Lopes, M. Beatriz S.

Abstract Diffuse hemispheric glioma, H3 G34‐mutant (DHG‐H3 G34) has been primarily molecularly characterized by methylation profiling and sequencing studies. We describe an integrated histomolecular evaluation including high‐resolution copy number profiling of a series of 60 DHG‐H3 G34 to further our understanding of the spectrum of genetic changes associated with this tumor type. Cases were clinically tested using an 187‐gene mutation and fusion targeted neuro‐oncology next‐generation sequencing panel ( n = 60) and Oncoscan chromosomal microarray ( n = 26) by a single laboratory (2018–2022). A subset of cases had immunohistochemical results for OLIG2 ( n = 42), p53 ( n = 48), and ATRX ( n = 46), and methylation array data ( n = 8). Median age at testing was 21 years (range, 12–50). No significant difference was noted in clinical, histopathological, and mutational profile between pediatric and adult patients. H3‐3A G34 mutations included G34R ( n = 56; 94%), G34V ( n = 3), and a non‐canonical G34E ( n = 1). Concurrent mutations most often involved TP53 ( n = 55; 92%), ATRX ( n = 50; 83%), and PDGFRA ( n = 34; 57%). A reportedly primary tumor was confirmed to be hypermutant and had a PMS2 mutation. A single case also showed an FGFR3::FAM184B fusion. All cases with available chromosomal microarray data had unbalanced genomes, which were often complex (14/26; 54%). The most frequent recurrent copy number abnormalities were losses involving 3q, 4q, 10q, 13q, and 18q, and 17p copy‐neutral loss of heterozygosity (cnLOH) encompassing TP53 . This copy number profile was reminiscent of that seen in Grade 4 IDH‐mutant astrocytomas. Collectively, a TP53 abnormality at copy number (12/26, all cnLOH), sequence (55/60) and protein expression (46/48) level was detected in all 60 cases. In conclusion, integrated high‐resolution copy number and histomolecular analysis expanded the spectrum of genetic changes associated with DHG‐H3 G34, including the presence of universal TP53 abnormalities with frequent cnLOH—a copy number abnormality that has been largely unrecognized—for this new 2021 World Health Organization central nervous system tumor type.

Abstract Background Methylation class pleomorphic xanthoastrocytoma (mcPXA) comprises tumors with the DNA methylation signature of classical PXA but with a wider histologic spectrum, including overlap with glioblastoma (GBM). Methods To clarify the histologic and molecular scope of mcPXA and characterize its clinical behavior, a cohort of 469 tumor samples from 458 patients matching to mcPXA by the DKFZ classifier (v12.6 score ≥0.85) was interrogated. Results Patient median age was 23 years (range 1-73 years) with a female predominance (259 female/199 male). CDKN2A/B homozygous deletion was observed in 406 of 469 (87%) samples. In samples tested for BRAF p.V600E mutations (n = 279), 240 (86%) harbored the mutation. A chr7+/chr10− pattern was observed in 103 of 469 (22%) samples. Among samples tested for TERT promoter mutations (n = 143), 32 (22%) harbored the mutation. Progression-free and overall survival of patients with mcPXA were comparable to patients with methylation class IDH-mutant astrocytoma, low grade, but a GBM-like subset (ie, cases with a pre-methylation working diagnosis of GBM) showed shorter survival. Histologic features of high grade, including palisading necrosis and microvascular proliferation, were prognostic in mcPXA. Compared to patients with BRAF p.V600E-altered GBM, patients with mcPXA were younger and had a lower frequency of TERT promoter mutations. Conclusion Tumors in mcPXA share molecular characteristics with histologically defined PXA, and high-grade histologic features can help predict their clinical behavior. The use of an epigenetic classification of PXA reveals that this group of tumors is more common than previously appreciated and warrants in-depth study to identify efficacious therapeutic options.

Abstract Abstract 995 PCNSL is an aggressive primary brain tumor characterized by a perivascular accumulation of malignant lymphoid cells. Most PCNSLs (90%) are diffuse large B-cell lymphoma (DLBCL); the remaining 10% are poorly characterized low-grade, Burkitt, and T-cell lymphomas. Since most patients are biopsed, genomic analyses are challenging. To determine the pattern of genetic alterations in PCNSL, frozen samples and formalin fixed embedded paraffin sections from 17 EBV and HIV negative and immunocompetent patients were studied by array-based comparative genomic hybridization (aCGH) using Sureprint G3 (1 million probes) array (Agilent). B-cell differentiation status was characterized by immunostains for CD10, MUM-1, and BCL-6. All cases were characterized by complex genomic aberrations with a median of 21 copy-number abnormalities (CNA) per patient (range 10–49). Overall, 22 minimal deleted regions (MDR) and 14 minimal amplified regions (MAR) were found in more than 20% of patients. Focal deletion affecting CDKN2A (9p21) was the most common CNA, found in 14 of 17 cases (82%); biallelic in six cases. Losses of 6q were observed in 71% of cases. Deletions of 6q23.3 (TNFAIP3) and 6q21 (PRDM1) were found in 59% (10/17) and 47% (8/17) of cases, respectively. Other common CNA were deletions of 6p21 (9/17; 53%), 3p21.1 (5/17; 29%), 3q26.32 (5/17; 29%), 8q12.1 (5/17; 29%), 10p14-p15.3 (5/17; 29%), 12q24.31 (5/17; 29%) and gains of 12q21-q24 (9/17; 53%), 7q21-q31 (6/17; 35%), 19q13 (6/17; 35%), 3q27.3 (5/17; 29%) and 11q24.1-q25 (5/17; 29%). Interestingly, several CNA were unique to PCNSL and were not identified in related entities as the typical DLBCL. Besides in CDKN2A, homozygous deletions were recurrently found in TMEM30A and TOX, the latter a regulator of T-cell development. Another 64 genes, including B2M, CD58, ETV6, LAPTM, MHC class II genes, PRDM1, TNFRSF10A and TNFRSF10B were also homozygously deleted. CD58, which encodes for a member of the immunoglobulin family and regulates the adhesion and activation of T lymphocytes, was also recurrently affected by focal monoallelic losses from 15 nucleotides to 1–2 exons, affecting the Ig-like C2-type domain as was confirmed by DNA resequencing. Focal heterozygous deletions affect TBL1XR1, a negative regulator of the NF-kB and Wnt pathways, and the putative tumor suppressor BCL7A in 29% of cases each. Pathway analysis done including the most commonly affected genes (Ingenuity Pathway Analysis) highlights the importance of networks associated with apoptosis and lymphocyte differentiation and proliferation, especially of T lymphocytes. In summary, this study showed evidence for a highly complex genome and identified target genes of potential relevance in the pathogenesis of PCNSL. The genomic profile described here is unique to PCNSL, thus helping to genetically differentiate this entity from the typical DLBCL and other related lymphomas. Disclosures: Fonseca: Genzyme: Consultancy; Medtronic: Consultancy; BMS: Consultancy; AMGEN: Consultancy; Otsuka: Consultancy; Celgene: Consultancy, Research Funding; Intellikine: Consultancy; Cylene: Research Funding; Onyx: Research Funding; FISH probes prognostication in myeloma: Patents & Royalties.

Abstract Primary central nervous system lymphoma (PCNSL) is an aggressive and incurable variant of non-Hodgkin lymphoma (NHL) that is confined to the central nervous system. Most PCNSL (90%) are part of the immune-privileged site-associated diffuse large B-cell lymphomas (DLBCL). Unlike nodal DLBCL, only a limited number of genetic studies have been performed in PCNSL, partly due to lack of available tissue specimens. Because of the fragmented knowledge of the genomic basis, it is still a matter of debate whether they differ from systemic DLBCL with respect to their molecular features and pathogenesis and also if there is a CNS specific signature. We performed a comprehensive genomic study in a cohort of 22 immunocompetent (HIV- and EBV-) PCNSL. DNA was extracted from FFPE tissues (N=15) and frozen tissue (N=7). Samples were analyzed using a combination of aCGH (N=18), exome sequencing (N=10), mate-pair genome sequencing (N=2) and targeted sequencing (N=8). We found a complex karyotype with a median of 21 copy-number abnormalities (range 10–47), 6 structural abnormalities, 6 frameshift indels and 67 nonsynonymous mutations. By integrating mutation and copy number data we found a group of genes recurrently mutated and/or deleted in PNCSL. Remarkable findings were the high prevalence of MYD88 activating mutations (L265P/M232T/V217F), found in 69% of cases and the biallelic loss of CDKN2A (60%). A subset of recurrent abnormalities was exclusively found in PCNSL, and not being previously identified in systemic DLBCL. Thus, 11% of PCNSL have biallelic inactivation of TOX (a regulator of T-cell development) and PRKCD (protein kinase C delta), while another 17% of cases show focal monoallelic deletions/mutations in these genes. Finally, recurrent mutations have been identified in ATM, which have not been found in nodal DLBCL. Several other genes affected have been previously identified in nodal DLBCL, such as biallelic loss of TNFAIP3 (16%), PRDM1 (16%), GNA13, TMEM30A, B2M and CD58 (11% each), activating mutations of CD79B (28%) and CARD11 (19%) and translocations of BCL6 (22%). Components of the NF-kB pathways were altered in >90% of PNCSL. Pathway analysis also showed an enrichment of networks associated with immune response, proliferation, regulation of apoptosis and lymphocyte differentiation and activation. Finally, we searched for associations between genetic alterations and clinical outcome. We showed that deletions of 6q21 (PRDM1) and 6q23 (TNFAIP3) were both associated with shorter overall survival (p=0.007 and p=0.03, respectively). In summary, we report a genomic background in PCNSL similar to post-GC DLBCL but reinforcing the existence of a subset of abnormalities specific to PCNSL, suggesting their potential relevance in the disease pathogenesis. Additionally, the results obtained from FFPE samples are encouraging and larger archival tissue collections can now be analyzed in order to complement the still fragmented knowledge we have of the genetic basis of the disease. Disclosures: Stewart: Onyx: Consultancy, Research Funding; Millenium: Honoraria, Research Funding; Celgene: Honoraria; BMS: Honoraria. Fonseca:Medtronic: Consultancy; Otsuka: Consultancy; Celgene: Consultancy; Genzyme: Consultancy; BMS: Consultancy; Lilly: Consultancy; Onyx: Consultancy, Research Funding; Binding Site: Consultancy; Millennium: Consultancy; AMGEN: Consultancy; Cylene: Research Funding; Prognostication of MM based on genetic categorization of the disease: Patents & Royalties.

Abstract Abstract 420 Primary central nervous system lymphoma (PCNSL) is a rare and aggressive non-Hodgkin lymphoma that is confined to the CNS because of a poorly understood neurotropism. Most of PCNSL (90%) are part of the immune-privileged site-associated DLBCL (IPDLBCL). IPDLBCL consist in late–germinal center or post–germinal center lymphoid cells but that show very distinct characteristics that separate them from systemic DLBCL. It is still a matter of debate whether the PCNSL differ from nodal DLBCL with respect to molecular features and pathogenesis and also if there is a genomic signature specific of PCNSL. Only few genetic studies have been performed in PCNSL, partly due to the rarity of the tumors and the limited amount of available tissue. To gain insight into the genomic basis of PCNSL, we performed an integrated, high-throughput, genomic analysis in 17 immunocompetent, EBV- and HIV- cases. B-cell differentiation status was characterized by immunostains for CD10, MUM-1, and BCL-6. Either frozen samples or formalin fixed embedded paraffin sections from 17 PCNSL were studied by array-based comparative genomic hybridization (aCGH) using Sureprint G3 (1 million probes) array. Massively parallel whole-exome sequencing was performed in 4 of these cases. Additionally, 2 cases were analyzed by mate-pair whole genome sequencing searching for chromosomal breakpoints. Sanger DNA sequencing was used for validation. All cases were characterized by complex genomic aberrations with a median of 21 copy-number abnormalities (CNA, range 10–49), 4 structural abnormalities, 6 frameshift indels and 99 nonsynonymous exonic mutations. Focal deletion affecting CDKN2A (9p21) was the most common CNA, found in 14 of 17 cases (82%); with 6 of these cases (35%) having homozygous deletion. The second most frequent CNA involved the HLA genes (6p21), found in 11 of 17 (65%) cases; 4 of them (23%) with homozygous deletions. We identified recurrent CNA and mutations in several genes previously found in systemic DLBCL. Thus, PRDM1 (BLIMP1) was deleted or mutated in 47% of cases and the translocation IgH-BCL6 was found in 30% of PCNSL. Furthermore, recurrent mutations were found in NF-kB genes CD79B (75%, 3 of 4 cases analyzed), MYD88 (70%, 7 of 10), TNFAIP3 (50%, 2 of 4) and CARD11 (50%, 2 of 4). Additionally, recurrent abnormalities were found in B2M, BCL7A, CD58, CIITA, ETV6, GNA13, PAX5, TMEM30A and TP53. Nevertheless, we identified several recurrent genetic alterations not described in systemic DLBCL. TOX (a regulator of T-cell development) and TBL1XR1 (a negative regulator of the NF-kB and Wnt pathways) were either deleted or mutated in 30% of PNCSL, something not previously described in systemic DLBCL. Additionally, chromosomal breakpoints either in DLGAP1 or DLGAP2 (play a role in neuronal cell signaling) were found in 18% of PCNSL but not in systemic DLBCL. Moreover, mutations in ATM (master controller of cell cycle checkpoint), BAI3 (inhibitor of brain angiogenesis), BTG2 (cell cycle arrest), KDM6B (histone demethylase), PKC family members PRKCD/PRKCDE, genes from the histone cluster, the protocadherin family and the WD repeat domain were found in 10% to 50% of PCNSL. Pathway analysis including the most commonly affected genes in PCNSL showed an enrichment of networks associated with immune response, NF-kB pathway, proliferation, regulation of the apoptosis and lymphocyte differentiation and activation. In summary, we show evidence of a highly complex genome and identified a subset of genes with potential relevance in PCNSL pathogenesis. The genomic profile described here reinforces the existence of a specific molecular signature in PCNSL, thus helping to genetically differentiate this entity from the nodal DLBCL and related lymphomas. Disclosures: Stewart: Millenium: Consultancy, Honoraria, Research Funding; Onyx: Consultancy; Celgene: Consultancy. Fonseca:Medtronic: Consultancy; Otsuka: Consultancy; Celgene: Consultancy; Genzyme: Consultancy; BMS: Consultancy; Lilly: Consultancy; Onyx: Consultancy; Binding site: Consultancy; Millenium: Consultancy; AMGEN: Consultancy; Celgene : Research Funding; Onyx: Research Funding; prognostication of MM based on genetic categorization of the disease: Prognostication of MM based on genetic categorization of the disease Patents & Royalties.

Abstract Purpose: Primary central nervous system lymphoma (PCNSL) is an aggressive non-Hodgkin lymphoma confined to the central nervous system. Whether there is a PCNSL-specific genomic signature and, if so, how it differs from systemic diffuse large B-cell lymphoma (DLBCL) is uncertain. Experimental Design: We performed a comprehensive genomic study of tumor samples from 19 immunocompetent PCNSL patients. Testing comprised array-comparative genomic hybridization and whole exome sequencing. Results: Biallelic inactivation of TOX and PRKCD was recurrently found in PCNSL but not in systemic DLBCL, suggesting a specific role in PCNSL pathogenesis. In addition, we found a high prevalence of MYD88 mutations (79%) and CDKN2A biallelic loss (60%). Several genes recurrently affected in PCNSL were common with systemic DLBCL, including loss of TNFAIP3, PRDM1, GNA13, TMEM30A, TBL1XR1, B2M, CD58, activating mutations of CD79B, CARD11, and translocations IgH-BCL6. Overall, B-cell receptor/Toll-like receptor/NF-κB pathways were altered in >90% of PNCSL, highlighting its value for targeted therapeutic approaches. Furthermore, integrated analysis showed enrichment of pathways associated with immune response, proliferation, apoptosis, and lymphocyte differentiation. Conclusions: In summary, genome-wide analysis uncovered novel recurrent alterations, including TOX and PRKCD, helping to differentiate PCNSL from systemic DLBCL and related lymphomas. Clin Cancer Res; 21(17); 3986–94. ©2015 AACR.

Abstract Major discoveries in the biology of nervous system tumors have raised the question of how non‐histological data such as molecular information can be incorporated into the next W orld H ealth O rganization ( WHO ) classification of central nervous system tumors. To address this question, a meeting of neuropathologists with expertise in molecular diagnosis was held in H aarlem, the N etherlands, under the sponsorship of the I nternational S ociety of N europathology ( ISN ). Prior to the meeting, participants solicited input from clinical colleagues in diverse neuro‐oncological specialties. The present “white paper” catalogs the recommendations of the meeting, at which a consensus was reached that incorporation of molecular information into the next WHO classification should follow a set of provided “ ISN ‐ H aarlem” guidelines. Salient recommendations include that (i) diagnostic entities should be defined as narrowly as possible to optimize interobserver reproducibility, clinicopathological predictions and therapeutic planning; (ii) diagnoses should be “layered” with histologic classification, WHO grade and molecular information listed below an “integrated diagnosis”; (iii) determinations should be made for each tumor entity as to whether molecular information is required, suggested or not needed for its definition; (iv) some pediatric entities should be separated from their adult counterparts; (v) input for guiding decisions regarding tumor classification should be solicited from experts in complementary disciplines of neuro‐oncology; and (iv) entity‐specific molecular testing and reporting formats should be followed in diagnostic reports. It is hoped that these guidelines will facilitate the forthcoming update of the fourth edition of the WHO classification of central nervous system tumors.

Abstract Introduction: Only few genetic studies have been performed in PCNSL, partly due to the rarity of the tumors and the limited amount of available tissue. Unlike nodal DLBCL, PCNSL is poorly understood in terms of its precise histogenetic origin and molecular pathogenesis. It is still a matter of debate whether PCNSL differs from nodal DLBCL with respect to their molecular features and pathogenesis and also if there is a genomic signature PCNSL-specific. Materials and Methods: To determine the pattern of genetic alterations, 17 immunocompetent (EBV-, HIV-) PCNSL were analyzed by aCGH. Seven cases were analyzed using fresh tumor samples and 10 using formalin-fixed paraffin embedded (FFPE) tissues. B-cell differentiation status was characterized by immunostains for CD10, MUM-1, and BCL-6. The aCGH profile from PCNSL cases was compared with aCGH data obtained from 59 nodal DLBCL. Results: We found a complex karyotype with a median of 21 copy-number abnormalities (CNA) per case (range 10-49). Overall, 18 regions (10 losses and 8 gains) were affected in >20% of cases. Loss of 9p21 (CDKN2A) was the commonest CNA (82% of cases). Other common CNAs were loss of 6q23.3 (TNFAIP3; 59%), 6p21 (HLA genes; 53%), 6q21 (PRDM1; 47%) and gain of 12q21-q24 (53%), 7q21-q31 (35%) and 19q13 (35%). Five novel recurrent focal CNAs were found in PCNSL; loss of 3p21.31, 3q26.32, 10p15.3-p14, 12q24.31 and 16q12.1-q21 (29% of cases each). All of them, excluding –16q12.1-q21, were found either in less than 5% or absent in nodal DLBCL, thus suggesting to be unique to PCNSL. Recurrent homozygous deletions were found in CDKN2A, TMEM30A, TOX and CD58, the latter two involved in T-cell development and activation. CD58 was also recurrently affected by small focal deletions in the Ig-like C2-type domain. Furthermore, DNA sequencing analysis showed missense mutations in the Toll/IL-1 receptor domain of MYD88 in 35% of PCNSLs. Another 34 genes, including B2M, ETV6, MHC class II genes, PRDM1, and TNFRSF10A were homozygously deleted. Bioinformatics analysis including the most commonly affected genes showed an enrichment of networks associated with immune response, NF-kB pathway, proliferation, regulation of the apoptosis and lymphocyte differentiation and activation. Conclusions: We showed evidence of a highly complex genome and identified a subset of genes with potential relevance in PCNSL pathogenesis. The genomic profile described here reinforces the existence of a specific molecular signature in PCNSL, thus helping to genetically differentiate this entity from the nodal DLBCL and related lymphomas. Additionally, the results obtained from FFPE samples are encouraging and larger archival tissue collections can now be analyzed in order to complement the still fragmented knowledge we have of the genetic basis of the disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5081. doi:1538-7445.AM2012-5081

Abstract Purpose: Only a limited number of genetic studies have been conducted in primary central nervous system lymphomas (PCNSL), partly due to the rarity of the tumors and the very limited amount of available tissue. In this report, we present the first molecular characterization of copy number abnormalities (CNA) of newly diagnosed PCNSL by array-based comparative genomic hybridization (aCGH) in formalin-fixed paraffin-embedded (FFPE) specimens and compare the results with matched, frozen tumor specimens. Experimental Design: We conducted aCGH in FFPE tissues from PCNSL. Results were compared with matched, paired, frozen tumors. Results: Our analysis confirmed the good to fair quality and reliability of the data generated from limited amounts of tumoral FFPE tissue. Overall, all PCNSL cases were characterized by highly complex karyotypes, with a median of 23 CNAs per patient (range, 17–47). Overall, 20 chromosomal regions were recurrently found in more than 40% of cases. Deletions of 6p21, 6q, and 9p21.3 and gain of 12q12-q24.33 were the commonest CNAs. Other minimal affected regions were defined, and novel recurrent CNAs affecting single genes were identified in 3q26.32 (TBL1XR1) and 8q12.1 (TOX). Conclusions: The results obtained are encouraging. Larger archival tissue collections can now be analyzed to complement the still fragmented knowledge we have of the genetic basis of the PCNSL. Clin Cancer Res; 17(13); 4245–53. ©2011 AACR.