Exome DNA Research Articles

Hemimegalencephaly associated with drug-resistant epilepsy and a rare molecular genetic alteration in the CPA6 gene: a clinical case

Hemimegalencephaly (HME) is one of the extremely rare congenital malformations of cortical development (MCD). It belongs to the MCD group of mTOR-related pathologies and can be the result of various genetic disorders. One of the main clinical manifestations of HME is drug-resistant epilepsy requiring surgical treatment.Case report. This article describes a clinical case of HME in a 4-year-old boy with frequent generalized tonic-clonic seizures and drug-resistant epilepsy; also, he had speech development delay. MRI revealed a HME of the right frontal lobe. Stereotaxic laser disconnection of the large cortical dysplasia in the right frontal lobe of the brain was performed. Morphological features of focal cortical dysplasia type IIb (FCD IIb) were reported. No seizures were observed in the hospital follow up after the operation for 14 days. The whole exome DNA sequencing showed the presence of a heterozygous state _000008.10^G 68419028del / 633del, pGlu212LysfsTers of the CPA6 gene.Discussion. A feature of the case is the identified association of HME, morphologically represented by FCD IIb, with a previously unknown heterozygous state in the 6th exon of the CPA6 gene. This association allows to expand our understanding of changes in the activation of PI3K/AKT/mTOR pathway as a key link in the pathogenesis of congenital anomaly of cortical development.

Clinical and genetic characteristics of the patients with hypertension and hypokalemia carrying a novel SCNN1A mutation

The objective of this study was to clinically and genetically characterize a pedigree with Liddle syndrome (LS). A LS pedigree comprising with one proband and seven family members was enrolled. The subjects’ symptoms, laboratory results and genotypes were analyzed. Peripheral venous samples were collected from the subjects, and genomic DNA was extracted. DNA library construction and exome capture were performed on an Illumina HiSeq 4000 platform. The selected variant sites were validated using Sanger sequencing. The mutation effects were investigated using prediction tools. The proband and her paternal male family members had mild hypertension, hypokalemia and muscle weakness, including the absence of low renin and low aldosterone. Genetic analysis revealed that the proband carried a compound heterozygous mutation in SCNN1A, a novel heterozygous mutation, c.1130T > G (p.Ile377Ser) and a previously characterized polymorphism, c.1987A > G (p.Thr633Ala). The novel mutation site was inherited in an autosomal dominant manner and was predicted by in silico tools to exert a damaging effect. Alterations in the SCNN1A domain were also predicted by protein structure modeling. After six months of follow-up, treatment had significantly improved the patient’s limb weakness and electrolyte levels. The novel mutation c.1130T > G of the SCNN1A gene was detected in the pedigree with LS. The clinical manifestations of the pedigree were described, which expand the phenotypic spectrum of LS. This result of this study also emphasizes the value of genetic testing for diagnosing LS.

P06.01.A Deconvolution of immunotherapy-treated glioblastoma identifies cellular heterogeneity and plasticity at the single-cell level

Abstract Background Glioblastoma is the most aggressive cancer originating in the brain with an average survival of 15 months. One of the characteristics of glioblastoma is the high level of intra-tumour heterogeneity (ITH), but the composition and complexity at the single-cell level is poorly understood. Here, we aimed to assess the effects and consequences of immune checkpoint inhibitor (ICI) on the cellular and molecular heterogeneity of glioblastoma tumours using at the single cell level. Material and Methods In collaboration with the phase I trials unit at Rigshospitalet, we performed paired molecular analysis of glioma cells from primary and relapse surgery after ICI treatment. Samples were analysed using single-cell RNA sequencing (scRNA-seq) as well as bulk RNA sequencing and whole exome DNA sequencing. Results In an effort to trace cellular lineages we developed and refined methods to a identify copy number changes using scRNA-seq. To this end, we identified clonal and subclonal tumour cell populations in each sample. We found high levels of ITH prior to treatment, both with respect to the glioblastoma subtype enrichment and the cell type-specific gene expression. Using expression-based cell-type classification, we found defined recurrent cell-type populations present at both surgery time points. The immune checkpoint treatment had consequences on the cellular phenotypes and proportions of tumour cells, suggesting a level of plasticity in the neoplastic cells. Moreover, we identified examples of clonal dynamics and sweeps following ICI treatment, pointing to potential treatment response and resistance in these population. Conclusion In summary, we pursued single cell-focused analysis of ICI treated glioblastoma patients to study the cellular and molecular heterogeneity within and between glioblastoma patients, which pointed to recurrent patterns of cellular responses following ICI treatment.

P05.05.B A new IDH-wildtype glioma subtype characterized by highly diffuse growth pattern, distinct epigenetic profile and relatively favorable prognosis

Abstract Background DNA methylation profiling has emerges as a powerful approach to CNS tumor classification and the discovery of novel, molecularly distinct entities. With the release of the 12.5 version of the Heidelberg Brain Tumor Classifier, some unclassifiable cases can be assigned to novel methylation classes. We retrospectively reviewed our databases and identified 16 previously unclassifiable cases, all of which belong to the provisional methylation class “adult-type diffuse high-grade glioma, IDH-wildtype, subtype F (HGG_F)”. Material and Methods We clinically, radiologically and morphologically characterized 16 HGG_F cases and compared them to 347 glioblastomas. We additionally analyzed copy-number alterations and performed DNA exome sequencing. Results Median age at diagnosis of the 12 males and 4 females was 65 years. Upon initial diagnostic workup, specimens were classified as CNS tissue with reactive changes (n=3) or suspicious for the infiltration zone of a diffuse glioma (n = 13). None of the cases demonstrated endothelial proliferation or necrosis and 10/16 tumors had flat copy number profiles. Radiological characteristics were reminiscent of gliomatosis cerebri in eight cases and 9/9 cases had normal FET-PET scans. Whole-exome sequencing revealed genetic alterations frequently found in IDH-wildtype glioblastomas, including TERT promoter mutations in 11/14 (78.6%) and PIK3 mutations (10/14, 71.4%). Outcome was significantly better compared to TCGA IDH-wildtype glioblastomas with a median progression-free survival of 58 months and overall survival of 73 months (both p<0.001). Conclusion We provide evidence that TERT promoter mutations in diffusely infiltrating gliomas without further morphological or molecular signs of high-grade glioma should be interpreted in the context of the clinico-radiological presentation as well as epigenetic prolife and may not be suitable as standalone diagnostic marker for glioblastoma, IDH wildtype.

Genetic and clinical phenotypic analysis of carney complex with external auditory canal myxoma.

Background: Mutations in PRKAR1A gene can lead to Carney complex (CNC), and most CNC patients develop cardiac and cutaneous myxomas. In particular, cardiac myxomas are a common cause of mortality in CNC patients. Cutaneous myxomas of the external ear are extremely rare, and do not have any specific clinical features Methods: In this retrospective study, we analyzed the clinical and genetic data of the proband and his family and fifty whole blood control samples selected from the molecular genetic database of our hospital. Whole exome DNA sequencing analysis was used to detect the mutation in the peripheral blood samples. Results: The results of the clinical analysis showed the presence of spotty skin pigmentation and external auditory canal myxoma in the proband as well as in his sister and mother. Whole-exome DNA sequencing showed a novel heterozygous mutation in the PRKAR1A gene i.e., c.824_825delAG (p.Gln275Leufs*2), in the proband and his sister and mother. Conclusion: In conclusion, the family members had the same autosomal dominant PRKAR1A mutation. DNA sequencing revealed a novel c.824_825delAG in exon 9 of PRKAR1A. This pathogenic mutation has not been reported previously, and may be related to the occurrence of external auditory canal myxomas and spotty pigmentation. This study broadens the genotypic spectrum of PRKAR1A mutations in CNC.

A Second Case With the V374A KCND3 Pathogenic Variant in an Italian Patient With Early-Onset Spinocerebellar Ataxia.

Background and ObjectivesTo date, approximately 20 heterozygous mainly loss-of-function variants in KCND3 have been associated with spinocerebellar ataxia (SCA) type 19 and 22, a clinically heterogeneous group of neurodegenerative disorders. We aimed at reporting the second patients with the V374A KCND3 mutation from an independent family, confirming its pathogenic role.MethodsWe describe the clinical history of a patient with SCA and conducted genetic investigations including mitochondrial DNA analysis and exome sequencing.ResultsThis male patient was reported to have unstable gait with tremors at the lower limbs and dysarthric speech since childhood. A neurologic examination also showed dysarthria, nystagmus, action tremor, dysmetria, and weak deep tendon reflexes. He had marked cerebellar atrophy at brain MRI, more evident at vermis. Molecular analysis, including exome sequencing and an in silico panel analysis of genes associated with SCA, revealed the c.1121T>C [p.V374A] mutation in KCND3.DiscussionThis report consolidates the pathogenicity of the V374A KCND3 mutation and suggests that the ataxic paroxysmal exacerbations are not a key phenotypic feature of this mutation.

Genetics of testicular cancer: a review.

Testicular germ cell tumours (TGCTs) are the most common solid malignant cancer diagnosed in young males and the incidence is increasing. Understanding the genetic basis of this disease will help us to navigate the challenges of early detection, diagnosis, treatment, surveillance, and long-term outcomes for patients. TGCTs are highly heritable. Current understanding of germline risk includes the identification of one moderate-penetrance predisposition gene, checkpoint kinase 2 ( CHEK2 ), and 78 low-to-moderate-risk single nucleotide polymorphisms identified in genome-wide-associated studies, which account for 44% of familial risk. Biomarker research in TGCTs has been challenging for multiple reasons: oncogenesis is complex, actionable mutations are uncommon, clonal evolution unpredictable and tumours can be histologically and molecularly heterogeneous. Three somatic mutations have thus far been identified by DNA exome sequencing, exclusively in seminomas: KIT, KRAS and NRAS . Several genetic markers appear to be associated with risk of TGCT and treatment resistance. TP53 mutations appear to be associated with platinum resistance. MicroRNA expression may be a useful biomarker of residual disease and relapse in future. The biology of testicular germ cells tumours is complex, and further research is needed to fully explain the high heritability of these cancers, as well as the molecular signatures which may drive their biological behaviour.

Oocyte maturation arrest due to compound heterozygous variants of the PATL2 gene in a case

To explore the genetic etiology of a patient with primary infertility and repeated failure of assisted reproductive technology. Peripheral blood samples of the patient and her husband were collected for the extraction of genomic DNA and clinical exome sequencing. Candidate variants were verified by Sanger sequencing. The patient was found to harbor compound heterozygous variants of the PATL2 gene, namely c.223-14_223-2del and c.1369G>T (p.G457*). Sanger sequencing has verified that they were respectively inherited from her father and mother. The patient was diagnosed with oocyte maturation defect type 4. Oocyte maturation arrest due to mutations of the PATL2 gene can result in primary female infertility. Discovery of the novel c.1369G>T (p.G457*) variant has expanded the spectrum of pathogenic variants of the PATL2 gene.

Functional genomic analysis of epithelioid sarcoma reveals distinct proximal and distal subtype biology.

BackgroundMetastatic epithelioid sarcoma (EPS) remains a largely unmet clinical need in children, adolescents and young adults despite the advent of EZH2 inhibitor tazemetostat.MethodsIn order to realise consistently effective drug therapies, a functional genomics approach was used to identify key signalling pathway vulnerabilities in a spectrum of EPS patient samples. EPS biopsies/surgical resections and cell lines were studied by next‐generation DNA exome and RNA deep sequencing, then EPS cell cultures were tested against a panel of chemical probes to discover signalling pathway targets with the most significant contributions to EPS tumour cell maintenance.ResultsOther biologically inspired functional interrogations of EPS cultures using gene knockdown or chemical probes demonstrated only limited to modest efficacy in vitro. However, our molecular studies uncovered distinguishing features (including retained dysfunctional SMARCB1 expression and elevated GLI3, FYN and CXCL12 expression) of distal, paediatric/young adult‐associated EPS versus proximal, adult‐associated EPS.ConclusionsOverall results highlight the complexity of the disease and a limited chemical space for therapeutic advancement. However, subtle differences between the two EPS subtypes highlight the biological disparities between younger and older EPS patients and emphasise the need to approach the two subtypes as molecularly and clinically distinct diseases.

Abstract 2945: Comprehensive analysis of tissue and plasma-related genetic alterations in Hungarian colorectal cancer patients

Abstract Background: Analysis of circulating cell-free DNA (cfDNA) of colorectal cancer (CRC) patients provides an ideal approach to explore genetic alterations in a minimally invasive way. Aims: We aimed to perform a comprehensive analysis of tissue-originated genomic DNA and plasma-derived cfDNA from CRC patients by whole-exome and targeted panel sequencing. Materials & methods: DNA was isolated from tissue and plasma samples of 55 [7 healthy (N), 16 adenomas (AD), and 32 CRC] patients using the High Pure PCR Template Kit (Roche) and the Quick cfDNA Serum & Plasma Kit (Zymo). cfDNA quality was assessed with the High Sensitivity DNA kit on BioAnalyzer 2100 and quantified by the Qubit dsDNA HS assay. Exome libraries from tissue samples were constructed by the Nextera DNA Exome Kit (Illumina). For the cfDNA samples, we used the QIAseq cfDNA All-in-One kit combined with the QIASeq Human Exome Kit, and in the case of 12 patients, CRC-specific oncogenes were further analyzed with a solution developed by QIAGEN for reliable calling of low frequency variants in cfDNA samples. Exome libraries were quantified with the KAPA Library Quantification Kit and the QIAseq™ Library Quant Assay Kit (Qiagen) and were sequenced using the NextSeq 500/550 High Output v2 kit on a NextSeq 500 Instrument (Illumina). Raw data analysis and demultiplexing were completed on the BaseSpace Sequence Hub. Variants of the tissue and cfDNA samples were determined by the Mutect2 and Haplotype caller algorithms of GATK 4.1.4.1, respectively. Clinical significance was evaluated according to the OncoKB database. Results: The most frequently mutated genes were APC, KRAS, DYNC1H1, KCNO5, and MARCH6 in the colorectal adenoma tissue samples, while those in CRC samples were APC, TP53, TTN, KRAS, and DYNCC2H1. CfDNA quantity was significantly higher in the CRC group compared to the AD (p<0.02) and N (p<0.005) patients. Based on the plasma exome results, 4.4-59.4% of the tumor somatic variants could be found in 12.5% of the patients. These patients were above 60 years of age and had Dukes D stage CRC. Panel sequencing of the circulating DNA samples detected tumor somatic variants in 8 out of the 12 enrolled patients. This method could identify 60% of all tumor somatic variants falling on its targeted regions, while whole-exome sequencing recovered only 20% of tumor somatic variants in the respective regions in cfDNA of the same patients. Conclusion: We have performed a comprehensive genetic analysis on CRC tissue and cfDNA samples in the Hungarian population. Exome sequencing offers a broad overview of the coding regions, however, targeted panel sequencing with a higher coverage depth can detect tumor somatic variants more reliably in cfDNA, therefore, it can hold a relevant clinical potential. Citation Format: Alexandra Kalmar, Gitta Szabo, Orsolya Galamb, Barbara Kinga Bartak, Zsofia Brigitta Nagy, Sara Zsigrai, Krisztina Andrea Szigeti, William Kothalawala, Peter Igaz, Istvan Takacs, Bela Molnar. Comprehensive analysis of tissue and plasma-related genetic alterations in Hungarian colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2945.

Genome-wide DNA methylation profiling and exome sequencing resolved a long-time misdiagnosed case.

The search for aetiology of Mendelian disorders is traditionally based on the observation of clinical phenotypes and molecular screening of associated genes. However, a disease-specific diagnosis can be challenging. In this study we detail how the combinatorial genomic and epigenomic assessment allowed to find the underlying molecular event of a clinical case that remained misdiagnosed for years. The individual was referred as affected by an atypical form of Kabuki syndrome with a variant of uncertain significance in the KMT2D gene. However, significant inconsistencies with this diagnosis emerged upon familial segregation of the variant and after the clinical re-evaluation. Therefore, we applied an epigenomic strategy by studying the DNA methylation profile which resulted not consistent with the Kabuki syndrome episignature or with any other disorder-specific episignature described so far, providing strong evidence that the Kabuki syndrome diagnosis does not apply. This result led us to further investigate for epigenetic machinery diseases by using a multigene panel for chromatinopathies. Since this analysis yielded negative results, we applied a whole exome sequencing and identified a de novo pathogenic variant in the CTNNB1 gene associated to NEDSDV syndrome, a neurodevelopmental disorder characterized by intellectual disability and craniofacial anomalies. Based on molecular results and the updated clinical features, we confirmed the NEDSDV diagnosis. Our findings show that the combination of genomic and epigenomics strategies, along with a deeper analysis of clinical phenotype, may provide a significant improvement in the diagnostic protocols for rare genetic disorders and help resolve long-time misdiagnosed and unsolved case.

A CTNNB1-altered medulloblastoma shows the immunophenotypic, DNA methylation and transcriptomic profiles of SHH-activated, and not WNT-activated, medulloblastoma.

Recent advancements in molecular characterisation have identified four principal molecular groups of medulloblastoma: WNT, SHH, group 3 and group 4. Each has its characteristic clinical features, signature genetic alterations and distinct DNA methylome profiles. Thus far, CTNNB1 mutations have been considered pathognomonic of WNT-activated medulloblastoma. Furthermore, it has been shown that CTNNB1 mutations dominantly drive the WNT-activated phenotype in medulloblastoma, even in the presence of alterations in the SHH pathway. We herein report an illustrative case that challenges this belief-a medulloblastoma with a pathogenic CTNNB1 mutation that otherwise showed the histopathology, immunophenotype and methylation and transcriptomic profiles of an SHH-activated medulloblastoma. Detailed molecular analyses, including whole exome sequencing, transcriptome analysis and DNA methylation profiling with DKFZ brain tumour classifier and St. Jude MLPnet neural network classifier analyses, have been performed on the tumour. Our example emphasises the diagnostic value of the immunohistochemistry panel with YAP1, GAB1 and β-catenin and DNA methylation profiling, combined with exome sequencing, in the characterisation of medulloblastoma. CTNNB1 mutations are not specific for WNT-activated medulloblastoma, and different CTNNB1 mutations have diverse oncogenic potential.

Abstract P1-05-01: The tumor immune microenvironment and HER2 landscape of high-risk ductal carcinoma in situ: The DEFENSE study

Abstract Introduction: Ductal carcinoma in situ (DCIS) of the breast is a premalignant lesion representing a spectrum of biology from indolent to aggressive. A minority of women present with clinically high-risk features associated with poor outcome. Yet even in patients with biologically aggressive DCIS, the risk of breast cancer mortality is only 3.3% compared to 30-40% in patients with biologically aggressive invasive cancer of the same size.1 We hypothesize that the tumor immune microenvironment could play a proactive role in preventing invasion in high grade clinically high-risk DCIS and that HER2 status, including specific HER2 isoform expression and post-translational modification of HER2, could impact progression. Methods: DCIS: Elaboration of Factors from Enlarged lesions that Nevertheless remain Stage 0 Entities (DEFENSE) is a study of high-risk DCIS, defined as having at least two of the following characteristics: large (>5cm), high grade, hormone receptor-negative status and/or HER2+ status. Slides obtained from FFPE tissue blocks were stained with fluorescence-based multiplex immunohistochemistry (mIHC) panels and imaged to characterize immune infiltrate within the ducts and the stromal compartments. mIHC was also used to detect extracellular and intracellular domains of HER2 with imaging analysis performed to identify HER2 isoforms of HER2+ specimens. Isoforms were characterized as 1) full-length/extracellular domain (ECD) intact, 2) pure/complete loss of ECD (p95 isoform), 3) subclonal populations of both full-length ECD and p95 and 4) gradient/representing partial loss of ECD per cell (reflecting post-translational cleavage). Clinical characteristics were correlated with molecular profile, tumor immune infiltrates, and HER2 isoform. Finally, expression profiling with a 44k array was conducted by Agendia, and MammaPrint and BluePrint results were generated. Results: Of 92 total patients, median age is 46 years. The average DCIS lesion size is 8.2cm, and 33% are hormone receptor negative (HR-). Based upon initial analysis, mIHC demonstrates significant heterogeneity in immune infiltrate populations of pathologically identical DCIS specimens and within regions of the same specimen. High-grade HR- disease has highly reactive stroma, characterized by dense CD3+, CD34+, and CD68+ infiltrate within the stromal compartment. HER2 testing of the first 51 cases demonstrates a high rate of positivity of 67% (34/51). Of those tested for HER2 isoform expression (n=21), none had homogeneous intact full-length HER2. Six (29%) demonstrate the pure p95 isoform, 3 (14%) demonstrate the subclonal isoform, and 12 (57%) demonstrate a gradient HER2 isoform phenotype. Preliminary data from expression profiling shows that the HER2+ cases are also HER2 intrinsic sub-type by BluePrint. Across all of the high-risk DCIS cases, all were scored as MammaPrint high risk, either Luminal B or HER2-type, with only 1 basal and no Luminal A. Additional analyses are ongoing, including completion of testing for the whole data set as well whole exome DNA sequencing and SMART-3SEQ RNA sequencing. Conclusions: Clinically high-risk and pathologically homogenous DCIS lesions demonstrate significant immune infiltrate heterogeneity. Nearly 70% of these large clinically high-risk DCIS lesions are HER2+ with HER2 isoforms most commonly representing either partial or complete loss of the HER2 ECD. This is significantly higher than what is reported for invasive HER2+ breast cancer. A comparison to size and molecularly matched invasive cancers, including from the I-SPY 2 trial, is underway in an effort to elucidate how molecularly aggressive lesions remain in situ despite their large size.

TB-2 Patient-derived meningioma organoid model demonstrates FOXM1 dependent tumor proliferation

Recent comprehensive studies have revealed several molecular alterations that are frequently found in meningiomas. However, effective treatment reagents targeting specific molecular alterations have not yet been identified because of the limited number of representative research models of meningiomas.We established 18 organoid models comprising of two malignant meningioma cells (HKBMM and IOMM-Lee), 10 benign meningiomas, four malignant meningiomas, and two solitary fibrous tumors (SFTs). Using immunohistochemistry and molecular analyses consisting of whole exome sequencing, RNA-seq, and DNA methylation analyses, we compared the histological findings and molecular profiling of organoid models with those of parental tumors. The organoids exhibited consistent histological features and molecular profiles with those of the parental tumors. Using a public database of meningioma, we identified that upregulated forkhead box M1 (FOXM1) was correlated with increased tumor proliferation. Overexpression of FOXM1 in benign meningioma organoids increased organoid proliferation; depletion of FOXM1 in malignant organoids decreased proliferation. Additionally, thiostrepton, a FOXM1 inhibitor combined with radiation therapy, significantly inhibited proliferation of malignant meningioma organoid models (P<0.01).An organoid model for meningioma enabled us to elucidate the tumor biology of meningioma along with potent treatment targets for meningioma.

Integrated Genomic Analysis Identifies ANKRD36 Gene as a Novel and Common Biomarker of Disease Progression in Chronic Myeloid Leukemia.

Simple SummaryChronic myeloid leukemia is a type of blood cancer that is regarded as a success story in determining the exact biological origin, pathogenesis and development of a molecularly targeted (mutation-specific) therapy that has led to successful treatment of this fatal cancer. It is caused by the BCR-ABL fusion gene, which is formed from the translocation between chromosomes 9 and 22. Anti-BCR-ABL drugs, known as tyrosine kinase inhibitors (TKIs), have led to long-term remissions in more than 80% of CML patients and even cure in about one-third of patients. Nevertheless, many patients face drug resistance, and disease progression occurs in about 30% of CML patients, leading to morbidities and mortality. Unfortunately, no biomarkers of CML progression are available due to a poor understanding of the mechanism of progression. Therefore, finding reliable molecular biomarkers of CML progression is one of the most attractive research areas in 21st-century cancer research. In this study, we report novel genomic variants exclusively found in all our advanced-phase CML patients. This study will help in identifying CML patients at risk of disease progression and timely therapeutic interventions to avoid or at least delay fatal disease progression in this cancer.Background: Chronic myeloid leukemia (CML) is initiated in bone marrow due to chromosomal translocation t(9;22) leading to fusion oncogene BCR-ABL. Targeting BCR-ABL by tyrosine kinase inhibitors (TKIs) has changed fatal CML into an almost curable disease. Despite that, TKIs lose their effectiveness due to disease progression. Unfortunately, the mechanism of CML progression is poorly understood and common biomarkers for CML progression are unavailable. This study was conducted to find novel biomarkers of CML progression by employing whole-exome sequencing (WES). Materials and Methods: WES of accelerated phase (AP) and blast crisis (BC) CML patients was carried out, with chronic-phase CML (CP-CML) patients as control. After DNA library preparation and exome enrichment, clustering and sequencing were carried out using Illumina platforms. Statistical analysis was carried out using SAS/STAT software version 9.4, and R package was employed to find mutations shared exclusively by all AP-/BC-CML patients. Confirmation of mutations was carried out using Sanger sequencing and protein structure modeling using I-TASSER followed by mutant generation and visualization using PyMOL. Results: Three novel genes (ANKRD36, ANKRD36B and PRSS3) were mutated exclusively in all AP-/BC-CML patients. Only ANKRD36 gene mutations (c.1183_1184 delGC and c.1187_1185 dupTT) were confirmed by Sanger sequencing. Protein modeling studies showed that mutations induce structural changes in ANKRD36 protein. Conclusions: Our studies show that ANKRD36 is a potential common biomarker and drug target of early CML progression. ANKRD36 is yet uncharacterized in humans. It has the highest expression in bone marrow, specifically myeloid cells. We recommend carrying out further studies to explore the role of ANKRD36 in the biology and progression of CML.

BIOM-40. TARGETED GENE EXPRESSION PROFILING PREDICTS MENINGIOMA OUTCOMES AND RADIOTHERAPY RESPONSES

Abstract BACKGROUND Surgery is the mainstay of meningioma treatment, but improvements in meningioma risk stratification are needed and indications for postoperative radiotherapy are controversial. DNA methylation profiling, copy number variants (CNVs), exome sequencing, and RNA sequencing have improved understanding of meningioma biology, but have not superseded histologic grading, or revealed biomarkers for radiotherapy responses. To address these unmet needs, we optimized and validated a targeted gene expression biomarker predicting meningioma outcomes and responses to radiotherapy. METHODS Targeted gene expression profiling was performed on a discovery cohort of 173 meningiomas (median follow-up 8.1 years) and a validation cohort of 331 meningiomas (median follow-up 6.1 years) treated with surgery (n=504) and postoperative radiotherapy (n=73) at independent, international institutions (70% WHO grade 1, 24% WHO grade 2, 6% WHO grade 3). Optimized targeted gene expression models predicting clinical outcomes (34 genes) or radiotherapy responses (12 genes) were developed from the discovery cohort, and compared to histologic and molecular classification systems by performing DNA methylation profiling, CNV analysis, exome sequencing, and RNA sequencing on the same meningiomas. RESULTS Targeted gene expression profiling achieved a concordance-index of 0.75 ± 0.03 (SEM) for local freedom from recurrence (LFFR) and 0.72 ± 0.03 for overall survival (OS) in the validation cohort, outperforming WHO grade (5-year LFFR delta-AUC 0.15, 95% CI 0.076-0.229, p=0.001) and DNA methylation grouping (delta-AUC 0.075, 95% CI 0.006-0.130, p=0.01) for LFFR, disease-specific survival, and OS. The biomarker was independently prognostic after accounting for WHO grade, extent of resection, primary versus recurrent presentation, CNV status, DNA methylation group, and Ki67 labeling index, and identified meningiomas benefiting from radiotherapy (interaction p-value=0.0008), suggesting postoperative radiotherapy could be refined in 30.2% of cases. CONCLUSIONS Targeted gene expression profiling of 504 meningiomas improves discrimination of meningioma local recurrence, disease-specific survival, and overall survival, and predicts radiotherapy responses.

TAMI-68. DECONVOLUTION OF IMMUNOTHERAPY-TREATED GLIOBLASTOMA IDENTIFIES CELLULAR HETEROGENEITY AND PLASTICITY AT THE SINGLE-CELL LEVEL

Abstract Glioblastoma is the most aggressive cancer originating in the brain with an average survival of 15 months. One of the characteristics of glioblastoma is the high level of intra-tumor heterogeneity (ITH), but the composition and complexity at the single-cell level is poorly understood. Here, we aimed to assess the effects and consequences of immune checkpoint inhibitor (ICI) on the cellular and molecular heterogeneity of glioblastoma tumors at the single cell level. In collaboration with the phase I trials unit at Rigshospitalet, we performed paired molecular analysis of glioma cells from primary and relapse surgery after ICI treatment. Samples were analyzed using single-cell RNA sequencing (scRNA-seq) as well as bulk RNA sequencing and whole exome DNA sequencing. In an effort to trace cellular lineages we developed and refined methods to a identify copy number changes using scRNA-seq. To this end, we identified clonal and subclonal tumor cell populations in each sample. We found high levels of ITH prior to treatment, both with respect to the glioblastoma subtype enrichment and the cell type-specific gene expression. Using expression-based cell-type classification, we found defined recurrent cell-type populations present at both surgery time points. The immune checkpoint treatment had consequences on the cellular phenotypes and proportions of tumor cells, suggesting a level of plasticity in the neoplastic cells. Moreover, we identified examples of clonal dynamics and sweeps following ICI treatment, pointing to potential treatment response and resistance in these populations. We additionally found a recurrent pattern of a small population showing high levels of cell cycle activation and simultaneously expressing markers of stem cell potential. In summary, we pursued single cell-focused analysis of ICI treated glioblastoma patients to study the cellular and molecular heterogeneity within and between glioblastoma patients, which pointed to recurrent patterns of cellular responses following ICI treatment.

Glioblastoma signature in the DNA of blood-derived cells.

Current approach for the detection of cancer is based on identifying genetic mutations typical to tumor cells. This approach is effective only when cancer has already emerged, however, it might be in a stage too advanced for effective treatment. Cancer is caused by the continuous accumulation of mutations; is it possible to measure the time-dependent information of mutation accumulation and predict the emergence of cancer? We hypothesize that the mutation history derived from the tandem repeat regions in blood-derived DNA carries information about the accumulation of the cancer driver mutations in other tissues. To validate our hypothesis, we computed the mutation histories from the tandem repeat regions in blood-derived exomic DNA of 3874 TCGA patients with different cancer types and found a statistically significant signal with specificity ranging from 66% to 93% differentiating Glioblastoma patients from other cancer patients. Our approach and findings offer a new direction for future cancer prediction and early cancer detection based on information derived from blood-derived DNA.

MED12 Mutation in Two Families with X-Linked Ohdo Syndrome.

X-linked intellectual deficiency (XLID) is a widely heterogeneous group of genetic disorders that involves more than 100 genes. The mediator of RNA polymerase II subunit 12 (MED12) is involved in the regulation of the majority of RNA polymerase II-dependent genes and has been shown to cause several forms of XLID, including Opitz-Kaveggia syndrome also known as FG syndrome (MIM #305450), Lujan-Fryns syndrome (MIM #309520) and the X-linked Ohdo syndrome (MIM #300895). Here, we report on two first cousins with X-linked Ohdo syndrome with a missense mutation in MED12 gene, identified through whole exome sequencing. The probands had facial features typical of X-linked Ohdo syndrome, including blepharophimosis, ptosis, a round face with a characteristic nose and a narrow mouth. Nextera DNA Exome kit (Illumina Inc., San Diego, CA, USA) was used for exome capture. The variant identified was a c.887G > A substitution in exon 7 of the MED12 gene leading to the substitution of a glutamine for a highly conserved arginine (p. Arg296Gln). Although the variant described has been previously reported in the literature, our study contributes to the expanding phenotypic spectrum of MED12-related disorders and above all, it demonstrates the phenotypic variability among different affected patients despite harboring identical mutations.

Multi-site tumor sampling highlights molecular intra-tumor heterogeneity in malignant pleural mesothelioma

BackgroundMalignant pleural mesothelioma (MPM) is a heterogeneous cancer. Better knowledge of molecular and cellular intra-tumor heterogeneity throughout the thoracic cavity is required to develop efficient therapies. This study focuses on molecular intra-tumor heterogeneity using the largest series to date in MPM and is the first to report on the multi-omics profiling of a substantial series of multi-site tumor samples.MethodsIntra-tumor heterogeneity was investigated in 16 patients from whom biopsies were taken at distinct anatomical sites. The paired biopsies collected from apex, side wall, costo-diaphragmatic, or highest metabolic sites as well as 5 derived cell lines were screened using targeted sequencing. Whole exome sequencing, RNA sequencing, and DNA methylation were performed on a subset of the cohort for deep characterization. Molecular classification, recently defined histo-molecular gradients, and cell populations of the tumor microenvironment were assessed.ResultsSequencing analysis identified heterogeneous variants notably in NF2, a key tumor suppressor gene of mesothelial carcinogenesis. Subclonal tumor populations were shared among paired biopsies, suggesting a polyclonal dissemination of the tumor. Transcriptome analysis highlighted dysregulation of cell adhesion and extracellular matrix pathways, linked to changes in histo-molecular gradient proportions between anatomic sites. Methylome analysis revealed the contribution of epigenetic mechanisms in two patients. Finally, significant changes in the expression of immune mediators and genes related to immunological synapse, as well as differential infiltration of immune populations in the tumor environment, were observed and led to a switch from a hot to a cold immune profile in three patients.ConclusionsThis comprehensive analysis reveals patient-dependent spatial intra-tumor heterogeneity at the genetic, transcriptomic, and epigenetic levels and in the immune landscape of the tumor microenvironment. Results support the need for multi-sampling for the implementation of molecular-based precision medicine.