analysis of tumor mutant variants and tumor-specific DNA methylation patterns in ctDNA from these patients. By bringing together LB experts from all DKTK partner sites and exploiting the diversity of their particular expertise, complementary skills and technologies, the EXLIQUID consortium addresses the challenges of translating LBs into the clinic. The DKTK structure provides EXLIQUID a unique position for the identification of liquid biomarkers even in less common tumor types, thereby extending the group of patients benefitting from non-invasive LB testing. Besides its scientific aims, EXLIQUID is building a valuable precision oncology cohort and LB platform which will be available for future collaborative research studies within the DKTK and beyond.

PURPOSE Clinical outcomes of patients with CNS lymphomas (CNSLs) are remarkably heterogeneous, yet identification of patients at high risk for treatment failure is challenging. Furthermore, CNSL diagnosis often remains unconfirmed because of contraindications for invasive stereotactic biopsies. Therefore, improved biomarkers are needed to better stratify patients into risk groups, predict treatment response, and noninvasively identify CNSL. PATIENTS AND METHODS We explored the value of circulating tumor DNA (ctDNA) for early outcome prediction, measurable residual disease monitoring, and surgery-free CNSL identification by applying ultrasensitive targeted next-generation sequencing to a total of 306 tumor, plasma, and CSF specimens from 136 patients with brain cancers, including 92 patients with CNSL. RESULTS Before therapy, ctDNA was detectable in 78% of plasma and 100% of CSF samples. Patients with positive ctDNA in pretreatment plasma had significantly shorter progression-free survival (PFS, P < .0001, log-rank test) and overall survival (OS, P = .0001, log-rank test). In multivariate analyses including established clinical and radiographic risk factors, pretreatment plasma ctDNA concentrations were independently prognostic of clinical outcomes (PFS HR, 1.4; 95% CI, 1.0 to 1.9; P = .03; OS HR, 1.6; 95% CI, 1.1 to 2.2; P = .006). Moreover, measurable residual disease detection by plasma ctDNA monitoring during treatment identified patients with particularly poor prognosis following curative-intent immunochemotherapy (PFS, P = .0002; OS, P = .004, log-rank test). Finally, we developed a proof-of-principle machine learning approach for biopsy-free CNSL identification from ctDNA, showing sensitivities of 59% (CSF) and 25% (plasma) with high positive predictive value. CONCLUSION We demonstrate robust and ultrasensitive detection of ctDNA at various disease milestones in CNSL. Our findings highlight the role of ctDNA as a noninvasive biomarker and its potential value for personalized risk stratification and treatment guidance in patients with CNSL. [Media: see text]

Of BCR-ABL negative myeloproliferative neoplasm (MPN) patients, 3-14 % display a concomitant monoclonal gammopathy (MGUS). Nonetheless, literature on co-occurring MPN and MGUS is scarce, the molecular underpinnings are unknown and it is unclear whether patients require a specific management. Here, we compared the clinical and genetic features of MPN patients with and without concomitant MGUS. Of 114 MPN patients prospectively studied by serum immunofixation (median age, 67 years; 36.0 % essential thrombocythemia [ET], 24.6 % polycythemia vera [PV], 11.4 % secondary myelofibrosis [sMF], 28.1 % primary myelofibrois [PMF]; 73.7 % JAK2 V617F positive), 10 (9 %) harbored an M-protein. No relevant clinical differences existed between MPN patients with or without M-protein. Seven additional MPN/MGUS patients were retrospectively identified in our MPN registry, yielding a total of 17 patients (7 ET, 3 PV, 3 sMF, 4 PMF). One patient developed multiple myeloma (MM) and one smoldering MM. Seven of 12 patients analyzed carried mutations (e.g. in ASXL1 or TET2) in addition to those in JAK2 or CALR, and 4 of 10 patients showed aberrant cytogenetics. M-protein was mainly IgG (12/17), followed by IgM (4/17). In the two patients that underwent allogeneic stem cell transplantation mutant JAK2 and M-protein were no longer detectable post-transplant. In conclusion, MGUS prevalence in our cohort was in the range of previous reports and at most slightly higher than expected in the general population. MGUS presence did not correlate with a specific MPN entity, clinical features or genetic alterations. Our observations suggest that there is no strong clinical or biological relationship between the occurrence of MGUS and MPN.

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.

We recently described the development of an inv(16) acute myeloid leukemia (AML) in a CBL mutated clonal hematopoiesis. Here, we further characterized the clonal composition and evolution of the AML based on the genetic information from the bulk specimen and analyses of individual bone marrow cells for mutations in CAND1, PTPRT, and DOCK6. To control for allele dropout, heterozygous polymorphisms located close to the respective mutation loci were assessed in parallel. The clonal composition concluded from exome sequencing suggested a proliferation advantage associated with the acquisition of mutations in CAND1, PTPRT, and DOCK6. Out of 102 single cell sequencing reactions on these mutations and the respective polymorphisms, analyses yielded conclusive results for at least 2 mutation sites in 12 cells. The single cell genotyping not only confirmed the co-occurrence of the PTPRT, CAND1 and DOCK6 mutations in the same AML clone but also revealed a clonal hierarchy, as the PTPRT mutation was likely acquired after the CAND1 and DOCK6 mutations. This insight had not been possible based solely on the exome sequencing data and suggests that the mutation in PTPRT, which encodes a STAT3-inhibiting protein tyrosine phosphatase, contributed to the AML development at a later stage by enhancing proliferation.

Knowledge of the molecular and clonal characteristics in the myelodysplastic syndromes (MDS) and during progression to acute myeloid leukaemia (AML) is essential to understand the disease dynamics and optimize treatment. Sequencing serial bone marrow samples of eight patients, we observed that MDS featured a median of 3 mutations. Mutations in genes involved in RNA-splicing or epigenetic regulation were most frequent, and exclusively present in the major clone. Minor subclones were distinguishable in three patients. As the MDS progressed, a median of one mutation was gained, leading to clonal outgrowth. No AML developed genetically independent of a pre-existing clone. The gained mutation mostly affected genes encoding signalling proteins. Additional acquisition of genomic aberrations frequently occurred. Upon treatment, emergence of new clones could be observed. As confirmed by single-cell sequencing, multiple mutations in identical genes in different clones were present within individual patients. DNA-methylation profiling in patients without identification of novel mutations in AML revealed methylation changes in individual genes. In conclusion, our data complement previous observations on the mutational and clonal characteristics in MDS and at progression. Moreover, DNA-methylation changes may be associated with progression in single patients. Redundancy of mutated genes in different clones suggests fertile grounds promoting clonal selection or acquisition.

Intratumoral genetic heterogeneity may impact disease outcome. Gold standard for dissecting clonal heterogeneity are single-cell analyses. Here, we present an efficient workflow based on an advanced Single-Cell Printer (SCP) device for the study of gene variants in single cancer cells. To allow for precise cell deposition into microwells the SCP was equipped with an automatic dispenser offset compensation, and the 384-microwell plates were electrostatically neutralized. The ejection efficiency was 99.7% for fluorescent beads (n = 2304) and 98.7% for human cells (U-2 OS or Kasumi-1 cancer cell line, acute myeloid leukemia [AML] patient; n = 150). Per fluorescence microscopy, 98.8% of beads were correctly delivered into the wells. A subset of single cells (n = 81) was subjected to whole genome amplification (WGA), which was successful in all cells. On empty droplets, a PCR on LINE1 retrotransposons yielded no product after WGA, verifying the absence of free-floating DNA in SCP-generated droplets. Representative gene variants identified in bulk specimens were sequenced in single-cell WGA DNA. In U-2 OS, 22 of 25 cells yielded results for both an SLC34A2 and TET2 mutation site, including cells harboring the SLC34A2 but not the TET2 mutation. In one cell, the TET2 mutation analysis was inconclusive due to allelic dropout, as assessed via polymorphisms located close to the mutation. Of Kasumi-1, 23 of 33 cells with data on both the KIT and TP53 mutation site harbored both mutations. In the AML patient, 21 of 23 cells were informative for a TP53 polymorphism; the identified alleles matched the loss of chromosome arm 17p. The advanced SCP allows efficient, precise and gentle isolation of individual cells for subsequent WGA and routine PCR/sequencing-based analyses of gene variants. This makes single-cell information readily accessible to a wide range of applications and can provide insights into clonal heterogeneity that were indeterminable solely by analyses of bulk specimens.

INTRODUCTION: We recently described the first case of the evolution of inv(16) AML on the background of a clonal hematopoiesis due to a germline CBL mutation (defining the CBL syndrome), and we identified possibly cooperating mutations by exome sequencing (Becker et al. Blood 2014;123:1883-6). Among the mutated genes was PTPRT, encoding a protein tyrosine phosphatase that inhibits STAT3 activity and is commonly mutated in cancer (reviewed by Zhao et al. Oncogene 2015;34:3885-94). Here, we investigated the co-occurrence of mutated PTPRT with other mutated genes by single cell genotyping in order gain insights into the clonal architecture and sequence of mutation acquisition. METHODS: Exome sequencing of the bulk specimens was previously described; germline or somatic origin of mutations was verified in skin fibroblasts (Becker et al. Blood 2014;123:1883-6). For single cell genotyping, Ficoll-enriched bone marrow aspirates were DAPI stained, and single cells were placed into each well of a PCR plate using a MoFlo high speed cell sorter (Beckman Coulter). Genomic DNA was amplified by whole genome amplification (WGA) using the REPLI-g Mini Kit (Qiagen) according to a modified protocol, and subjected to PCR and Sanger sequencing of the respective mutation loci. As WGA can lead to allele dropout (ADO), we also sequenced single nucleotide polymorphisms (SNPs), that were identified by CytoScan HD array (Affymetrix) to be heterozygous in the sample and that were located nearby the respective mutation loci. RESULTS: Exome sequencing allows prediction of a possible clonal architecture based on the variant allele frequencies (VAFs). VAFs of the mutations identified in the AML were as follows: KIF14 p.V341I (VAF 51%), TMEM125 p.D113N (51%), MIOX p.W225R (46%), CAND1 p.E584* (39%), NID2 p.D319N (38%), ARF3 p.N101S (36%), PRSS16 p.R491C (36%), PTPRT p.T844M (33%), DOCK6 p.R1872_K1873insP (33%), ADAM12 p.A222V (21%), CMIP p.T323M (15%) and MYOCD p.D283N (7%); due to its germline nature, all leukemic cells harbored the CBL p.D390V mutation. In order to verify the co-occurrence of mutations in a clone and thus the clonal architecture, we performed single cell genotyping of the mutations in PTPRT as well as CAND1 and DOCK6. CAND1 and DOCK6 were selected in addition to PTPRT since their comparable VAFs did not allow identifying the sequence of acquisition. Moreover, CAND1 and DOCK6 were affected by likely deleterious mutations and were previously found mutated in AML. To control for ADO, we included the heterozygous SNPs rs2867061 (PTPRT), rs1252402 (C AND1), and rs12980863 (DOCK6) in our analyses. We analyzed 19 single cells for the 6 mutations and SNPs. This resulted in 102 successful sequencing reactions, and yielded informative results for at least 2 mutations in 12 cells and for all 3 mutations in 5 cells. Based on the concurrent presence of the wild-type allele at the mutation locus and ADO at the SNP site, 18 mutation analyses were judged to be inconclusive. Overall, our analyses confirmed that the mutations in CAND1, PTPRT and DOCK6 occurred together in the same clone. Moreover, based on the identification of cells with the presence of both CAND1 and DOCK6 mutations but presence or absence of PTPRT mutations, respectively, we concluded that PTPRT mutations were acquired after the mutations in DOCK6 and CAND1. CONCLUSION: Single cell genotyping verified the co-occurrence of PTPRT, CAND1 and DOCK6 mutations in the same AML clone and revealed a clonal hierarchy, as the PTPRT mutation was acquired after the mutations in CAND1 and DOCK6. These insights into the clonal architecture and evolution had not been possible solely based on exome sequencing and suggest that the sequential expression of mutated PTPRT may cooperate with mutated CBL and inv(16) at a late stage of AML development. Disclosures No relevant conflicts of interest to declare.