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Contains fulltext : 282509.pdf (Publisher’s version ) (Open Access) 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]

Large B-cell lymphoma of immune-privileged sites (LBCL-IP) arise in immune sanctuaries including the testis and central nervous system (CNS). After initially reaching complete response, relapses occur in almost 50% of patients, typically at other immune-privileged sites. Resolution of the clonal relationships and evolutionary patterns of LBCL-IP is required to understand the unique clinical behavior. We collected a unique set of 33 primary–relapse LBCL-IP sample pairs and performed next-generation sequencing for copy number, mutation, translocation, and immunoglobulin clonality analysis. All LBCL-IP sample pairs were clonally related, and both tumors developed from a common progenitor cell (CPC) with MYD88 and TBL1XR1 mutations and/or BCL6 translocations in 30/33 cases, indicating that these are early genetic events. This was succeeded by intermediate genetic events including shared, as well as unique alterations in targets of aberrant somatic hypermutation (aSHM), CD79B mutations, and 9p21.3/CDKN2A loss. Genetic alterations in genes involved in immune escape (HLA, CD274/PDCD1LG2) were predominantly unique in primary and relapse samples and thus considered late genetic events. Together, this study indicates that primary and relapsed LBCL-IP follow an early parallel evolutionary pattern where the CPC contains genetic alterations that support prolonged survival/proliferation and retention in a memory B-cell state, followed by germinal center reentry, aSHM and immune escape. Significance: Genomic analyses reveal that primary and relapse LBCL-IP originate from a common progenitor cell with a small set of genetic alterations, followed by extensive parallel diversification, elucidating the clonal evolution of LBCL-IP.

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Large B-cell lymphoma of immune-privileged sites (LBCL-IP) arise in immune sanctuaries including the testis and central nervous system (CNS). After initially reaching complete response, relapses occur in almost 50% of patients, typically at other immune-privileged sites. Resolution of the clonal relationships and evolutionary patterns of LBCL-IP is required to understand the unique clinical behavior. We collected a unique set of 33 primary–relapse LBCL-IP sample pairs and performed next-generation sequencing for copy number, mutation, translocation, and immunoglobulin clonality analysis. All LBCL-IP sample pairs were clonally related, and both tumors developed from a common progenitor cell (CPC) with MYD88 and TBL1XR1 mutations and/or BCL6 translocations in 30/33 cases, indicating that these are early genetic events. This was succeeded by intermediate genetic events including shared, as well as unique alterations in targets of aberrant somatic hypermutation (aSHM), CD79B mutations, and 9p21.3/CDKN2A loss. Genetic alterations in genes involved in immune escape (HLA, CD274/PDCD1LG2) were predominantly unique in primary and relapse samples and thus considered late genetic events. Together, this study indicates that primary and relapsed LBCL-IP follow an early parallel evolutionary pattern where the CPC contains genetic alterations that support prolonged survival/proliferation and retention in a memory B-cell state, followed by germinal center reentry, aSHM and immune escape.Significance:Genomic analyses reveal that primary and relapse LBCL-IP originate from a common progenitor cell with a small set of genetic alterations, followed by extensive parallel diversification, elucidating the clonal evolution of LBCL-IP.

Somatic copy number alterations can be detected in cell-free DNA (cfDNA) by shallow whole genome sequencing (sWGS). PCR is typically included in library preparations, but a PCR-free method could serve as a high-throughput alternative. To evaluate a PCR-free method for research and diagnostics, archival peripheral blood or bone marrow plasma samples, collected in EDTA- or lithium-heparin–containing tubes, were collected from patients with non–small-cell lung cancer (n = 10 longitudinal samples; 4 patients), B-cell lymphoma (n = 31), and acute myeloid leukemia (n = 15), or from healthy donors (n = 14). sWGS was performed on PCR-free and PCR library preparations, and the mapping quality, percentage of unique reads, genome coverage, fragment lengths, and copy number profiles were compared. The percentage of unique reads was significantly higher for PCR-free method compared with PCR method, independent of the type of collection tube: EDTA PCR-free method, 96.4% (n = 35); EDTA PCR method, 85.1% (n = 32); heparin PCR-free method, 94.5% (n = 25); and heparin PCR method, 89.4% (n = 10). All other evaluated metrics were highly comparable for PCR-free and PCR library preparations. These results demonstrate the feasibility of somatic copy number alteration detection by PCR-free sWGS using cfDNA from plasma collected in EDTA- or lithium-heparin–containing tubes and pave the way for an automated cfDNA analysis workflow for samples from cancer patients.

To understand the clinical significance of the tumor microenvironment (TME), it is essential to study the interactions between malignant and non-malignant cells in clinical specimens. Here, we established a computational framework for a multiplex imaging system to comprehensively characterize spatial contexts of the TME at multiple scales, including close and long-distance spatial interactions between cell type pairs. We applied this framework to a total of 1,393 multiplex imaging data newly generated from 88 primary central nervous system lymphomas with complete follow-up data and identified significant prognostic subgroups mainly shaped by the spatial context. A supervised analysis confirmed a significant contribution of spatial context in predicting patient survival. In particular, we found an opposite prognostic value of macrophage infiltration depending on its proximity to specific cell types. Altogether, we provide a comprehensive framework to analyze spatial cellular interaction that can be broadly applied to other technologies and tumor contexts.

Large-scale chromosomal deletions are a prevalent and defining feature of cancer. A high degree of tumor-type and subtype specific recurrencies suggest a selective oncogenic advantage. However, due to their large size it has been difficult to pinpoint the oncogenic drivers that confer this advantage. Suitable functional genomics approaches to study the oncogenic driving capacity of large-scale deletions are limited. Here, we present an effective technique to engineer large-scale deletions by CRISPR-Cas9 and create isogenic cell line models. We simultaneously induce double-strand breaks (DSBs) at two ends of a chromosomal arm and select the cells that have lost the intermittent region. Using this technique, we induced large-scale deletions on chromosome 11q (65 Mb) and chromosome 6q (53 Mb) in neuroblastoma cell lines. A high frequency of successful deletions (up to 30% of selected clones) and increased colony forming capacity in the 11q deleted lines suggest an oncogenic advantage of these deletions. Such isogenic models enable further research on the role of large-scale deletions in tumor development and growth, and their possible therapeutic potential.

In routine diagnostic pathology, cancer biopsies are preserved by formalin-fixed, paraffin-embedding (FFPE) procedures for examination of (intra-) cellular morphology. Such procedures inadvertently induce DNA fragmentation, which compromises sequencing-based analyses of chromosomal rearrangements. Yet, rearrangements drive many types of hematolymphoid malignancies and solid tumors, and their manifestation is instructive for diagnosis, prognosis, and treatment. Here, we present FFPE-targeted locus capture (FFPE-TLC) for targeted sequencing of proximity-ligation products formed in FFPE tissue blocks, and PLIER, a computational framework that allows automated identification and characterization of rearrangements involving selected, clinically relevant, loci. FFPE-TLC, blindly applied to 149 lymphoma and control FFPE samples, identifies the known and previously uncharacterized rearrangement partners. It outperforms fluorescence in situ hybridization (FISH) in sensitivity and specificity, and shows clear advantages over standard capture-NGS methods, finding rearrangements involving repetitive sequences which they typically miss. FFPE-TLC is therefore a powerful clinical diagnostics tool for accurate targeted rearrangement detection in FFPE specimens., Preservation of cancer biopsies by FFPE introduces DNA fragmentation, hindering analysis of rearrangements. Here the authors introduce FFPE Targeted Locus Capture for identification of translocations in preserved samples.

TO THE EDITOR: In rare instances, malignant lymphomas present as a body cavity–based effusion, without an identifiable tumor mass. Most frequently, this concerns primary effusion lymphoma (PEL), a human herpes virus-8 (HHV8)-positive B-cell lymphoma that typically occurs in HIV-positive patients

Ependymomas (EPN) are neuroepithelial tumors of the central nervous system occurring both in adults and children. DNA methylation profiling has identified ten distinct molecular groups. In the supratentorial (ST) compartment, ZFTA-fusion positive and YAP1-fusion positive ST-EPN represent the two main groups in pediatric patients that are histologically very similar but have a completely different clinical behavior. While most ST-EPN-YAP1 patients survive, only 50% of ZFTA positive patients survive longer than 5 years. Improving survival of ZFTA positive patients requires better understanding of the tumor biology. Despite having different oncogenic fusions, it is unclear whether the cellular origin and tumor biology is related or not and why they respond differently to treatment. The development of human stem cell-derived brain organoids provides a novel opportunity to model these ST-EPN in vitro. These organoids mimic embryonal brain development, during which the cells-of-origin of ST-EPN as well as their micro-environment are present. To induce the development of ST-EPN in vitro, we genetically manipulated cerebral organoids to overexpress the group-specific oncogenic fusion proteins. Electroporating constructs expressing the different fusion proteins into organoids early in brain development (day 11), induced ectopic growth after approximately 30 days. Based on immunohistochemistry, we observed a different phenotype using either the ZFTA or YAP1 fusion constructs, as well as (over)expression of group-specific markers in the organoids. Preliminary transcriptomic analyses suggest that these genetically modified brain organoids represent models with features of human ST-EPN tumors. Ongoing single-cell RNA-sequencing and epigenomic analyses during normal organoid and tumor development will further reveal how well these models represent human primary tumors, how they develop in time, and possibly whether they originate from similar cells or different cells within these organoids. Hence, these models will uniquely contribute to the molecular and biological understanding of ependymomas and can be used to identify new targeted therapies.

Over the last decade, molecular characterization has resulted in many tumors previously classified as central nervous system primitive neuroectodermal tumors (CNS-PNETs) now being classified into their own distinct tumor types. These novel types are often characterized by very specific genomic aberrations. For instance, embryonal tumors with multilayered rosettes (ETMR) harbor amplifications of miRNA cluster C19MC or complex DICER1 mutations, while in CNS neuroblastoma with FOXR2 activation structural aberrations result in aberrant FOXR2 expression. Despite the presence of distinct oncodrivers, our understanding of these tumors is still limited. To elucidate tumor biology and to discover tumor specific treatments, we need to uncover how these oncodrivers contribute to tumorigenesis. However, a bottleneck in basic and translational research of these novel tumor types, is the lack of representative preclinical models, especially in vitro. To overcome this hurdle, we aim to mimic tumor development in genetically modified brain organoids. Human brain organoids derived from pluripotent stem cells are generated to represent either the developing forebrain or cerebellum. To mimic oncodriving events, DNA plasmids are introduced via electroporation into the proposed cell-of-origin populations to knockout tumor suppressor genes or overexpress oncogenes. By detecting fluorescent proteins encoded by the plasmids, electroporated cells are followed over time. Based on our preliminary data, for instance, overexpression of C19MC results in ectopic expansion of the electroporated cells. Ongoing histological and molecular characterizations, including (single cell) transcriptomic and epigenomic analyses, will reveal to which extend these organoid models resemble the specific human tumor types. Although further validation is required, these organoid models provide a novel avenue to study especially brain tumor types with distinct oncodriving events for which patient-derived models have not yet been established. They also allow for in-depth analyses of the potential cells of origin and the contribution of different mutations to tumor biology.

Four main medulloblastoma (MB) molecular subgroups are known, including the sonic hedgehog (SHH) subgroup, which represents ~25% of MB cases. The 5-year overall survival of SHH-MB is ~80%. However, survival between patients is highly diverse and dependent on the driver mutation(s) of the tumor. Patients with TP53 mutated tumors (often accompanied with MYCN and/or GLI2 amplifications) don’t respond well to current therapies and have a 10-year overall survival below 20%. Therefore, there is a need for new and more tailored therapies for these patients. In this study we aim to screen patient-derived organoid models of TP53-mutated SHH MB with a library of ~200 different compounds. We have optimized the cultures of two PDX-derived and one patient-derived organoid line in vitro. The lines will be screened in a high-throughput manner and the best hits and combinations will be validated in corresponding in vivo PDX models. To further assess the role of specific mutations in therapy outcome of TP53-mutated SHH MB, cerebellar organoids generated from human iPSCs were genetically modified with overexpression of dominant-negative P53 (DNP53) alone or in combination with MYCN and/or GLI2. Introduction of DNP53 and MYCN overexpression in cerebellar organoids at day 28/35 leads to the outgrowth of a Ki67-positive proliferating mass after three weeks, indicating tumor growth. Further analyses are ongoing to see how they match SHH-MB patient tumors. These genetically engineered organoid models may elucidate the role of specific mutations in therapy response and/or resistance. In addition, as tumors in these genetically engineered cerebellar organoids arise in a microenvironment of normal cerebellar cell types, initial safety of drugs on cerebellar cells can be assessed. In conclusion, different organoid models of TP53-mutated SHH MB will enable us to find more effective treatments and to better understand how to treat patients with different mutation combinations.

KWF Kankerbestrijding, Grant/AwardNumber: KWF-5510; Cancer Center Amsterdam Foundation, Grant/AwardNumber: CCA-2013; The Hodgkin Lymphoma MRD Foundation; Technology Foundation STW, Grant/AwardNumber: CANCER-ID Project, Minimally-invasive tools to assess tumour presence and burden may improve clinical management. FDG-PET (metabolic) imaging is the current gold standard for interim response assessment in patients with classical Hodgkin Lymphoma (cHL), but this technique cannot be repeated frequently. Here we show that microRNAs (miRNA) associated with tumour-secreted extracellular vesicles (EVs) in the circulation of cHL patients may improve response assessment. Small RNA sequencing and qRT-PCR reveal that the relative abundance of cHL-expressed miRNAs, miR-127-3p, miR-155- 5p, miR-21-5p, miR-24-3p and let-7a-5p is up to hundred-fold increased in plasma EVs of cHL patients pre-treatmentwhen compared to completemetabolic responders (CMR). Notably, in partial responders (PR) or treatment-refractory cases (n = 10) the EV-miRNA levels remain elevated. In comparison, tumour specific copy number variations (CNV) were detected in cell-free DNA of 8 out of 10 newly diagnosed cHL patients but not in patients with PR. Combining EV-miR-127-3p and/or EV-let- 7a-5p levels, with serum TARC (a validated protein cHL biomarker), increases the accuracy for predicting PET-status (n = 129) to an area under the curve of 0.93 (CI: 0.87-0.99), 93.5% sensitivity, 83.8/85.0% specificity and a negative predictive value of 96%. Thus the level of tumour-associated miRNAs in plasma EVs is predictive of metabolic tumour activity in cHL patients. Our findings suggest that plasma EV-miRNA are useful for detection of small residual lesions and may be applied as serial response prediction tool., KWF Kankerbestrijding KWF-5510, Cancer Center Amsterdam Foundation CCA-2013, Hodgkin Lymphoma MRD Foundation, Technologiestichting STW

Breast implant–associated anaplastic large cell lymphoma (BIA-ALCL) is a very rare type of T-cell lymphoma that is uniquely caused by a single environmental stimulus. Here, we present a comprehensive genetic analysis of a relatively large series of BIA-ALCL (n = 29), for which genome-wide chromosomal copy number aberrations (CNAs) and mutational profiles for a subset (n = 7) were determined. For comparison, CNAs for anaplastic lymphoma kinase (ALK)− nodal anaplastic large cell lymphomas (ALCLs; n = 24) were obtained. CNAs were detected in 94% of BIA-ALCLs, with losses at chromosome 20q13.13 in 66% of the samples. Loss of 20q13.13 is characteristic of BIA-ALCL compared with other classes of ALCL, such as primary cutaneous ALCL and systemic type ALK+ and ALK− ALCL. Mutational patterns confirm that the interleukin-6–JAK1–STAT3 pathway is deregulated. Although this is commonly observed across various types of T-cell lymphomas, the extent of deregulation is significantly higher in BIA-ALCL, as indicated by phosphorylated STAT3 immunohistochemistry. The characteristic loss of chromosome 20 in BIA-ALCL provides further justification to recognize BIA-ALCL as a separate disease entity. Moreover, CNA analysis may serve as a parameter for future diagnostic assays for women with breast implants to distinguish seroma caused by BIA-ALCL from other causes of seroma accumulation, such as infection or trauma.

Background & Aims Drugs that inhibit the erb-b2 receptor tyrosine kinase 2 (ERBB2 or HER2) are the standard treatment of patients with different types of cancer, including HER2-overexpressing gastroesophageal tumors. Unfortunately, cancer cells become resistant to these drugs, so overall these drugs provide little benefit to patients with these tumors. We investigated mechanisms that mediate resistance of esophageal adenocarcinoma (EAC) cells and patient-derived xenograft tumors to ERBB inhibitors. Methods We cultured primary tumor cells, isolated from EAC patient samples, and OE19 and OE33 EAC cell lines with trastuzumab (an inhibitor of HER2), with or without pertuzumab (which inhibits dimerization of HER2 with HER3) or a specific antibody against HER3 (anti-HER3). HER2 was knocked down by expression of small hairpin RNAs. In addition, cells were incubated with NRG1-β, a mediator of HER2–HER3 signaling, or A83-01, an inhibitor of transforming growth factor beta (TGFβ) signaling. Cells were analyzed for markers of the epithelial to mesenchymal transition (EMT) using flow cytometry, immunofluorescence, and quantitative reverse transcription polymerase chain reaction. We performed limiting dilution, transwell, and cell viability assays to study the functional effects of HER2 and HER3 inhibition and reactivation. We analyzed publicly available EAC gene expression datasets to correlate expression of ERBB genes with genes encoding epithelial and mesenchymal proteins. NOD.Cg- Prkdc scid Il2rg tm1Wjl /SzJ (NSG) mice were given subcutaneous injections of AMC-EAC-007B cells and also given injections of single or combined inhibitors; growth of these patient-derived xenograft tumors was quantified. Results EAC cells incubated with trastuzumab decreased expression of epithelial markers (CD24, CD29, and CDH1) and increased expression of mesenchymal markers (CXCR4, VIM, ZEB1, SNAI2, and CDH2), compared with cells not exposed to trastuzumab, indicating induction of EMT. Addition of NRG1-β to these cells restored their epithelial phenotype. Incubation of EAC cells with trastuzumab and pertuzumab accelerated the expression of EMT markers, compared with cells incubated with trastuzumab alone. EAC cells cultured for 2 months with a combination of trastuzumab and pertuzumab became resistant to chemotherapeutic agents (5-fluoruracil, carboplatin, cisplatin, eribulin, and paclitaxel), based on their continued viability, which was accompanied with an enhanced migratory capacity in transwell assays and clonogenicity in limiting dilution analyses. In comparisons of EAC gene expression patterns, we associated high expression of ERBB3 with an epithelial gene expression signature; expression of TGFβ correlated with expression of EMT-related genes, and we found an inverse correlation between expression of TGFB1 and ERBB3. EAC cells incubated with ERBB inhibitors began to secrete ligands for the TGFβ receptor and underwent EMT. Incubation of EAC cells with trastuzumab, followed by 10 days of incubation with the TGFβ receptor inhibitor in the presence of trastuzumab, caused cells to regain an epithelial phenotype. EAC patient-derived xenograft tumors grew more slowly in mice given the combination of trastuzumab, pertuzumab, and the TGFβ inhibitor than in mice given single agents or a combination of trastuzumab and pertuzumab. Tumors exposed to trastuzumab and pertuzumab expressed EMT markers and were poorly differentiated, whereas tumors exposed to the combination of trastuzumab, pertuzumab, and the TGFβ inhibitor expressed epithelial markers and were more differentiated. Conclusions EAC cells become resistant to trastuzumab and pertuzumab by activating TGFβ signaling, which induces EMT. Agents that block TGFβ signaling can increase the anti-tumor efficacies of trastuzumab and pertuzumab.