3041 Background: Profiling of targetable genetic alterations within molecular tumor boards (MTB) guides for personalized treatment selection in patients with advanced cancers. During therapy, response is typically assessed by CT scans or MRI, which often have suboptimal sensitivity and specificity. Circulating tumor DNA (ctDNA) from blood plasma has emerged as a promising biomarker for noninvasive profiling of tumor mutational landscapes and disease monitoring. Here, we applied a pan-cancer next-generation sequencing (NGS) technology to assess the role of ctDNA for comprehensive tumor genotyping, early response prediction, and characterization clonal heterogeneity in patients receiving MTB recommended therapies. Methods: We developed and applied a custom targeted NGS approach (ExTARGET), which covers 266 genes across a 540 kb genomic region, to 157 plasma samples obtained at distinct milestones from 57 patients with diverse solid cancers. Plasma samples from healthy individuals ( n = 24) were used to determine the specificity of our technology. Results: We identified variants in 96% of baseline plasma samples by ctDNA profiling, with a median of 7 mutations per patient (range: 1-41). Most frequently mutated genes included KRAS (35%), BRAF (24%), ERBB2 (22%) and TP53 (22%). Targetable tumor variants that led to treatment recommendations within the MTB were found non-invasively in 69% of patients. Longitudinal monitoring of baseline ctDNA variants in on-treatment samples, obtained early during therapy ( n = 21), revealed that ctDNA dynamics were predictive of disease progression and preceded radiological/clinical progression in 8/19 (42%) patients. All patients with increasing ctDNA levels early during treatment showed radiologic disease progression in subsequent CT scans. On the other hand, an early decrease of ctDNA levels was associated with durable disease control in most patients and significantly favorable progression-free survival ( p = 0.008; HR = 0.1, 95%CI: 0.02-0.6). Next, we explored temporal clonal heterogeneity in plasma samples collected from 16 patients with disease progression following MTB-recommended therapies. We observed substantial clonal evolution over time, with all samples harboring at least one emerging variant. Among these emerging alterations, 19% were classified as ‘oncogenic’ and 5% were identified as potentially targetable. Conclusions: We here developed an NGS-based technology for ctDNA profiling in heavily pretreated patients receiving MTB-recommended therapies. Non-invasive genotyping from plasma robustly identifies targetable aberrations and allows comprehensive tumor genotyping. Monitoring of ctDNA during treatment and at disease progression facilitates early prediction of treatment response and profiling of temporal clonal heterogeneity that could enable subsequent treatment selection.

Abstract Circulating tumor DNA (ctDNA) from blood plasma has emerged as a promising biomarker for noninvasive profiling of tumor mutational landscapes and disease monitoring across cancers. In this study, we developed a targeted next-generation sequencing approach to explore the role of ctDNA for comprehensive tumor genotyping, early response prediction, and characterization of clonal heterogeneity in patients with advanced and rare cancers treated within molecular tumor boards. We applied our technology to 157 plasma specimens from 57 patients at distinct disease milestones and detected tumor variants in 96% of baseline samples, with 65% of them harboring actionable aberrations. Longitudinal monitoring of baseline mutations in on-treatment plasma revealed that ctDNA dynamics were significantly associated with clinical outcomes and enabled early prediction of disease progression. Finally, we observed substantial clonal heterogeneity over time, identifying emerging mutations in all analyzed plasma samples obtained at progression, including potentially targetable variants for subsequent personalized therapies.

Biological mechanisms underlying clinical heterogeneity in central nervous system lymphoma (CNSL) are largely unknown. While previous studies suggest the chemokine CLL19 as a crucial factor for the formation of CNSL in murine models, its role in human disease remains elusive. Here, we performed in-depth genetic and transcriptomic profiling of 82 CNSL specimens and identified distinct genetic aberrations and tumor cell compositions in lymphomas with high CCL19 expression, both of which were associated with immunosuppressive and anti-apoptotic signatures. CCL19 levels varied widely across CNSL patients. High CCL19 expression was significantly and independently associated with inferior progression-free and overall survival. Spatial and single-nucleus analyses as well as immunohistochemistry revealed pericytes within vessel-rich areas as the predominant source of CCL19, accompanied by significant co-localization of CCL19 with its primary receptor CCR7 that was enriched in plasmablast-like malignant B cells, as well as dendritic cells, NK cells, and CD4+ T cells. Collectively, our study identified pericyte-derived CCL19 as a novel prognostic marker in CNSL that is associated with unfavorable genetic aberrations and modifications of the immune landscape towards a resting tumor microenvironment. Spatial CCR7 co-localization suggests avenues for future therapeutic strategies targeting the CCL19-CCR7 axis.

Abstract Cancer treatment has shifted toward personalized therapy based on molecular profiling, particularly in advanced disease. Existing circulating tumor DNA panels are often broad, generating many non-actionable variants and incurring costs that limit routine use in molecular tumor boards. We developed and validated a manufacturer-independent, 109-gene liquid biopsy-centered pan-cancer open next generation sequencing panel (LION panel), combined with an in-house bioinformatic pipeline to support clinical decision-making. A total of 87 samples were analyzed, including 17 reference samples, 21 healthy blood donor controls, and 49 patient samples including nine tumor entities. The LION panel achieved 92% sensitivity and 99% specificity in reference samples, with high concordance to digital droplet PCR (r = 0.99). It detected variant allele frequencies as low as 0.05% (tumor-informed) and 0.5% (tumor-uninformed). Clinical concordance reached 82% with blood-based digital droplet PCR and 75% with whole exome tissue sequencing. In representative cases, variant dynamics correlated with disease progression and revealed additional targetable variants. Overall, the LION panel supports clinical decision-making by enabling identification of targetable variants, disease monitoring, and detection of treatment resistance, particularly when tumor tissue is unavailable.

Introduction Primary central nervous system lymphomas (PCNSL) are aggressive extranodal non-Hodgkin lymphomas confined to the CNS, accounting for 5–7% of primary brain tumors. PCNSL are biologically unique among brain malignancies due to their extracerebral origin and their specific migration and proliferation in the CNS compartment. Somatic mutations in genes involved in the B-cell receptor and Toll-like receptor signaling pathways are frequently found in PCNSL, suggesting a common genetic mechanism underlying CNS tropism. However, the developmental stage at which these genetic aberrations arise and their phylogenetic hierarchies remain unknown. Here, we applied ultrasensitive targeted capture sequencing to directly characterize tumor-specific mutational profiles in circulating and bone marrow- (BM-) resident cells to map the cellular hierarchies of lymphoma-specific genetic alterations and elucidate potential processes involved in PCNSL tropism. Methods We collected peripheral blood (PB) and BM mononuclear cell (PBMC/BMMC) samples at baseline from nine patients with PCNSL, all of whom showed no evidence of systemic lymphoma involvement by conventional imaging and PB/BM assessment. PBMCs and BMMCs were FACS-sorted into purified B-cell and T-cell populations. We then performed targeted capture next-generation sequencing (CAPP-Seq), covering immunoglobulin (Ig) regions and 100 additional genes recurrently mutated in PCNSL, to profile the mutational landscape in all nine tumor samples. These somatic alterations were tracked in bulk PBMC and BMMC samples (n=18) as well as the corresponding sorted B-cell and T-cell subsets (n=36). To control for sequencing errors and to evaluate the specificity of the monitoring approach, the same sorting and sequencing strategy was applied to PBMC/BMMC samples from four patients in complete remission one year after allogeneic stem cell transplantation for myeloid neoplasia. Results We detected a median of 256 somatic mutations in PCNSL tumor samples (range: 24-327), with PIM1 (78%), MYD88 (67%), and CD79B (67%)being the most frequently mutated genes. At a predefined specificity threshold of 95%, we identified tumor-specific alterations in 7 of 9 PCNSL patients (78%) by ultrasensitive tracking of mutations in PB or BM samples. While tumor mutations were detectable in 2 of 9 bulk PBMC and 4 of 9 bulk BMMC samples (median allele frequency [AF]: 0.003%), detection rates and allelic fractions were substantially higher in purified B-cell subsets, with 7 of 9 PB B-cell (78%) and 6 of 9 BM B-cell samples (67%) yielding positive monitoring results with a median AF of 0.03%. Notably, no PCNSL-specific somatic variants were found in any T-cell subset from either compartment. Furthermore, tumor-specific clonal VDJ rearrangements were observed in most PB B-cell samples (5/9, 56%), but only in 2 of 9 BM B-cell samples and in none of the bulk specimens or T-cell populations. Next, we focused on the PB B-cell compartment to delineate PCNSL-specific genetic patterns in circulating cells. The majority of detected variants were located in immunoglobulin regions (median of 81%) and corresponded to subclonal events in matched tumor biopsies. However, we also identified various pathogenic somatic mutations in key driver genes, including MYD88 (n=2), CD79B (n=1), and TBL1XR1 (n=1), all of which represented truncal variants in corresponding tumors. Of note, the most frequently observed aberrations involved the 5′UTR region of DTX1 (5/9 cases, 56%), a gene implicated in extranodal dissemination of germinal center (GC) B-cells. Conclusion Our findings reveal the presence of circulating and BM-resident malignant or premalignant cells in the majority of PCNSL patients, and the detection of mutations in driver genes commonly associated with PCNS lymphomagenesis. Tumor-specific mutations were exclusively identified in the B-cell compartment, while T-cell subsets remained unaffected. This absence of somatic alterations in T-cell populations together with an accumulation of Ig mutations and the presence of clonal VDJ rearrangements in B cells suggests that (pre-)malignant clones originate outside the CNS at the GC-experienced B-cell stage, without involvement of earlier hematopoietic precursors.

Background Central nervous system lymphomas (CNSL) display remarkable clinical heterogeneity, yet accurate prediction of outcomes remains challenging. The IPCG criteria are widely used in routine practice for the assessment of treatment response. However, the value of the IPCG criteria for ultimate outcome prediction is largely unclear, mainly due to the uncertainty in delineating complete from partial responses during and after treatment. Methods We explored various MRI features including semi-automated 3D tumor volume measurements at different disease milestones and their association with survival in 93 CNSL patients undergoing curative-intent treatment. Results At diagnosis, patients with more than 3 lymphoma lesions, periventricular involvement, and high 3D tumor volumes showed significantly unfavorable PFS and OS. At first interim MRI during treatment, the IPCG criteria failed to discriminate outcomes in responding patients. Therefore, we randomized these patients into training and validation cohorts to investigate whether 3D tumor volumetry could improve outcome prediction. We identified a 3D tumor volume reduction of ≥97% as the optimal threshold for risk stratification (=3D early response, 3D_ER). Applied to the validation cohort, patients achieving 3D_ER had significantly superior outcomes. In multivariate analyses, 3D_ER was independently prognostic of PFS and OS. Finally, we leveraged prognostic information from 3D MRI features and circulating biomarkers to build a composite metric that further improved outcome prediction in CNSL. Conclusions We developed semi-automated 3D tumor volume measurements as strong and independent early predictors of clinical outcomes in CNSL patients. These radiologic features could help improve risk stratification and help guide future treatment approaches.

Introduction: The Bruton's tyrosine kinase inhibitor (BTKi) ibrutinib has shown clinical efficacy as monotherapy and in combination with immunochemotherapy in patients with central nervous system lymphoma (CNSL). In addition to its direct impact on lymphoma cells, there is growing evidence that ibrutinib regulates the tumor microenvironment (TME) and T-cell immunity in systemic B-cell lymphomas such as chronic lymphocytic leukemia. However, the effect of ibrutinib on the unique TME of the brain in CNSL patients has not been explored yet. Here, we used human slice cultures from a CNSL patient undergoing complete brain tumor resection due to a radiologically suspected glioblastoma to investigate the influence of ibrutinib treatment on the myeloid compartment of the brain TME. Methods: Slices of human CNSL tissue were cultured and treated over five consecutive days either with ibrutinib in physiological concentrations observed in the cerebrospinal fluid (CSF) of lymphoma patients (ibrutinib-treated) or with DMSO (DMSO-treated). Untreated slice cultures served as additional controls. Single nuclei suspensions from the slice cultures of all three conditions were analyzed using 10x single nucleus RNA sequencing (snRNA-Seq), with the primary focus of investigating the cellular composition and transcriptional profiles of tumor cells and the TME. In addition, we performed shallow whole genome sequencing and targeted capture NGS (CAPP-Seq) of the bulk tumor as well as the slice cultures to characterize their genetic profiles. To validate our results, we performed snRNA-Seq of cells isolated from CSF of an additional patient undergoing ibrutinib therapy for progressive CNSL. Results: Genetic profiling of the slice cultures revealed the presence of characteristic mutations in MYD88, CD79B, PIM1, and TBL1XR1 genes as well as copy number alterations that are associated with immune evasion, including losses of 6p21 (HLA-D) and 1p13 (CD58). These genetic alterations were identical to those identified in the tumor bulk and their allelic representation mirrored the fraction of B-cells in the slice cultures, confirming the presence of the same malignant B-cell clone. Next, we successfully delineated nine cellular components of the brain from slice cultures by snRNA-Seq, including B-cells, T-cells, myeloid cells, endothelial cells, stromal cells, oligodendrocytes, astrocytes, and neurons. By integrating the myeloid cell compartment from all three conditions, we identified five distinct subclusters based on their transcriptional signatures. In ibrutinib-treated slices, the proportion of myeloid cells with an antigen-presenting expression pattern, defined by high expression of CIITA, CD74, and other HLAs, increased compared to those in DMSO-treated or untreated slice cultures. Conversely, the cluster of SPP1-expressing myeloid cells substantially decreased after ibrutinib treatment compared to the controls. Pseudotime analysis indicated that these two expression patterns are the result of two distinct polarization states of myeloid cells within the TME of CNSL. Finally, snRNA-Seq of CSF cells from an additional CNSL patient receiving ibrutinib identified the same upregulated antigen-presenting expression pattern in the myeloid cell compartment, validating these findings. Conclusion: Based on a unique human slice culture model from primary CNSL tissue, our data suggest a modulating effect of ibrutinib on the myeloid compartment in human CNSL by shifting myeloid cells from an immunosuppressive phenotype expressing SPP1 to an antigen-presenting phenotype. Despite being inherently limited by this single-case analysis, these results indicate an unknown immune-activating effect of ibrutinib on the brain TME in CNSL patients.

Introduction: Refractory or relapsed central nervous system lymphoma (r/r CNSL) remains a therapeutic challenge with poor overall survival. The biological mechanisms underlying clinical heterogeneity in response to standard immunochemotherapy remain largely unknown. Previous publications suggest a critical role of CCL19 for parenchymal retention of malignant B-cells and formation of CNSL in the brain of mice (O'Connor et al., Cancer Cell, 2019). Here, we characterized CCL19 in primary CNSL tissue, explored its prognostic role, the impact on the tumor microenvironment (TME), and its spatial/cellular origins in patients with CNSL using a multi-omics approach. Methods: We comprehensively profiled 82 tumor specimens obtained through stereotactic biopsies (n=74) or brain tumor resections (n=8) from primary CNSL (PCNSL) patients by bulk RNA sequencing (RNA-Seq), targeted capture next-generation sequencing (CAPP-Seq), and shallow whole-genome sequencing. CIBERSORTx was used to define immunological and CNS-specific cell subsets. The immune cell landscape and genetic/transcriptional features were further correlated with clinical and radiological parameters. To investigate the cellular and spatial origin of CCL19, 10x single nucleus RNA-Seq (snRNA-Seq, n=7, 150,300 cells) and spatial transcriptomics (n=7) of primary CNSL samples were performed. Results: CLL19 expression was significantly correlated with tumor volumes measured by magnetic resonance imaging (MRI, p=0.02, r=0.4), supporting the results observed in mouse models. Furthermore, CCL19 was a strong prognostic marker in patients undergoing curative-intent high-dose methotrexate-based immunochemotherapy, with high CCL19 expression levels being associated with significantly unfavorable progression-free survival (p=0.009, HR=2.4, CI: 1.2-4.7) and overall survival (p=0.02, HR=2.5, CI: 1.1-5.8). We further observed that CNSL tumor biopsies revealing high CCL19 expression levels were significantly enriched for CD4+ memory T cells and NK cells in a resting state and tended to be associated with a higher number of macrophages in the TME. CCL19-high tumors did not present with a specific mutational or copy number profile; yet, all EBV-positive cases (n=5) showed high CCL19 levels. We further delineated the unique cellular components of the brain from seven CNSL tissue samples by snRNA-Seq and found that the stromal cell compartment displayed the highest expression of CCL19 in the CNSL TME. Finally, spatial transcriptomics from seven CNSL tumor slices showed particularly high CCL19 expression in vessel-rich areas and decreasing CCL19 levels in less-vascularized regions. Conclusion: Collectively, we identified CCL19 as a prognostic molecular marker in CNSL, predicting unfavorable clinical outcomes. Our results indicate that CCL19, derived from stromal cells in vessel-rich areas, might cause retention of malignant B-cells in the brain parenchyma of CNSL patients and may be associated with modifications of the immune landscape towards a cold microenvironment.

Introduction Patients with relapsed or refractory diffuse large B-cell lymphoma (r/r DLBCL) are characterized by a particularly poor prognosis. Anti-CD19 chimeric antigen receptor (CAR) T-cell therapy has improved outcomes of patients with r/r DLBCL, yet a substantial proportion of patients still experiences relapse or progression after CAR T-cell treatment. Identifying patients at high risk of CAR T-cell therapy resistance or future lymphoma progression remains challenging. Here, we explored the value of circulating tumor DNA (ctDNA) as a prognostic biomarker at baseline and early into treatment in DLBCL patients undergoing standard-of-care CAR T-cell therapy, without the need for matched tumor genotypes. We further investigated associations between ctDNA and other known clinical risk factors. Methods We applied targeted next-generation sequencing (NGS) to a total of 53 samples from r/r DLBCL patients receiving CAR T-cell therapy (axicabtagene ciloleucel and tisagenlecleucel, n=16) at the University Medical Center Freiburg (Germany), using a modified version of the AVENIO ctDNA analysis workflow (Roche; Research Use Only) that covered ~314kb and targeted 466 distinct genes recurrently mutated in DLBCL (based on the CAPP-Seq workflow, Kurtz DM et al. J Clin Oncol 2018). Tumor genotypes were assessed noninvasively from plasma samples obtained at baseline before lymphodepletion with matched germline controls. Concentrations of ctDNA were quantified at baseline and early after CAR T-cell therapy at day 7-10. Initial treatment response was assessed by conventional PET-CT obtained 4-6 weeks after treatment based on the Lugano criteria. Results At baseline prior to lymphodepletion, we detected somatic mutations in 100% of plasma samples by noninvasive genotyping, with a median of 19.5 mutations per patient (range: 3-97). The most frequently mutated genes were IGHV, IGLL5, BCL2, MYC, BTG1, CREBBP, and TP53. Patients with high baseline ctDNA concentrations (mean allele frequency [AF] >= 1%) showed shorter progression-free survivial (PFS) and overall survival (OS) than patients with ctDNA levels below this threshold (median PFS: 86.5 days vs. not reached, p=0.04, HR: 6.3, 95% CI: 1.7-23.6; median OS: 219 days vs. not reached, p=0.048, HR: 4.1, 95% CI 1.0-16.6, Figure 1). Pretreatment ctDNA levels were significantly correlated with LDH concentrations ( p=0.049, r=0.5). We further found significantly higher ctDNA concentrations in patients with transformed DLBCL (vs. non-transformed, p=0.008) and in those with 3 or more prior treatment lines (vs. 1-2, p=0.04). At day 7-10 after CAR T-cell therapy, ctDNA levels dropped a median of 10-fold compared to the pretreatment time point ( p=0.008). Patients achieving a complete (CMR) or partial metabolic response (PMR) by PET-CT after 4-6 weeks showed a significant decrease of ctDNA ( p=0.001), while those with a stable (SD) or progressive disease (PD) had no significant ctDNA reduction at day 7-10 after CAR T-cell infusion. Vice versa, while 100% of patients with a >1.5-log-fold drop of ctDNA level between the pretreatment and interim time point revealed a CMR or PMR in early PET-CT scans, only 62% of patients with a smaller ctDNA decline responded to treatment according to PET-CT. Conclusions Our data suggests, together with previously published studies (Sworder BJ et al., Cancer Cell, 2023; Frank MJ et al. J Clin Oncol, 2021), that ctDNA profiling may allow robust noninvasive genotyping, accurate risk-stratification, and early prediction of clinical outcomes in r/r DLBCL patients undergoing CAR T-cell therapy. Although limited by small sample size, this pilot study highlights a potential future role of ctDNA measurements for personalized treatment selection and guidance in clinical trials.

Abstract 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.