CDKN2A Homozygous Deletion Research Articles

Correlation of MTAP Immunohistochemistry With CDKN2A Status Assessed by Fluorescence In Situ Hybridization and Clinicopathological Features in CNS WHO Grade 2 and 3 Meningiomas: A Single Center Cohort Study.

CDKN2A homozygous deletion has occasionally been reported in atypical and anaplastic meningiomas and is considered as one of the genetic alterations commonly involved in their recurrence and malignant progression. Methylthioadenosine phosphorylase (MTAP) immunohistochemistry is a promising surrogate marker for CDKN2A homozygous deletion in different cancers but has not been examined in meningiomas. We performed CDKN2A FISH and MTAP immunohistochemistry on specimens from 30 patients with CNS WHO grade 2 (n = 27) and 3 (n = 3) meningiomas, including specimens from primary and recurrent tumors and then determined whether MTAP immunohistochemistry correlated with CDKN2A homozygous deletion and clinicopathological features. CDKN2A homozygous deletion was detected in 12% (3/26) of CNS WHO grade 2 and 67% (2/3) of CNS WHO grade 3 meningiomas; 3 cases exhibited temporal and/or spatial heterogeneity. MTAP loss was in excellent concordance with CDKN2A homozygous deletion (sensitivity; 100%, specificity; 100%). MTAP loss/CDKN2A homozygous deletion correlated with cellular proliferation (mitotic rate; p = 0.001, Ki-67 labeling index; p = 0.03) and poor prognosis (overall survival; p = 0.01, progression free survival; p < 0.001). Thus, MTAP immunostaining can be a surrogate marker for CDKN2A homozygous deletion in meningiomas, and MTAP loss/CDKN2A homozygous deletion may be an important prognostic factor for meningiomas.

Clinicopathologic and Genetic Features of Primary T-cell Lymphomas of the Central Nervous System: An Analysis of 11 Cases Using Targeted Gene Sequencing.

Primary central nervous system lymphoma (PCNSL) of peripheral T-cell lineage (T-PCNSL) is rare, and its genetic and clinicopathologic features remain unclear. Here, we present 11 cases of T-PCNSL in immunocompetent individuals from a single institute, focusing on their genetic alterations. Seven cases were subject to targeted panel sequencing covering 120 lymphoma-related genes. Nine of the eleven cases were classified as peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS), of which one was of γδT-cell lineage. There was one case of anaplastic lymphoma kinase-positive anaplastic large cell lymphoma and another of extranodal natural killer (NK)/T-cell lymphoma (ENKTL) of αβT-cell lineage. The male to female ratio was 7 : 4 and the age ranged from 3 to 75 years (median, 61 y). Most patients presented with neurological deficits (n=10) and showed multifocal lesions (n=9) and deep brain structure involvement (n=9). Tumor cells were mostly small-to-medium, and T-cell monoclonality was detected in all nine evaluated cases. PTCL-NOS was CD4-positive (n=4), CD8-positive (n=3), mixed CD4-positive and CD8-positive (n=1), or CD4/CD8-double-negative (n=1, γδT-cell type). Cytotoxic molecule expression was observed in 4 (67%) of the 6 evaluated cases. Pathogenic alterations were found in 4 patients: one PTCL-NOS case had a frameshift mutation in KMT2C, another PTCL-NOS case harbored a truncating mutation in TET2, and another (γδT-cell-PTCL-NOS) harbored NRAS G12S and JAK3 M511I mutations, and homozygous deletions of CDKN2A and CDKN2B. The ENKTL (αβT-cell lineage) case harbored mutations in genes ARID1B, FAS, TP53, BCOR, KMT2C, POT1, and PRDM1. In conclusion, most of the T-PCNSL were PTCL-NOS, but sporadic cases of other subtypes including γδT-cell lymphoma, anaplastic lymphoma kinase-positive anaplastic large cell lymphoma, and ENKTL were also encountered. Immunophenotypic analysis, clonality test, and targeted gene sequencing along with clinicoradiologic evaluation, may be helpful for establishing the diagnosis of T-PCNSL. Moreover, this study demonstrates genetic alterations with potential diagnostic and therapeutic utility in T-PCNSL.

Genetic alterations associated with malignant transformation of sporadic vestibular schwannoma

IntroductionMalignant peripheral nerve sheath tumor of the vestibulocochlear nerve (VN-MPNST) is exceedingly rare and carries a poor prognosis. Little is known about its underlying genetics and in particular the process of malignant transformation. There is an ongoing debate on whether the transformation is initiated by ionizing radiation. We present here the analysis and comparison of two post-radiation VN-MPNST and one undergoing spontaneous transformation.MethodsFour tumors from three patients (radiation-naïve vestibular schwannoma before (VS) and after (VN-MPNST) malignant transformation in addition to two post-radiation VN-MPNST) were subjected to DNA whole-genome microarray and whole-exome sequencing and tumor-specific mutations were called. Mutational signatures were characterized using MuSiCa.ResultsThe tumor genomes were characterized predominantly by copy-number aberrations with 36–81% of the genome affected. Even the VS genome was grossly aberrated. The spontaneous malignant transformation was characterized by a near-total whole-genome doubling, disappearance of NF2 mutation and new mutations in three cancer-related genes (GNAQ, FOXO4 and PDGFRB). All tumors had homozygous loss of the tumor suppressor CDKN2A. Neither mutational signature nor copy number profile was associated with ionizing radiation.ConclusionThe VN-MPNST genome in our cases is characterized by large copy-number aberrations and homozygous deletion of CDKN2A. Our study demonstrates a VS with genetic alterations similar to its malignant counterpart, suggesting the existence of premalignant VS. No consistent mutational signature was associated with ionizing radiation.

EPCO-09. LONGITUDINAL ANALYSIS OF DIFFUSE GLIOMA REVEALS CELL STATE DYNAMICS AT RECURRENCE ASSOCIATED WITH CHANGES IN GENETICS AND THE MICROENVIRONMENT

Abstract Diffuse glioma is characterized by a poor prognosis and a universal resistance to therapy, though the evolutionary processes behind this resistance remain unclear. The Glioma Longitudinal Analysis (GLASS) Consortium has previously demonstrated that therapy-induced selective pressures shape the genetic evolution of glioma in a stochastic manner. However, single-cell studies have revealed that malignant glioma cells are highly plastic and transition their cell state in response to diverse challenges, including changes in the microenvironment and the administration of standard-of-care therapy. To interrogate the factors driving therapy resistance in diffuse glioma, we collected and analyzed RNA- and/or DNA-sequencing data from temporally separated tumor pairs of over 300 adult patients with IDH-wild-type or IDH-mutant glioma. In a subset of these tumor pairs, we complemented these data with single-nucleus RNAseq and multiplex imaging mass cytometry at each time point. Recurrent tumors exhibited diverse changes that were attributable to changes in histological features, somatic alterations, and microenvironment interactions. IDH-wild-type tumors overall were more invasive at recurrence and exhibited increased expression of neuronal signaling programs that reflected a possible role for neuronal interactions in promoting glioma progression. In contrast, recurrent IDH-mutant tumors exhibited a significant increase in proliferative expression programs that correlated with discrete genetic changes. Hypermutation and acquired CDKN2A homozygous deletions associated with an increase in proliferating stem-like malignant cells at recurrence in both glioma subtypes, reflecting active tumor expansion. A transition to the mesenchymal phenotype was associated with the presence of a specific myeloid cell state defined by unique ligand-receptor interactions with malignant cells, providing opportunities to target this transition through therapy. Collectively, our results uncover recurrence-associated changes in genetics and the microenvironment that can be targeted to shape disease progression following initial diagnosis.

EXTH-55. TARGETING RECURRENT IDH MUTANT GLIOMA WITH CDK4/6 INHIBITION

Abstract Despite initial responsiveness to standard treatments like radiation and chemotherapy, IDH mutant gliomas inevitably recur, become more clinically aggressively and lead to untimely death. Recurrent IDH mutant tumors are less responsive to conventional treatments, highlighting the need for improved therapeutic strategies at this stage of the disease. At least 20% of recurrent IDH mutant gliomas exhibit homozygous loss of CDKN2A, which results in aberrant signaling through the CDK-RB pathway. We hypothesized that CDKN2A loss leads to enhanced sensitivity to CDK4/6 inhibitors, which are approved for use in a variety of other cancer types. We examined the relationship between CDK4/6 inhibitor sensitivity and CDKN2A loss using patient-derived models of IDH mutant glioma with endogenous CDKN2A homozygous deletion as well as with CRIPSR-mediated gene deletion. We observed enhanced cytotoxicity in glioma models with CDKN2A loss in vitro. Studies to examine the efficacy of CDK4/6 inhibitor treatment on slowing tumor growth in patient-derived xenograft models are ongoing. These preclinical results provide foundational data for design of a biomarker-driven clinical trial of CDK4/6 inhibitors in patients with recurrent IDH mutant glioma.

Comparative Analysis of Clinicopathological Features and Genetic Landscape of Relapsed/ Refractory and Effectively Treated Extranodal Nasal NK/ T Cell Lymphoma

BackgroundExtranodal natural killer/T-cell lymphoma (ENKTL) is an Epstein-Bar virus (EBV)-associated aggressive lymphoma that is endemic in East Asia, Central and South America, and sporadic in Western countries. L-asparaginase regimens have significantly improved prognosis and are the current most commonly used initial therapy. Unfortunately, about 20% to 40% patients had inadequate responses to L-asparagine regimens, and diagnosed as relapsed or refractory (R/R). Due to the lack of study investigating on R/R-ENKTL, the differences in clinicopathological features and gene mutations between R/R-ENKTL and effectively treated ENKTL (ET-ENKTL) remain unclear.Material and methodThis study included 21 R/R-ENKTL cases and 10 ET-ENKTL cases from June 2015 to December 2020. The clinical data, pathological features, and the genomic variations detected by whole exome sequence (WES), were compared between the two groups.ResultThe clinical characteristics of R/R and ET ENKTL were summarized in Table 1.Compared with ET patients, more R/R-ENKTL patients had B symptoms (P = 0.038) and elevated LDH (P = 0.041). Pathological features such as the diffuse distribution of medium-sized cells and necrosis were observed in both groups. It is noteworthy that angiocentric and angiodestructive growth pattern is more frequently present in the R/R group (P = 0.032). Immunohistochemistry showed that R/R-ENKTL patients had higher CD30 positive rate (P = 0.045) and lower PD-L1 expression rate (P = 0.030)（Figure 1. A）.A significant difference in WES-based tumor mutation burden (TMB) between the two groups was observed, with a median TMB of 3.7 Muts/Mb in the R/R group and 2.3 Muts/Mb in the ET group. Mutated genes were mainly involved in the biological processes (BP) with extracellular structure/ matrix organization in the R/R group, but not the ET group, in the gene ontology (GO) analysis (Figure 1. B). As the change of extracellular matrix will restrict the entry of drugs into target tissues, this may be one of the reasons that led to the resistance of R/R-ENKTL.Among all the 1738 mutant genes in the R/R-ENKTL groups, none of substitution mutations were found significantly higher frequency than the ET group. However, when all of the epigenetic regulatory genes were gathered and compared, the mutation frequencies of them were significantly higher in the R/R-ENKTL group, especially for the genes encoding histone methylation including KMT2D, KMT2C and KDM4C etc. Mutations of KMT2D were found in 5 patients (23.8%), including three substitutions and two truncations in the R/R group. In contrast, only a truncation of KMT2D was found in a patient of the ET group (10.0%) (Figure 1. C). It suggested that histone methylation abnormality might play a role in ENKTL resistance, which is worthy of further study.Compared with R/R groups, the mutation frequencies of three mutant genes (RELN, LAMA5 and TPTE, all with P = 0.027 respectively) were more prevalent in the ET-ENKTL (Figure 1. C). Whether the abnormalities of these genes were related to the effectiveness of drugs like L-asparagine and acted as the potential biomarker in ENKTL needs to be further investigated.The patterns of structural variations (SVs) were also different between the two groups. NRG1 was amplified in 19.0% (4/21) patients of the R/R group, while no abnormality was found in the ET group. Besides, SVs that only appeared in the R/R group included homozygous deletion of CDKN2A (9.5%) and CDKN2B (4.8%), and so on. In addition, the PD-L1 encoding gene CD274 had a higher rate of fusions in the R/R-ENKTL group (n=3) than the ET group (n=1).Discussion and ConclusionOur results showed that more R/R-ENKTL patients had B symptoms and elevated LDH. For pathological features, R/R-ENKTL patients was more likely to have angiocentric and angiodestructive growth pattern, higher CD30 positive rate and lower PD-L1 expression rate. Based on tumor genetics, frequently mutated genes encoding histone methylation and enriched in the GO term of extracellular structure/ matrix organization might play a role in the drug resistance of R/R-ENKTL. Furthermore, the high prevalence of NRG1 amplification and CD274 fusions suggested their potential as therapeutic targets in R/R-ENKTL treatment. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Work in progress: Changes to who brain tumor classification

The WHO classification of central nervous system (CNS) tumors had been revised in 2016 to include for the first time molecular biomarkers that are important for tumor classification and clinical decision making. Thereafter, the cIMPACT-NOW (Consortium to Inform Molecular and Practical Approaches to CNS Tumor Taxonomy – not officially WHO) formed to further refine CNS tumor classification through specific recommendations that addressed issues of classification still controversial in 2016. Several cIMPACT-NOW proposals have shaped the new 2021 WHO classification of brain tumors. Mutations in the isocitrate dehydrogenase (IDH) 1 or 2 genes continue to play a major role in glioma classification beyond 2021. Among IDH-mutant gliomas, loss of ATRX expression identifies IDH-mutant astrocytomas without necessity for 1p/19q codeletion testing. Conversely, etection of a 1p/19q codeletion in an IDH-mutant glioma defines an oligodendroglial tumor. The nomenclature for IDH-mutant glioblastoma has been changed to astrocytoma, IDH-mutant, WHO grade 4, with CDKN2A homozygous deletion representing a molecular marker for these tumors. IDH-wildtype astrocytomas that lack microvascular proliferation or necrosis but exhibit telomerase reverse transcriptase promoter mutation, epidermal growth factor receptor amplification and/or a + 7/−10 genotype are now classified as IDH-wildtype glioblastoma. H3.3 G34-mutant diffuse hemispheric gliomas have been proposed as a new entity separate from IDH-wildtype glioblastoma. The new WHO classification increases the diagnostic accuracy and refines clinical care by changing treatment recommendations, e.g. for patients with IDH-wildtype astrocytomas showing molecular features of glioblastoma. It also has major implications for clinical trial design, notable inclusion and exclusion criteria for ongoing and upcoming clinical trials.

PL03.2.A Radiotherapy is associated with a deletion signature that contributes to poor outcomes in glioma patients

Abstract BACKGROUND Diffuse gliomas are highly aggressive brain tumors that invariably relapse despite treatment with chemo- and radiotherapy. Treatment with alkylating chemotherapy can drive tumors to develop a hypermutator phenotype. In contrast, the genomic effects of radiation therapy (RT) remain largely unknown. MATERIAL AND METHODS We analyzed the mutational spectra following treatment with RT in whole genome or exome sequencing data from 190 paired primary-recurrent gliomas from the Glioma Longitudinal Analysis (GLASS) dataset and 3693 post-treatment metastatic tumors from the Hartwig Medical Foundation (HMF). RESULTS We identified a significant increase in the burden of small deletions following radiation therapy that was independent of other factors (P = 3e-03, multivariable log-linear regression). These novel deletions demonstrated distinct characteristics when compared to pre-existing deletions present prior to RT-treatment and deletions in RT-untreated tumors. Radiation therapy-acquired deletions were characterized by a larger deletion size (GLASS and HMF, P = 1.5e-04 and P = 6e-16, respectively; Mann-Whitney U test), an increased distance to repetitive DNA elements (P &amp;lt; 2.2e-16, Kolmogorov-Smirnov test) and a lack of microhomology at breakpoints (P = 6.6e-05, paired Wilcoxon signed-rank test). Furthermore, mutational signature analysis confirmed the distinct genomic characteristics of RT-associated deletions when compared to deletions arising via homologous recombination deficiency or microsatellite instability. These observations suggested that canonical non-homologous end joining (c-NHEJ) was the preferred pathway for DNA double strand break repair of RT-induced DNA damage. Furthermore, RT resulted in frequent chromosomal deletions and significantly increased frequencies of CDKN2A homozygous deletions in IDHmut glioma (P= 1.9e-05, Fisher’s exact test). Finally, a high burden of RT-associated deletions was associated with worse clinical outcomes (GLASS and HMF, P = 3.4e-02 and P &amp;lt; 1e-04, respectively; log-rank test). CONCLUSION Our results collectively suggest that effective repair of RT-induced DNA damage is detrimental to patient survival and that inhibiting c-NHEJ may be a viable strategy for improving the cancer-killing effect of radiotherapy. Furthermore, CDKN2A homozygous deletion at recurrence may be leveraged as a promising clinical biomarker of RT-resistance in IDHmut glioma. Taken together, the identified genomic scars as a result of RT reflect a more aggressive tumor with increased levels of resistance to follow up treatments.

Malignant pleural mesothelioma showing rare morphology indistinguishable from myxofibrosarcoma concomitant with EGFR-mutated lung adenocarcinoma: A case report.

Introduction and importancePrimary tumors of the pleura are rare, with malignant mesothelioma being the most common of these neoplasms. Pathological diagnosis of sarcomatoid mesothelioma can be more challenging than that of epithelioid malignant mesothelioma because of its similarities with true sarcomas and restricted or inconsistent expression of mesothelial markers in immunohistochemistry analysis.Presentation of caseHere, we present an unusual case of malignant pleural mesothelioma concomitant with lung adenocarcinoma in a 72-year-old Japanese man, a smoker with no family history of cancer and asbestos exposure. Malignant pleural mesothelioma is composed of epithelial and spindle-shaped cells. Spindle-shaped cells with scant eosinophilic cytoplasm and hyperchromatic nuclei proliferated in abundant myxoid stroma containing thin-walled blood vessels, mimicking myxofibrosarcoma. The loss of BAP1 (BRCA1-associated protein 1) expression, as assessed by immunohistochemistry, and homozygous deletions of CDKN2A, detected using fluorescence in situ hybridization (FISH), were observed in both components. Targeted sequencing revealed that lung adenocarcinoma harbored EGFR mutations, whereas no mutations were detected in either component of biphasic mesothelioma.DiscussionAlthough alcian blue-stained mucins were detected in biphasic mesothelioma subsets, the clinicopathological significance of myxoid stroma in biphasic and sarcomatoid mesothelioma remains largely unknown.ConclusionOur case presented a unique morphology mimicking myxofibrosarcoma in a sarcomatoid component of biphasic mesothelioma; therefore, it raises a question on the clinicopathological significance of myxoid stroma in sarcomatous areas of biphasic and sarcomatoid mesothelioma.

OMRT-3. Longitudinal analysis of diffuse glioma reveals cell state dynamics at recurrence associated with changes in genetics and the microenvironment

Diffuse glioma is an aggressive brain cancer that is characterized by a poor prognosis and a universal resistance to therapy. The evolutionary processes behind this resistance remain unclear. Previous studies by the Glioma Longitudinal Analysis (GLASS) Consortium have indicated that therapy-induced selective pressures shape the genetic evolution of glioma in a stochastic manner. However, single-cell studies have revealed that malignant glioma cells are highly plastic and transition their cell state in response to diverse challenges, including changes in the microenvironment and the administration of standard-of-care therapy. Interactions between these factors remain poorly understood, making it difficult to predict how a patient’s tumor will evolve from diagnosis to recurrence. To interrogate the factors driving therapy resistance in diffuse glioma, we collected and analyzed RNA- and/or DNA-sequencing data from temporally separated tumor pairs of 292 adult patients with IDH-wild-type or IDH-mutant glioma. Recurrent tumors exhibited diverse changes that were attributable to changes in anatomic composition, somatic alterations, and microenvironment interactions. Hypermutation and acquired CDKN2A homozygous deletions associated with an increase in proliferating stem-like malignant cells at recurrence in both glioma subtypes, reflecting active tumor expansion. IDH-wild-type tumors were more invasive at recurrence, and their malignant cells exhibited increased expression of neuronal signaling programs that reflected a possible role for neuronal interactions in promoting glioma progression. Mesenchymal transition was associated with the presence of a specific myeloid cell state defined by unique ligand-receptor interactions with malignant cells, providing opportunities to target this transition through therapy. Collectively, our results uncover recurrence-associated changes in genetics and the microenvironment that can be targeted to shape disease progression following initial diagnosis.

Abstract 2068: Radiotherapy is associated with a deletion signature that contributes to poor cancer patient outcomes

Abstract Diffuse gliomas are highly aggressive brain tumors that invariably relapse despite treatment with chemo- and radiotherapy. Treatment with alkylating chemotherapy can drive tumors to develop a hypermutator phenotype. In contrast, the genomic effects of radiation therapy (RT) remain unknown. We analyzed the mutational spectra following treatment with ionizing radiation in sequencing data from 190 paired primary-recurrent gliomas from the Glioma Longitudinal Analysis (GLASS) dataset and 2116 post-treatment metastatic tumors from the Hartwig Medical Foundation. We identified a significant increase in the burden of small deletions following radiation therapy that was independent of other factors and was significantly associated with the clinically applied RT-dosage in Gy (P = 1e-02, multivariable log-linear regression). These novel deletions demonstrated distinct characteristics when compared to pre-existing deletions present prior to RT-treatment and deletions in RT-untreated tumors. Radiation therapy-acquired deletions were characterized by a larger deletion size (GLASS and metastatic cohort, P = 1.2e-02 and P = 8e-11, respectively; Mann-Whitney U test), an increased distance to repetitive DNA elements (P &amp;lt; 2.2e-16, Kolmogorov-Smirnov test) and a reduction in microhomology at breakpoints (P = 3.2e-02, paired Wilcoxon signed-rank test). These observations suggested that canonical non-homologous end joining (c-NHEJ) was the preferred pathway for DNA double strand break repair of RT-induced DNA damage. Furthermore, radiotherapy resulted in frequent chromosomal deletions and significantly increased frequencies of CDKN2A homozygous deletions. Finally, a high burden of RT-associated deletions was associated with worse clinical outcomes (GLASS and metastatic cohort, P &amp;lt; 1e-04 and P = 2.6e-02, respectively; Wald test). Our results collectively suggest that effective repair of RT-induced DNA damage is detrimental to patient survival and that inhibiting c-NHEJ may be a viable strategy for improving the cancer-killing effect of radiotherapy. Taken together, the identified genomic scars as a result of radiation therapy reflect a more aggressive tumor with increased levels of resistance to follow up treatments. Citation Format: Emre Kocakavuk, Kevin J. Anderson, Kevin C. Johnson, Frederick S. Varn, Samirkumar B. Amin, Erik P. Sulman, Floris P. Barthel, Roel G. Verhaak. Radiotherapy is associated with a deletion signature that contributes to poor cancer patient outcomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2068.

Homozygous Co-Deletion of Type I Interferons and CDKN2A Genes in Thoracic Cancers: Potential Consequences for Therapy.

Homozygous deletion (HD) of the tumor suppressor gene CDKN2A is the most frequent genetic alteration in malignant pleural mesothelioma and is also frequent in non-small cell lung cancers. This HD is often accompanied by the HD of the type I interferons (IFN I) genes that are located closed to the CDKN2A gene on the p21.3 region of chromosome 9. IFN I genes encode sixteen cytokines (IFN-α, IFN-β…) that are implicated in cellular antiviral and antitumor defense and in the induction of the immune response. In this review, we discuss the potential influence of IFN I genes HD on thoracic cancers therapy and speak in favor of better taking these HD into account in patients monitoring.

TERT promoter mutation status is necessary and sufficient to diagnose IDH-wildtype diffuse astrocytic glioma with molecular features of glioblastoma.

The Consortium to Inform Molecular and Practical Approaches to CNS Tumor Taxonomy (cIMPACT-NOW) update 3 recommends that histologic grade II and III IDH-wildtype diffuse astrocytic gliomas that harbor EGFR amplification, the combination of whole chromosome 7 gain and whole chromosome 10 loss (7 + /10 -), or TERT promoter (pTERT) mutations should be considered as glioblastomas (GBM), World Health Organization grade IV. In this retrospective study, we examined the utility of molecular classification based on pTERT status and copy-number alterations (CNAs) in IDH-wildtype lower grade gliomas (LGGs, grade II, and III). The impact on survival was evaluated for the pTERT mutation and CNAs, including EGFR gain/amplification, PTEN loss, CDKN2A homozygous deletion, and PDGFRA gain/amplification. We analyzed 46 patients with IDH-wildtype/pTERT-mutant (mut) LGGs and 85 with IDH-wildtype/pTERT-wildtype LGGs. EGFR amplification and a combination of EGFR gain and PTEN loss (EGFR + /PTEN -) were significantly more frequent in pTERT-mut patients (p < 0.0001). Cox regression analysis showed that the pTERT mutation was a significant predictor of poor prognosis (hazard ratio [HR] 2.79, 95% confidence interval [CI] 1.55-4.89, p = 0.0008), but neither EGFR amplification nor EGFR + /PTEN - was an independent prognostic factor in IDH-wildtype LGGs. PDGFRA gain/amplification was a significant poor prognostic factor in IDH-wildtype/pTERT-wildtype LGGs (HR 2.44, 95% CI 1.09-5.27, p = 0.03, Cox regression analysis). The IDH-wildtype LGGs with either pTERT-mut or PDGFRA amplification were mostly clustered with GBM by DNA methylation analysis. Thus, our study suggests that analysis of pTERT mutation status is necessary and sufficient to diagnose IDH-wildtype diffuse astrocytic gliomas with molecular features of glioblastoma. The PDGFRA status may help further delineate IDH-wildtype/pTERT-wildtype LGGs. Methylation profiling showed that IDH-wildtype LGGs without molecular features of GBM were a heterogeneous group of tumors. Some of them did not fall into existing categories and had significantly better prognoses than those clustered with GBM.

CDKN2A or CDKN2B homozygous deletion with high-immune infiltration as a favorable prognostic factor for lung adenocarcinoma.

e20543 Background: CDKN2A and CDKN2B both acted as tumor suppressor genes by regulating the cell cycle, which in humans were located at chromosome 9, band p21.3. The frequencies of homozygous deletion (HomDel) in CDKN2A and CDKN2B in lung adenocarcinoma (LUAD) were 12.5% and 12.1%, respectively. However, the genomic, immunogenomic features and impact on the prognosis of LUAD patients with CDKN2A/B HomDel were still unclear. Methods: The cohort of this study was from The Cancer Genome Atlas (TCGA). A total of 508 LUAD patients, including 99 CDKN2A/B HomDel (homdel) and 509 CDKN2A/B wild (wild). This study explored the difference of genomic and immunogenomic landscape between homdel and wild by analysis of whole-exome sequencing (WES) and RNA sequencing data. Results: The most frequently mutated genes were TP53, TTN, MUC16, and CSMD3. Their frequencies in homdel and wild are 46% and 48%, 43% and 46%, 35% and 41%, 33% and 38%, respectively. There was no significant difference of tumor mutational burden (TMB) between homdel and wild (median TMB, 133 in homdel vs 177 in wild; Wilcoxon test, p = 0.11), and clinical characteristics including age, gender, smoking history, and tumor stage were not significantly different between homdel and wild. Homdel had a shorter overall survival (OS) than wild (Log-rank test, p = 0.04, Hazard Ratio: 0.7, 95% CI: 0.49-1.02), but there was no significant difference in progression-free survival (PFS) (Log-rank test, p = 0.05, Hazard Ratio: 0.73, 95% CI: 0.51-1.04). We used single sample gene set enrichment analysis (ssGSEA) to calculate the enrichment score (ES) of 25 immune-related pathways such as antigen presentation and T cell-mediated immunity, and then used the consensus clustering algorithm (ConsensusClusterPlus) to cluster homdel and wild respectively, and both clustered into low and high immune infiltration groups. For the high immune infiltration and low immune infiltration in homdel and wild, high immune infiltration had a longer OS (Log-rank test, p = 0.009, Hazard Ratio: 2.19, 95% CI: 1.22-3.94) and PFS (Log-rank test, p = 0.044, Hazard Ratio: 1.8, 95% CI: 1.01-3.2) than low immune infiltration in homdel. However, there was no significant heterogeneity between high and immune infiltration in terms of PFS (Log-rank test, p = 0.28, Hazard Ratio: 1.21, 95% CI: 0.87-1.68) and OS (Log-rank test, p = 0.96, Hazard Ratio: 1.01, 95% CI: 0.71-1.44) in the wild group, the wild group had longer OS than homdel group with low immune infiltration (Log-rank test, p = 0.003, Hazard Ratio: 0.5, 95% CI: 0.29-0.88), while had the same OS with homdel with high immune infiltration, irrespective of immune infiltration. And so was PFS (Log-rank test, p = 0.005, Hazard Ratio: 0.48, 95% CI: 0.27-0.82). Conclusions: CDKN2A/B homdel was an unfavorable prognostic factor for LUAD, but which with high immune infiltration might improve patient survival time.

Molecular Characterization of "True" Low-Grade IDH-Wildtype Astrocytomas.

Numerous recent studies have demonstrated that the vast majority of IDH-wildtype astrocytomas with WHO grade II/III histology have clinical outcomes equivalent to IDH-wildtype glioblastomas. This has called into question the existence of an IDH-wildtype lower-grade astrocytoma (LGA) category, and the cIMPACT-NOW study group has suggested 3 molecular features which, if present, warrant upgrading IDH-wildtype LGA to glioblastoma: EGFR amplification, 7+/10-, and TERT promoter mutation. Herein, we evaluate the clinical, histologic, and molecular features of IDH-wildtype low-grade astrocytomas, defined here as infiltrative adult astrocytoma lacking histologic features of glioblastoma (microvascular proliferation and/or necrosis), IDH1/2 mutation, and all 3 of the cIMPACT-NOW update 3 factors. Compared with their counterparts with cIMPACT-NOW features of glioblastoma (LGA-C+; n = 108), IDH-wildtype LGAs lacking these features (LGA-C0; n = 36) occur in significantly younger patients, are more frequently WHO grade II, have less total copy number variation distributed across the entire genome, less frequent homozygous deletion of CDKN2A, less frequent PTEN and PIK3CA alterations, and more frequent NF1 alterations. These results suggest that although rare, a "true" IDH-wildtype LGA category does exist, and has distinct clinical and molecular features consistent with relatively beneficial clinical outcomes in these patients.

Abstract PO-019: Radiotherapy in cancer is associated with a deletion signature that contributes to poor patient outcomes

Abstract Diffuse gliomas are highly aggressive brain tumors that invariably relapse despite treatment with chemo- and radiotherapy. Treatment with alkylating chemotherapy can drive tumors to develop a hypermutator phenotype. In contrast, the genomic effects of radiation therapy (RT) remain unknown. We analyzed the mutational spectra following treatment with ionizing radiation in sequencing data from 190 paired primary-recurrent gliomas from the Glioma Longitudinal Analysis (GLASS) dataset and 3693 post-treatment metastatic tumors from the Hartwig Medical Foundation (HMF). We identified a significant increase in the burden of small deletions following radiation therapy that was independent of other factors. These novel deletions demonstrated distinct characteristics when compared to pre-existing deletions present prior to RT-treatment and deletions in RT-untreated tumors. Radiation therapy-acquired deletions were characterized by a larger deletion size (GLASS and metastatic cohort, P=1.2e-02 and P=8e-11, respectively; Mann-Whitney U test), an increased distance to repetitive DNA elements (P&amp;lt;2.2e-16, Kolmogorov-Smirnov test) and a reduction in microhomology at breakpoints (P=3.2e-02, paired Wilcoxon signed-rank test). These observations suggested that canonical non-homologous end joining (c-NHEJ) was the preferred pathway for DNA double strand break repair of RT-induced DNA damage. Furthermore, radiotherapy resulted in frequent chromosomal deletions and significantly increased frequencies of CDKN2A homozygous deletions. Finally, a high burden of RT-associated deletions was associated with worse clinical outcomes (GLASS and metastatic cohort, P &amp;lt; 1e-04 and P = 2.6e-02, respectively; Wald test). Our results suggest that effective repair of RT-induced DNA damage is detrimental to patient survival and that inhibiting c-NHEJ may be a viable strategy for improving the cancer-killing effect of radiotherapy. Taken together, the identified genomic scars as a result of radiation therapy reflect a more aggressive tumor with increased levels of resistance to follow up treatments. Citation Format: Emre Kocakavuk, Kevin J. Anderson, Frederick S. Varn, Kevin C. Johnson, Samirkumar B. Amin, Erik. P. Sulman, Martijn Lolkema, Floris P. Barthel, Roel G.W. Verhaak. Radiotherapy in cancer is associated with a deletion signature that contributes to poor patient outcomes [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-019.

Clinical significance of CDKN2A homozygous deletion in combination with methylated MGMT status for IDH-wildtype glioblastoma.

ObjectiveAccumulating evidence from recent molecular diagnostic studies has indicated the prognostic significance of various genetic markers for patients with glioblastoma (GBM). To evaluate the impact of such genetic markers on prognosis, we retrospectively analyzed the outcomes of patients with IDH‐wildtype GBM in our institution. In addition, to assess the impact of bevacizumab (BEV) treatment, we compared overall survival (OS) between the pre‐ and post‐BEV eras.MethodsWe analyzed the data of 100 adult patients (over 18 years old) with IDH‐wildtype GBM from our database between February 2006 and October 2018. Genetic markers, such as MGMT methylation status, EGFR amplification, CDKN2A homozygous deletion, and clinical factors were analyzed by evaluating the patients’ OS.Results CDKN2A homozygous deletion showed no significant impact on OS in patients with methylated MGMT status (p = 0.5268), whereas among patients with unmethylated MGMT status, there was a significant difference in OS between patients with and without CDKN2A homozygous deletion (median OS: 14.7 and 16.9 months, respectively, p = 0.0129). This difference was more evident in the pre‐BEV era (median OS: 10.1 and 15.6 months, respectively, p = 0.0351) but has become nonsignificant in the post‐BEV era (median OS: 16.0 and 16.9 months, respectively, p = 0.1010) due to OS improvement in patients with CDKN2A homozygous deletion. However, these findings could not be validated in The Cancer Genome Atlas cohort.Conclusions MGMT and CDKN2A status subdivided our cohort into three race‐specific groups with different prognoses. Our findings indicate that BEV approval in Japan led to OS improvement exclusively for patients with concurrent unmethylated MGMT status and CDKN2A homozygous deletion.

Establishment and genomic characterization of a sporadic malignant peripheral nerve sheath tumor cell line

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive Schwann cell-derived neoplasms that occur sporadically or in patients with neurofibromatosis type 1 (NF1). Preclinical research on sporadic MPNSTs has been limited as few cell lines exist. We generated and characterized a new sporadic MPNST cell line, 2XSB, which shares the molecular and genomic features of the parent tumor. These cells have a highly complex karyotype with extensive chromothripsis. 2XSB cells show robust invasive 3-dimensional and clonogenic culture capability and form solid tumors when xenografted into immunodeficient mice. High-density single nucleotide polymorphism array and whole exome sequencing analyses indicate that, unlike NF1-associated MPNSTs, 2XSB cells have intact, functional NF1 alleles with no evidence of mutations in genes encoding components of Polycomb Repressor Complex 2. However, mutations in other genes implicated in MPNST pathogenesis were identified in 2XSB cells including homozygous deletion of CDKN2A and mutations in TP53 and PTEN. We also identified mutations in genes not previously associated with MPNSTs but associated with the pathogenesis of other human cancers. These include DNMT1, NUMA1, NTRK1, PDE11A, CSMD3, LRP5 and ACTL9. This sporadic MPNST-derived cell line provides a useful tool for investigating the biology and potential treatment regimens for sporadic MPNSTs.

ES17.04 Updates in Mesothelioma

The histologic subtype of malignant mesothelioma of the pleura has an impact on prognosis and treatment decisions and therefore has to be documented in diagnostic pathology reports. Epithelioid morphology is associated with a better prognosis when compared to sarcomatoid and biphasic subtypes. Surgical treatment may also have a prognostic impact in epithelioid subtype, while the same benefit is not seen in biphasic and sarcomatoid mesotheliomas. The 2020/21 WHO classification divides mesothelial proliferations into benign and preinvasive (adenomatoid tumor, well-differentiated papillary mesothelial tumor, mesothelioma in situ) and mesothelioma (localized pleural mesothelioma, diffuse pleural mesothelioma). The main change is the introduction of mesothelioma in situ (MIS) in the classification. Until recently the consensus was that MIS cannot be separated from reactive surface mesothelial proliferations on morphology alone. However, the concept of MIS was recently reintroduced for surface, usually flat or simple papillary, mesothelial proliferations with loss of BAP1 nuclear staining by immunohistochemistry or CDKN2A homozygous deletion, in the absence of radiologic or clinical gross evidence of the disease.1-3 Clinically, MIS is suspected in patients with unexplained recurrent effusions in the setting of heavy asbestos exposure, after irradiation and in patients with familial predisposition. The diagnosis requires sufficient tissue usually large biopsy samples. Small biopsy and cytology samples are not appropriate. Patients with MIS have a high incidence of eventual development of invasive mesothelioma. The updated classification of epithelioid mesotheliomas includes architectural patterns (tubular, papillary, tubulopapillary, trabecular, solid, adenomatoid and microcystic), stromal (myxoid) and cytologic (pleomorphic ,rhabdoid, deciduoid, small cell, clear cell, signet ring) features. Their identification is important for correct diagnosis, and some have prognostic values. Two cytologic features, transitional and pleomorphic, were previously classified as epithelioid patterns. They have shown a similar survival to biphasic and sarcomatoid mesothelioma and should be reclassified.5-8 Transitional features have appearance between epithelioid and sarcomatoid morphology, and have genomic characteristics similar to sarcomatoid subtype, and therefore were reclassified as cytologic feature of sarcomatoid mesothelioma. 6. Epithelioid mesotheliomas with transitional features should be reclassified as biphasic mesothelioma. Transitional features within biphasic mesothelioma carry negative prognostic significance, with a median survival of 6 months for cases with transitional features compared to 12 months for those without transitional features .6 Pleomorphic features, as described in epithelioid mesothelioma, can also be seen in sarcomatoid mesotheliomas. In contrast to transitional features, emerging genomic data suggest that a mesothelioma with pleomorphic features genomically can cluster with either sarcomatoid or epithelioid subtypes. Therefore, mesotheliomas with pleomorphic features should be classified based on the most predominant morphology as either sarcomatoid or epithelioid. A two-tier grading system for epithelioid mesothelioma allows the identification of more aggressive mesotheliomas and can be applied to biopsy and resection specimens 9, 10 It is primarily based on the combination of nuclear features, mitotic rate and the presence or absence of necrosis (Figure 1). Molecular testing in mesothelioma compared to other cancers is very limited. Recently, there have been several sequencing efforts to provide insights into the genomic characteristics and improved prognostic classification of mesothelioma. 10

HGG-40. EXCEPTIONAL SYNCHRONOUS OCCURENCE OF A BRAF V600E MUTANT GLIOBLASTOMA AND A H3.3K27M MUTANT DIFFUSE INTRINSIC PONTINE GLIOMA: A CASE REPORT

We report herein the case of a 17-year-old female who presented with intracranial hypertension and diplopia. Magnetic resonance imaging showed a large left cystic and solid temporoparietal lesion, associated with an infiltrating lesion of the brainstem, hypointense in T1 and hyperintense in FLAIR sequences, without enhancement after injection of gadolinium. Complete resection of the parietal mass and biopsy of the brainstem lesion were performed. Histopathological analysis of the parietal mass showed glioblastoma (WHO grade IV) with no IDH1/2 or H3.3/H3.1 gene mutation detected by Sanger sequencing. Immunohistochemistry found the expression of the proteins of mismatch repair system. Whole exome and RNA sequencing identified a BRAF-V600E mutation. The brainstem lesion was a diffuse midline glioma, H3K27M-mutant (grade IV) according to the 2016 WHO classification. Pan-genomic SNP arrays of the 2 tumors showed distinct genetic alterations. The parietal glioblastoma displayed complex genomic alterations whereas the brainstem glioma harbored chromosome 7q gain, chromosome 9p and 10 losses, and RB, TP53 and CDKN2A homozygous deletions. The patient was treated by concomitant radiochemotherapy (according to Stupp protocol). After 12 cycles of temozolomide, there was complete remission persistant in the parietal lobe. The brainstem tumor was stable but progressed after 3 months of temozolomide discontinuation. Treatment with mTOR inhibitors was initiated. At 21-month follow-up, the patient remains with few symptoms. No predisposition syndrome was identified in the patient or her family. Concurrent glioblastomas with distinct driver gene mutations are exceptional.