Erik van Dijk

The evolutionary processes that drive malignant progression of IDH-mutant astrocytomas remain unclear. Here, we performed multiomics on matched initial and recurrent tumor samples from a cohort of 105 patients and overlaid the data with detailed clinical annotation. We identified overlapping features associated with malignant progression that are derived from three molecular mechanisms: cell cycling, tumor cell (de)differentiation and remodeling of the extracellular matrix. Together, they provide a rationale of the underlying biology of tumor malignancy. DNA methylation levels decreased over time, predominantly in tumors with malignant transformation, and co-occurred with poor prognostic genetic events. We identified a DNA methylation-based signature strongly associated with survival, which allows objective, molecular-based grading of IDH-mutant astrocytomas to aid clinical decision making. Our findings were validated on large, independent cohorts of IDH-mutant astrocytoma samples. Lastly, in this retrospective study, we found little effect of radiotherapy or chemotherapy on the molecular features associated with malignant progression.

To study the evolutionary processes that drive malignant progression of IDH-mutant astrocytomas, we performed multi-omics on a large cohort of matched initial and recurrent tumor samples. The overlay of genetic, epigenetic, transcriptomic and proteomic data, combined with single-cell analysis, have identified overlapping features associated with malignant progression. These features are derived from three molecular mechanisms and provide a rationale of the underlying biology of tumor malignancy: cell-cycling, tumor cell (de-)differentiation and remodeling of the extracellular matrix. Specifically, DNA-methylation levels decreased over time, predominantly in tumors with malignant transformation and co-occurred with poor prognostic genetic events. DNA-methylation was lifted from specific loci associated with DNA replication and was associated with an increased RNA and protein expression of cell cycling associated genes. All results were validated on samples of newly diagnosed IDH-mutant astrocytoma patients included the CATNON randomized phase 3 clinical trial. Importantly, malignant progression was hardly affected by radio- or chemotherapy, indicating that treatment does not affect the course of disease. Our results culminate in a DNA-methylation based signature for objective tumor grading.

Background: The T2-FLAIR mismatch sign is defined by signal loss of the T2-weighted hyperintense area with Fluid-Attenuated Inversion Recovery (FLAIR) on magnetic resonance imaging, causing a hypointense region on FLAIR. It is a highly specific diagnostic marker for IDH-mutant astrocytoma and is postulated to be caused by intercellular microcystic change in the tumor tissue. However, not all IDH-mutant astrocytomas show this mismatch sign and some show the phenomenon in only part of the lesion. The aim of the study is to determine whether the T2-FLAIR mismatch phenomenon has any prognostic value beyond initial noninvasive molecular diagnosis. Methods: Patients initially diagnosed with histologically lower-grade (2 or 3) IDH-mutant astrocytoma and with at least 2 surgical resections were included in the GLASS-NL cohort. T2-FLAIR mismatch was determined, and the growth pattern of the recurrent tumor immediately before the second resection was annotated as invasive or expansive. The relation between the T2-FLAIR mismatch sign and tumor grade, microcystic change, overall survival (OS), and other clinical parameters was investigated both at first and second resection. Results: The T2-FLAIR mismatch sign was significantly related to Grade 2 (80% vs 51%), longer post-resection median OS (8.3 vs 5.2 years), expansive growth, and lower age at second resection. At first resection, no relation was found between the mismatch sign and OS. Microcystic change was associated with areas of T2-FLAIR mismatch. Conclusions: T2-FLAIR mismatch in IDH-mutant astrocytomas is correlated with microcystic change in the tumor tissue, favorable prognosis, and Grade 2 tumors at the time of second resection.

Although the genomic and immune microenvironmental landscape of follicular lymphoma (FL) has been extensively investigated, little is known about the potential biological differences between stage I and stage III/IV disease. Using next-generation sequencing and immunohistochemistry, 82 FL nodal stage I cases were analyzed and compared with 139 FL stage III/IV nodal cases. Many similarities in mutations, chromosomal copy number aberrations, and microenvironmental cell populations were detected. However, there were also significant differences in microenvironmental and genomic features. CD8+ T cells (P = .02) and STAT6 mutations (false discovery rate [FDR] <0.001) were more frequent in stage I FL. In contrast, programmed cell death protein 1–positive T cells, CD68+/CD163+ macrophages (P < .001), BCL2 translocation (BCL2trl+) (P < .0001), and KMT2D (FDR = 0.003) and CREBBP (FDR = 0.04) mutations were found more frequently in stage III/IV FL. Using clustering, we identified 3 clusters within stage I, and 2 clusters within stage III/IV. The BLC2trl+ stage I cluster was comparable to the BCL2trl+ cluster in stage III/IV. The two BCL2trl– stage I clusters were unique for stage I. One was enriched for CREBBP (95%) and STAT6 (64%) mutations, without BLC6 translocation (BCL6trl), whereas the BCL2trl– stage III/IV cluster contained BCL6trl (64%) with fewer CREBBP (45%) and STAT6 (9%) mutations. The other BCL2trl– stage I cluster was relatively heterogeneous with more copy number aberrations and linker histone mutations. This exploratory study shows that stage I FL is genetically heterogeneous with different underlying oncogenic pathways. Stage I FL BCL2trl– is likely STAT6 driven, whereas BCL2trl– stage III/IV appears to be more BCL6trl driven.

A significant proportion of colorectal cancer (CRC) patients develop peritoneal metastases (PM) in the course of their disease. PMs are associated with a poor quality of life, significant morbidity and dismal disease outcome. To improve care for this patient group, a better understanding of the molecular characteristics of CRC-PM is required. Here we present a comprehensive molecular characterization of a cohort of 52 patients. This reveals that CRC-PM represent a distinct CRC molecular subtype, CMS4, but can be further divided in three separate categories, each presenting with unique features. We uncover that the CMS4-associated structural protein Moesin plays a key role in peritoneal dissemination. Finally, we define specific evolutionary features of CRC-PM which indicate that polyclonal metastatic seeding underlies these lesions. Together our results suggest that CRC-PM should be perceived as a distinct disease entity.

Introduction: In gastric cancer, studies of tumor infiltrating lymphocytes as a prognostic biomarker show contradictory results. These results may be caused by use of variable immunohistochemistry (IHC) quantification techniques, different marker selections and particularly inconsistency in the evaluated tumor regions. To overcome these issues, we performed a comprehensive digital image analysis of 5 immune cell markers for their prognostic value in a cohort of gastric cancer patients who were treated with surgery only in the Dutch D1/D2 trial. Methods: All available surgical resection specimens of gastric cancer patients were included in this study (N=251). IHC for T-cell markers CD3, CD45RO, CD8, FOXP3 and Granzyme B was performed on serial slides. After manual annotation of the tumor area, an invasive margin was defined as 0.5 mm into the tumor still containing tumor cells (inner margin, IM) and 0.5 mm outside of the tumor not containing tumor cells (outer margin, OM). The density of positive immune cells (cells/mm²) was digitally quantified using QuPath for each 0.5x0.5 mm² square across tumor center (TC), IM and OM, separately. A classification and regression tree (CART) model was employed to identify an optimal combination of prognostic markers from the continuous immune cell density variables with cancer specific survival (CSS) as outcome. Results: The CART decision tree identified CD8 OM (≥798 cells/mm²) as most dominant prognostic factor, followed by FOXP3 TC (≥20 cells/mm²) in the CD8 OM low subset. This resulted in 3 CART branches in the decision tree: CD8 OM high with best prognosis, CD8 OM low/FOXP3 TC high with intermediate prognosis, and CD8 OM low/FOXP3 TC low with worst prognosis (Log-rank P-value Citation Format: Tanya T. Soeratram, Hedde D. Biesma, Jacqueline M. Egthuijsen, Elma Meershoek-Klein Kranenbarg, Henk H. Hartgrink, Cornelis J. van de Velde, Erik van Dijk, Yongsoo Kim, Bauke Ylstra, Hanneke W. van Laarhoven, Nicole C. van Grieken. CD8+ T cells in the invasive margin combined with FOXP3+ T cells in the tumor center significantly associate with survival in resectable gastric cancer, a post-hoc analysis of the Dutch D1/D2 trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1727.

INTRODUCTION. Diffuse gliomas are the most frequent and devastating primary CNS tumors in adults. Standard treatment has limited efficacy and without exception these gliomas recur. The evolutionary processes that drive progression in glioma of the IDH-mutant astrocytoma subgroup, remain unclear. The GLASS-NL consortium was initiated to gain insight into the molecular mechanisms underlying glioma evolution and to identify markers of progression in IDH-mutant astrocytomas. Such markers can ultimately assist clinical decision making. Here, we present the results of genome wide DNA-methylation profiling of samples included in the GLASS-NL study. METHODS. 110 adult patients were identified with an IDH-mutant, 1p19q non-codeleted, astrocytoma at first diagnosis. All patients underwent surgical resection of the tumor at least twice, separated by >6 months with a median of 41.9 months (IQR:26.5-65.9). After surgical resection of the initial tumor, 63% and 22% of the patients were treated with radiotherapy or chemotherapy respectively. DNA-methylation profiling was performed on 235 samples from 103 patients, using the Illumina Infinium MethylationEPIC BeadChip array. Copy number alterations (CNAs) were extracted from these data. Methylation subclasses were determined according to Capper et al. (Nature, 2018). Overall survival (OS) was measured from date of initial surgery. RESULTS. Of all primary tumors, 85 (87%) of the tumor samples were assigned to the A_IDH (‘low grade’) methylation subclass and 10 (10%) to the A_IDH_HG (‘high grade’) subclass. The relative proportion of high grade tumors increased ~three-fold at tumor recurrence (32/101, 32%) and even further in the second recurrence (15/29, 52%). The high grade subclass of the recurrent, but not the initial tumor sample, was negatively associated with OS (p < 0.0001). The overall DNA-methylation levels of recurrent samples were lower than that of initial samples. This difference is explained by the increased number of high grade samples at recurrence, since near identical DNA-methylation levels were observed in samples that remained low grade. Analysis of CNAs revealed several chromosomal arms and bands that were more frequently altered in samples of the high grade methylation subclass compared to low grade. In addition, gains and losses of specific genes, such as homozygous deletion (HD) of CDKN2A/B, were more frequent in high grade samples. Overall DNA-methylation levels of recurrent samples with CDKN2A/B HD were lower than that of samples without this deletion. However, CDKN2A/B HD alone does not fully explain DNA-demethylation at malignant progression and other molecular aberrations are likely to contribute as well. CONCLUSION. Longitudinal methylation profiling analysis of IDH-mutant astrocytoma reveals a shift towards a higher grade at tumor recurrence coinciding with reduced genome-wide DNA-methylation levels. Citation Format: Wies Rijan Vallentgoed, Anneke Niers, Karin van Garderen, Martin van den Bent, Erik van Dijk, Kaspar Draaisma, Paul van Eijk, Iris de Heer, Mathilde Kouwenhoven, Johan Kros, Wendy de Leng, Ivonne Martin, Pierre Robe, Marion Smits, Mircea Tesileanu, Hinke van Thuijl, Roel Verhaak, Bart Westerman, Mark van de Wiel, Bauke Ylstra, Pieter Wesseling, Pim French. Methylation analysis of matched primary and recurrent IDH-mutant astrocytoma; an update from the GLASS-NL consortium [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4020.