An Integrated Epigenomic and Genomic View on Phyllodes and Phyllodes-Like Breast Tumors

Fibroepithelial lesions (FL) of the breast, in particular Phyllodes tumors (PT) and fibroadenomas, pose a significant diagnostic challenge. There are no generally accepted criteria that distinguish benign, borderline, malignant PT, and FA. Combined genome-wide DNA methylation and copy number variant (CNV) profiling is an emerging strategy to classify tumors. We compiled a series of patient-derived archival biopsy specimens reflecting the FL spectrum and histological mimickers including clinical follow-up data. DNA methylation and CNVs were determined by well-established microarrays. Comparison of the patterns with a pan-cancer dataset assembled from public resources including "The Cancer Genome Atlas" (TCGA) and "Gene Expression Omnibus" (GEO) suggests that FLs form a methylation class distinct from both control breast tissue as well as common breast cancers. Complex CNVs were enriched in clinically aggressive FLs. Subsequent fluorescence in situ hybridization (FISH) analysis detected respective aberrations in the neoplastic mesenchymal component of FLs only, confirming that the epithelial component is non-neoplastic. Of note, our approach could lead to the elimination of the diagnostically problematic category of borderline PT and allow for optimized prognostic patient stratification. Furthermore, the identified recurrent genomic aberrations such as 1q gains (including MDM4), CDKN2a/b deletions and EGFR amplifications may inform therapeutic decision-making.

RB1 loss triggers dependence on ESRRG in retinoblastoma

Retinoblastoma (Rb) is a deadly childhood eye cancer that is classically initiated by inactivation of the RB1 tumor suppressor. Clinical management continues to rely on nonspecific chemotherapeutic agents that are associated with treatment resistance and toxicity. Here, we analyzed 103 whole exomes, 16 whole transcriptomes, 5 single-cell transcriptomes, and 4 whole genomes from primary Rb tumors to identify novel Rb dependencies. Several recurrent genomic aberrations implicate estrogen-related receptor gamma (ESRRG) in Rb pathogenesis. RB1 directly interacts with and inhibits ESRRG, and RB1 loss uncouples ESRRG from negative regulation. ESRRG regulates genes involved in retinogenesis and oxygen metabolism in Rb cells. ESRRG is preferentially expressed in hypoxic Rb cells in vivo. Depletion or inhibition of ESRRG causes marked Rb cell death which is exacerbated in hypoxia. These findings reveal a novel dependency of Rb cells on ESRRG, and they implicate ESRRG as a potential therapeutic vulnerability in Rb.

Nanopore sequencing for the screening of myeloid and lymphoid neoplasms with eosinophilia and rearrangement of PDGFRα, PDGFRβ, FGFR1 or PCM1-JAK2

Eosinophilia represents a group of diseases with heterogeneous pathobiology and clinical phenotypes. Among the alterations found in primary Eosinophilia, gene fusions involving PDGFRα, PDGFRβ, FGFR1 or JAK2 represent the biomarkers of WHO-defined “myeloid and lymphoid neoplasms with eosinophilia”. The heterogeneous nature of genomic aberrations and the promiscuity of fusion partners, may limit the diagnostic accuracy of current cytogenetics approaches. To address such technical challenges, we exploited a nanopore-based sequencing assay to screen patients with primary Eosinophilia. The comprehensive sequencing approach described here enables the identification of genomic fusion in 60 h, starting from DNA purified from whole blood.

Understanding Monoclonal B Cell Lymphocytosis: An Interplay of Genetic and Microenvironmental Factors

The term monoclonal B-cell lymphocytosis (MBL) describes the presence of a clonal B cell population with a count of less than 5 × 109/L and no symptoms or signs of disease. Based on the B cell count, MBL is further classified into 2 distinct subtypes: ‘low-count’ and ‘high-count’ MBL. High-count MBL shares a series of biological and clinical features with chronic lymphocytic leukemia (CLL), at least of the indolent type, and evolves to CLL requiring treatment at a rate of 1-2% per year, whereas ‘low-count’ MBL seems to be distinct, likely representing an immunological rather than a pre-malignant condition. That notwithstanding, both subtypes of MBL can carry ‘CLL-specific’ genomic aberrations such as cytogenetic abnormalities and gene mutations, yet to a much lesser extent compared to CLL. These findings suggest that such aberrations are mostly relevant for disease progression rather than disease onset, indirectly pointing to microenvironmental drive as a key contributor to the emergence of MBL. Understanding microenvironmental interactions is therefore anticipated to elucidate MBL ontogeny and, most importantly, the relationship between MBL and CLL.

Specific T Cell Receptor Gene Repertoire Profiles in Subgroups of CLL Patients with Distinct Genomic Aberrations

Chronic lymphocytic leukemia (CLL) B cells engage in multifaceted bi-directional interactions with bystander cells, including T cells. Immunogenetic studies in CLL revealed clonal expansions of T cells and shared T cell clonotypes between different patients, strongly implying clonal selection by antigens. Although the exact nature of these antigens remains largely elusive, evidence exists that the clonotypic B cell receptor immunoglobulin (BcR IG) may serve as a source of antigenic epitopes for T cells. That said, recurrent genomic aberrations associated with distinct abnormal expression profiles could represent an alternative, non mutually exclusive, source of potent immunogenic onco-antigens that might shape the T cell repertoire in CLL. On these grounds, here we interrogated the T cell receptor (TR) gene repertoire of CLL patients with different genomic aberration profiles with the aim to identify unique signatures that would allude to distinct antigen selection pressures. The study group included 46 patients with CLL, sampled before treatment initiation, who were categorized in 5 subgroups defined by a unique genomic aberration, as follows: +12, n=18; del(11q), n=10; del(13q), n=7; del(17p)/TP53mut, n=6; NOTCH1mut, n=5. Confounding effects of multiple aberrations have been minimized, as we previously established through comprehensive characterization (including FISH, SNP arrays and gene panels) that the analyzed patients carried only one of the above aberrations. Starting material was RNA extracted from blood mononuclear cells. TRBV-TRBD-TRBJ gene rearrangements were RT-PCR amplified and subjected to paired-end next generation sequencing (NGS). Raw NGS reads (n=13,213,563| median: 294,757/sample) were processed through a purpose-built bioinformatics pipeline. Only productive rearrangements (n=9,249,546 | median=199,184/sample) were taken into consideration for the computation of clonotypes i.e. TRB rearrangements with identical TRBV gene usage and amino acid complementarity-determining region 3 (CDR3) sequence. Overall, 513,984 distinct clonotypes (median=10,304 clonotypes/sample) were assessed. The main measure of clonality employed in this study was the median cumulative frequency of the 10 most expanded T cell clonotypes/sample (MCF-10). For comparisons of the clonality profiles, a group of 17 aged-matched healthy individuals were used as controls. All patients displayed oligoclonal T cell expansions with the following MCF-10 values: del(11q): 21.6%, +12: 25%, del(13q): 20.6%, NOTCH1mut: 9.1%, del(17p)/TP53mut: 12.9%; the difference between the del(11q) and +12 groups versus the NOTCH1mut group was statistically significant (p<0.05). The MCF-10 value of the control group was estimated at 17.5%, supporting the notion of age-related decrease in TR repertoire diversity. However, the del(11q), +12 and del(13q) CLL groups displayed elevated clonality, reaching statistical significance (p<0.002) in the case of +12. TRBV gene repertoire analysis revealed that the TRBV12-3 gene predominated in most groups, except for the del(17p)/TP53mut, where the predominant gene was TRBV10-3. Clonotype comparisons disclosed the presence of shared TR clonotypes both within a particular group but also between groups. Overall, 446/513,984 clonotypes were found to be shared by at least two patients across all groups; the vast majority (392/446, 88%) of shared clonotypes appeared to be CLL-biased since they did not match entries in public databases of TR clonotypes from various contexts. Subgroup-specific clonotypes were identified for all aberrations examined; these emerged as unique to the particular subgroups, as revealed by extensive comparisons against both public databases but also a large TR clonotype database from CLL available to us from our previous studies. In conclusion, recurrent genomic aberrations, especially large chromosomal abnormalities, display an oligoclonal TR gene repertoire. The distinct immunogenetic profile of each group examined here and, most importantly, the existence of subgroup-specific clonotypes, suggest that abnormal protein expression and gene dosage effects likely represent a relevant source of CLL-specific selecting antigens. Disclosures Scarfo: Janssen: Honoraria, Other: Travel grants; Astra Zeneca: Honoraria; Abbvie: Honoraria. Anagnostopoulos: Abbvie: Other: clinical trials; Sanofi: Other: clinical trials ; Ocopeptides: Other: clinical trials ; GSK: Other: clinical trials; Incyte: Other: clinical trials ; Takeda: Other: clinical trials ; Amgen: Other: clinical trials ; Janssen: Other: clinical trials; novartis: Other: clinical trials; Celgene: Other: clinical trials; Roche: Other: clinical trials; Astellas: Other: clinical trials . Ghia: AbbVie: Consultancy, Honoraria, Research Funding; Acerta/AstraZeneca: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria, Research Funding; ArQule/MSD: Consultancy, Honoraria; BeiGene: Consultancy, Honoraria; Celgene/Juno/BMS: Consultancy, Honoraria; Gilead: Consultancy, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria; Sunesis: Research Funding. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Rosenquist: Roche: Honoraria; Janssen: Honoraria; Illumina: Honoraria; AstraZeneca: Honoraria; Abbvie: Honoraria. Stamatopoulos: Gilead: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Baliakas: Janssen: Honoraria; Gilead: Honoraria, Research Funding; Abbvie: Honoraria. Chatzidimitriou: Abbvie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding.

PATH-18. INTEGRATING TUMOR MICROENVIRONMENT WITH GENOMIC ABERRATIONS AND TUMOR CLASS IN ADULT GLIAL/GLIONEURONAL TUMORS BY DECONVOLUTION OF BULK METHYLATION DATA

It is increasingly recognized that the tumor microenvironment (TME) plays a critical role in the biology of cancer. To better understand the role of non-neoplastic immune cellular components in CNS tumors, we applied a deconvolution approach to bulk DNA methylation array data using methylCIBERSORT on 450k/850k methylation data from a set (n= 4057) of high- and low-grade glial and glioneuronal tumors. Using the cell type proportion data as input, we used dimension reduction (UMAP) to visualize sample-wise patterns of that emerge from the cell type proportion estimations. In glioblastomas (n= 2076) we identified distinct tumor clusters based on immune cell proportion and, interestingly, TME-based cluster groups demonstrated an association with specific genetic alterations such as EGFR amplification and/or CDKN2A/B homozygous deletion. Among 1178 IDH-mutant gliomas, clustering of tumors according to immune cell proportions led to 2 major subgroups, which largely aligned with 1p/19q co-deletion status. Among the non-codeleted tumors (IDH-mutant astrocytomas, N=734), clustering of immune cell decomposition revealed clusters which showed distinct proportions of a key genomic aberration in these tumors (CDKN2A/B loss). To investigate the possible role of monocyte proportion-relative gene expression and promoter methylation of the immune checkpoint PD-L1 and PD-L2 genes, we used a data subset (n=594) samples with matched gene expression profiles. We observed significantly high positive correlations (R=0.54 and 0.68, respectively) between monocyte proportion and expression of PD-L1 and PD-L2, in line with prior reports that monocytic cells can express these immune markers. Consistent with this, we found high negative correlations (R= -0.51 and -0.61, respectively) between monocytes and promoter methylation of PD-L1 and PD-L2, respectively. Overall, the findings highlight specific roles of the TME in biology and classification of adult CNS tumors, where specific immune cell admixtures correlate with tumor types and genomic aberrations.

PATH-20. SPATIAL MAPPING OF THERAPY-INDUCED, PATHOLOGICAL CHANGES IN GLIOBLASTOMA AT SINGLE-CELL RESOLUTION

Glioblastoma (GBM) remains a highly malignant, intrinsically resistant and inevitably recurring brain tumor with dismal prognosis. The aggressiveness and lack of effective GBM treatments can be attributed to the highly heterogeneous and plastic nature of GBM tumor cells, which easily confer resistance to standard-of-care (SOC) therapy. While tumor progression has also been attributed to interactions with the tumor microenvironment, quantitative data describing these interactions are still largely missing. Here, we used high-dimensional, multiplexed immunohistochemistry to map evolutions in the spatial, single-cell tissue architecture of 120 paired adult GBM tumor samples derived from 60 patients at diagnosis (ND) and upon recurrence (REC) following SOC treatment. We mapped the spatial distribution of a multitude of GBM tumoral subtypes across this multicentric cohort, through which we identified a high level of heterogeneity defined by specific tumoral niches within and across patients and which evolved when subjected to SOC therapy. In addition, we describe the relationship of the various tumoral niches with their local immune-infiltrates, highlighting an even more immunosuppressive environment following SOC resistance. Finally, by aligning these findings to the observed genomic aberrations and the clinical data of the patients, we are now able to more precisely describe the heterogeneous landscape of glioblastoma and how it evolves under SOC treatment at spatial, single-cell resolution.

High Expression of PPM1D Induces Tumors Phenotypically Similar to TP53 Loss-of-Function Mutations in Mice

PPM1D is a negative regulator of p53 and genomic aberrations resulting in increased activity of PPM1D have been observed in cancers of different origins, indicating that PPM1D has oncogenic properties. We established a transgenic mouse model overexpressing PPM1D and showed that these mice developed a wide variety of cancers. PPM1D-expressing mice developed tumors phenotypically and genetically similar to tumors in mice with dysfunctional p53. T-cell lymphoblastic lymphoma was the most frequent cancer observed in these mice (55%) followed by adenocarcinomas (24%), leukemia (12%) and other solid tumors including neuroblastoma. Characterization of T-cell lymphomas in mice overexpressing PPM1D demonstrates Pten-deletion and p53-accumulation similar to mice with p53 loss-of-function. Also, Notch1 mutations which are recurrently observed in T-cell acute lymphoblastic lymphoma (T-ALL) were frequently detected in PPM1D-transgenic mice. Hence, PPM1D acts as an oncogenic driver in connection with cellular stress, suggesting that the PPM1D gene status and expression levels should be investigated in TP53 wild-type tumors.

The Modes of Dysregulation of the Proto-Oncogene T-Cell Leukemia/Lymphoma 1A

Incomplete biological concepts in lymphoid neoplasms still dictate to a large extent the limited availability of efficient targeted treatments, which entertains the mostly unsatisfactory clinical outcomes. Aberrant expression of the embryonal and lymphatic TCL1 family of oncogenes, i.e., the paradigmatic TCL1A, but also TML1 or MTCP1, is causally implicated in T- and B-lymphocyte transformation. TCL1A also carries prognostic information in these particular T-cell and B-cell tumors. More recently, the TCL1A oncogene has been observed also in epithelial tumors as part of oncofetal stemness signatures. Although the concepts on the modes of TCL1A dysregulation in lymphatic neoplasms and solid tumors are still incomplete, there are recent advances in defining the mechanisms of its (de)regulation. This review presents a comprehensive overview of TCL1A expression in tumors and the current understanding of its (dys)regulation via genomic aberrations, epigenetic modifications, or deregulation of TCL1A-targeting micro RNAs. We also summarize triggers that act through such transcriptional and translational regulation, i.e., altered signals by the tumor microenvironment. A refined mechanistic understanding of these modes of dysregulations together with improved concepts of TCL1A-associated malignant transformation can benefit future approaches to specifically interfere in TCL1A-initiated or -driven tumorigenesis.