NGS Panel Research Articles

Identification of Predictive Models Including Polymorphisms in Cytokines Genes Associated with Post-Transplant Complications after Identical HLA-Allogeneic Stem Cell Transplantation

Introduction Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative treatment for patients with hematologic malignances. Immune system genes like cytokines play a role in inflammatory process that occur during graft versus host disease (GVHD). Therefore, the analysis of polymorphisms that affect the expression or activity of these genes could be used as biomarkers to predict the development of complications after allo-HSCT. The aim of this study was to identify new polymorphisms in genes related to the immune response, mainly cytokines, that could be related to the development of post-transplant complications. Methods We retrospectively selected 90 patients with hematological malignancies who received an allo-HSCT from an HLA-identical sibling donor from 2000 to 2015. The genotyping was performed using a custom NGS panel (73 interleukins and 59 chemokines genes) in a HiSeq platform (Illumina, USA). The bioinformatics analysis was carried out with the GeneSystems software (Sistemas Genómicos, Spain). We analyzed variants located in coding regions, splicing sites, UTR, and 5’ upstream and 3'downstream regions (+/-200pb). The polymorphisms selected corresponding to non-synonymous variants, with depth ≥30X in the canonical isoform with an allele frequency ≥0.4, with a minor allele frequency ≥1% and represented in at least 5% in our cohort. Bayesian logistic regression models (BLR) were used to select the most relevant variables for each post-transplant complication. The models with the highest AUC value, sensitivity and specificity value and the lowest number of genetic variants used were selected. Finally, the score obtained from the chosen predictive model for each complication was used to classify patients as low or high risk according to the selected cutoff point. Results The cumulative incidence (CI) rates for aGVHD of grades II-IV and III-IV at 100 days after transplantation were 56.7% and 23.3%, respectively. The CI for cGVHD and moderate-severe cGVHD was 43.3% and 27.8%, respectively, at 3 years after allo-HSCT. The CI of TRM at 3 years was 24.4% and OS at 5 years was 30%. Using filters defined previously, we detected 737 polymorphisms (644 in non-coding regions and 93 in coding regions). We developed BLR models for II-IV and III-IV aGVHD, cGVHD, moderate-severe cGVHD, TRM, relapse and OS (Table 1). Stratification of the cohort of patients according to the risk of develop each complication is showed in Figure 1. Most polymorphisms found are located in regulatory regions, which until have been little studied, but these regions are involved in the expression genes, therefore could be affecting the function of these genes. Conclusions We have identified new genetic polymorphisms correlate with the risk of developing complications after allo-HSCT from an HLA-identical sibling donor. Based on these data, we have developed a genetic score that encompasses polymorphisms of greater relevance. In this way, patients at greatest risk for developing post-transplant complication who could benefit from personalized management through immunosuppression and other drugs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Evolutionary Portrait of Adult Core-Binding Factor Leukemia Patients Treated with a Continuation Therapy with Midostaurin: Preliminary Results

Genetic and Clinical Background The clinical outcome of Core Binding Factor Leukemia (CBFL) has been reported to be influenced by KIT mutations, that was frequent in CBFL, and by co-occurring different spectra of cooperating mutations. In addition, gene expression studies found KIT to be highly expressed in CBFL regardless of its mutational status. In vitro experiments revealed that Midostaurin (Mido) is effective in inhibiting both wild type (WT) and a range of KIT mutants. In vivo studies demonstrate the efficacy in KIT-positive malignancies such as Aggressive Systemic Mastocytosis, Mast Cell Leukemia, and SM with Associated Hematological Neoplasm. Therefore, we designed a Phase II trial to evaluate the safety and efficacy of Mido, in association with Intensive Chemotherapy (IC), in CBFL regardless of KIT mutational status. (EudraCT Number 2017-002094-18; ClinicalTrials ID: NCT 03686345). 34 adult patients with t(8;21) (n=16) and inv(16) (n=18) were enrolled between December 2018 to July 2022. Method Patients with de-novo CBFL received standard induction therapy with an anthracycline containing regimen ("7+3"-like) + Mido, three cycles of post-remission consolidation chemotherapy with high-dose cytarabine + Mido, and 12 months of Mido as Maintenance. At diagnosis, 32 out of 34 patients were studied by a comprehensive NGS panel targeting 40 DNA genes and 29 RNA fusion driver genes associated with major myeloid disorders (Oncomine Myeloid Research Assay). Aim To study the clonal evolution of CBFLs, NGS and MRD status (performed by qPCR and high-resolution multicolor flow cytometry) were assessed at established check-points during consolidation, maintenance therapy, and at relapse. ResultsKIT was the most frequently mutated gene (28%) and there was an enrichment of FLT3 and NRAS (Figure 1). Additionally, the spectrum of recurrent mutations was different, as codon 816 KIT mutations and FLT3-ITD were common only in RUNX1::RUNX1T1 cases. Overall, the CR rate was 97%. At a median follow-up of 18 months (range 2-43 months), we recorded a RI of 37.5 %, an OS of 85 %, and a DFS of 60%. We performed targeted next generation sequencing for six out of 12 relapsed samples in the cohort t(8;21) (n=5) and inv(16) (n=1), showing loss, retention or gain of somatic mutations (Figure 2). Three out of six relapsed patients showed at diagnosis a CEBPA mutation (Gly104del) co-occurring with KIT in RUNX1::RUNX1T1 cases and with NRAS in CBFB::MYH11. Conclusion In patients with CBFL, we found FLT3 and/or KIT mutations actionable by a regimen consisting of intensive chemotherapy and Mido in 47% of cases. In two out of three relapsed cases the KIT mutation is maintained, although frequency of the mutant allele is lower at relapse, while all FLT3 and NRAS mutated patients lose the mutation upon disease relapse. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

BIOM-51. ELUCIDATION OF THE TRANSCRIPTOMIC CONSEQUENCES OF KLF4 AND TRAF7 MUTATIONS IN SECRETORY MENINGIOMAS

Abstract Secretory meningioma (SM) is a benign histological meningioma subtype frequently located in the central skull-base, complicating surgical removal, highlighting the need of gentle targeting treatment alternatives. SM tumors arise from the arachnoid layer in the meninges and are associated with a hotspot p.K409Q mutation in KLF4 combined with different mutations in TRAF7. The objective of this study was to identify how KLF4 and TRAF7 mutations alter signaling in SM tumors.The study includes 16 FFPE samples from SM tumors and three arachnoid cysts as normal-tissue reference. All tissues were histologically confirmed, followed by genomic sequencing of KLF4, TRAF7, NF2 and TERT-promoter (C220T/C228T) using a customized NGS panel. Gene expression profiles were analyzed using NanoString Pan-cancer Progression and Pan-cancer Pathways panels, which resulted in measurement of 1,202 unique transcripts over background level.All 16 SM tumors presented the KLF4p.K409Q mutation, of which 12 had mutation in TRAF7 (four being p.N520S), while no cysts had mutations in these genes. Neither tumors nor cysts had NF2 or TERT-promoter mutations. PCA analysis showed different RNA expression profiles for SM tumors and cysts, while TRAF7 mutation was without influence on the overall gene expression patterns in the tumors. Sub-analysis found increased KLF4 expression in TRAF7-mutated SM tumors versus non-mutated tumors and cysts at the RNA level, while TRAF7 expression was stable in all samples. In total 98 genes were found differentially expressed (54 up- and 44 downregulated) with a log2-fold change over 1 and an adjusted p-value under 0.05 in tumors versus cysts. Further analyses are planned to explore and validate these results. In conclusion, we find that the KLF4p.K409Q mutation correlates more strongly with SM histology, than the TRAF7 mutation, and that SM tumors have gene expressions profiles that are distinct from normal arachnoid tissue for a number of disease relevant genes.

PATH-12. DIFFUSE HEMISPHERIC GLIOMA, H3 G34-MUTANT: A COMPREHENSIVE SEQUENCING AND HIGH-RESOLUTION GENOME-WIDE COPY NUMBER ANALYSIS

Abstract Diffuse hemispheric glioma, H3 G34-mutant (DHG_H3G34) is a novel tumor type in the 2021 WHO CNS classification. We describe a comprehensive sequencing and high-resolution genome-wide copy number analysis of a series of cases clinically tested by a single laboratory (2018-2022). Cases included tumors from 47 unique patients (1 reportedly recurrent) that had an H3-3A G34 mutation detected using an 187-gene mutation and fusion targeted neuro-oncology NGS panel (n=47). A subset (n=18) of cases was also tested Oncoscan chromosomal microarray. Median age at testing was 20 years (range, 12-50). H3-3A G34 mutations included G34R (n=44; 94%), G34V (n=2) and a novel G34E variant considered to be likely clinically relevant (n=1). All DHG_H3G34 were hemispheric tumors, and one tumor was multifocal with midline involvement. Concurrent mutations recurrently involved ATRX (n=38), TERT promoter (n=3; mutually exclusive with ATRX mutations), TP53 (n=43), PDGFRA (n=26), PTEN (n=5), NF1 (n=4, all patients over 20 years), PIK3CA (n=4) and CDKN2A (n=3). The single recurrent tumor also had an MSH6 mutation, and in addition to 3 other cases, had increased number of sequence variants suggestive of a high tumor mutational burden. A single case also showed an FGFR3::FAM184B (exon 17::exon 2) fusion. All cases with available chromosomal microarray data had unbalanced genomes with multiple chromosomal gains/losses, and regions of copy-neutral loss of heterozygosity. The most frequent recurrent (at least 50% of cases) abnormalities were losses involving 3q, 4q, 10q, 13q and 18q, and 17p copy-neutral loss of heterozygosity encompassing TP53 (all but one case had concomitant TP53 mutation), a pattern reminiscent of IDH-mutant astrocytomas. Oncogene amplifications (PDGFRA and PIK3CA) were detected in 3 (of 18) cases. CDKN2A/B deletion was observed in 8 cases (2 homozygous). In conclusion, we describe additional sequence and copy-number abnormalities in DHG_H3G34, expanding the spectrum of genetic changes associated with this novel tumor type.

A novel homozygous mutation in TRAPPC9 gene causing autosomal recessive non-syndromic intellectual disability

BackgroundThe etiology of intellectual disabilities is diverse and includes both genetic and environmental factors. The genetic causes of intellectual disabilities range from chromosomal aberrations to single gene disorders. The TRAPPC9 gene has been reported to cause autosomal recessive forms of intellectual disabilities in 56 patients from consanguineous and non-consanguineous families around the world.MethodsWe analyzed two siblings with intellectual disability, microcephaly and delayed motor and speech development from a consanguineous Sudanese family. Genomic DNA was screened for mutations using NGS panel (NextSeq500 Illumina) testing 173 microcephaly associated genes in the Molecular Genetics service in Robert Debre hospital in Paris, France.ResultsA novel homozygous mutation (NM_031466.7 (TRAPPC9):c.2288dup, p. (Val764Glyfs*7) in exon 14 of TRAPPC9 gene was found in the two patients. The mutation was predicted to cause nonsense mediated decay (NSMD) using SIFT prediction tool. The variant has not been found in either gnomAD or Exac databases. Both parents were heterozygous (carriers) to the mutation.ConclusionThis is the first study to report patients with TRAPPC9-related disorder from Sub-Saharan Africa.

Application of NGS molecular classification in the diagnosis of endometrial carcinoma: A supplement to traditional pathological diagnosis.

This study aims to demonstrate the advantages of NGS molecular classification in EC diagnosis and to assess whether molecular classification could be performed on curettage specimens and its concordance with subsequent hysterectomy specimens. 80 patients with hysterectomy specimens and 35/80 patients with paired curettage specimens were stratified as POLE mut, MSI-H, TP53 wt, or TP53 abn group by NGS panel. Histotype, tumor grade, IHC results, and other pathological details were taken from original pathological reports. The correlation analysis of 80 patients with hysterectomy specimens between NGS molecular classification and clinicopathological characters displayed that the POLE mut group was associated with EEC (87.5%) and TP53 abn subtype was correlated to a later stage (Stage II-IV, 47.6%), G3 (76.2%), serous histology (61.9%) and myometrial invasion ≥50% (47.6%). A favorable concordance (31/32, 96.9%) was shown in MSI analysis and MMR IHC results, and the agreement rate of p53 IHC and TP53 mutation was 81.5% (53/65). Compared with the p53 IHC abnormal group, the TP53 mutation group had a higher correlation with high-risk factors. A high level of concordance (31/35, 88.0%) of NGS molecular classification was achieved between curettage specimens and hysterectomy specimens while grade and histotype (including unclassified group) from curettage specimens and hysterectomy specimens showed only moderate levels of agreement, 54.3% (19/35) and 68.6% (24/35), respectively. NGS molecular classification achieved on curettage samples showed high concordance with the final hysterectomy specimens, demonstrating superior to the conventional pathological assessment of grade and histotype and potential utilization in clinical practice.

114. Comparison of optical genome mapping, CMA, and 523-gene NGS panel for Homologous Recombination Deficiency calculation

Homologous Recombination Deficiency (HRD) is characterized by the inability of a cell to repair the double-stranded breaks using the homologous recombination repair (HRR) pathway. The deficiency of the HRR pathway results in defective DNA repair, leading to genomic instability and tumorigenesis. The presence of HRD has been found to make tumors sensitive to ICL-inducing platinum-based therapies and poly(adenosine diphosphate [ADP]?ribose) polymerase (PARP) inhibitors (PARPi). However, there are no standardized methods to define, measure, and report HRD in diagnostic laboratories. Herein, we compare optical genome mapping (OGM), chromosomal microarray (CMA), and 523-gene NGS panel for HRD scar calculations. Traditionally, CMA has been used to calculate either loss of heterozygosity (LOH), or a combination of LOH, telomeric allelic imbalance (TAI), and large scale transitions (LST), where a combination is more accurate than LOH score alone for HRD calculation. We demonstrate the use of a 523-gene NGS panel to detect HRD using NxClinical software with automated HRD calculation using LOH, TAI, and LST. More interestingly, we compare OGM with CMA and NGS panel and highlight the potential advantages of using OGM, which reveals the genomic instability with a much higher resolution compared to other methodologies. For example, in a case of glioblastoma, the HRD score with CMA was 7 (TAI:1, HRD-LOH: 2, LST: 4) compared to 29 with OGM (TAI:1, HRD-LOH:2, LST:25). The HRD definition, measurement, and reporting criteria need to be standardized for each platform, while OGM might be the most sensitive platform for HRD calculation.

Immunophenotypic and molecular characterization of pancreatic neuroendocrine tumors producing serotonin

AbstractSerotonin producing pancreatic neuroendocrine tumors (SP-PanNET) account for 0.58–1.4% of all pancreatic neuroendocrine tumors (PanNET). They may present with atypical symptoms, such as acute pancreatitis and are often radiologically characterized by main pancreatic duct dilatation. SP-PanNET are well differentiated neuroendocrine tumors (NET) distinct from classical PanNET by atypical serotonin secretion and abundant dense stroma deposition, like serotonin producing ileal NET leading in some cases to difficulties to reliably distinguish SP-PanNET from ileal NET metastases. The biology and molecular profile of SP-PanNET remain poorly characterized and the cell of origin within the pancreas is unclear. To address these questions, we analyzed a large cohort of SP-PanNET by immunohistochemistry (n = 29; ATRX, DAXX, MENIN, Islet1, PAX6, PDX1, ARX, CDX2), whole genome copy number array (Oncoscan™) and a large NGS panel (NovoPM™) (n = 10), FISH (n = 13) and RNA sequencing (n = 24) together with 21 ileal NET and 29 nonfunctioning PanNET (NF-PanNET). These analyses revealed a unique genomic profile with frequent isolated loss of chromosome 1 (14 cases-61%) and few pathogenic mutations (KMT2C in 2 cases, ARID1A in 1 case). Unsupervised RNAseq-based clustering showed that SP-PanNET were closer to NF-PanNET than ileal NET with an exclusive beta cell-like signature. SP-PanNET showed TGF-β pathway activation signatures associated with extracellular matrix remodeling and similar signature were reproduced in vitro when pancreatic stellate cells were exposed to serotonin. SP-PanNET immunohistochemical profile resemble that of ileal NET except for PDX1 and PAX6 expression to a lesser extend suggesting that these two markers may be useful to diagnose SP-PanNET. Taken together, this suggests that SP-PanNET are a very specific PanNET entity with a peculiar biology leading to the characteristic fibrotic aspect.

134. Novel fusions in aggressive infant sarcomas: Expanding the scope of 'CIC-rearranged' sarcoma without CIC rearrangement

CIC-rearranged sarcomas are newly defined undifferentiated soft tissue tumors with CIC-associated fusions, mostly with DUX4, and dismal prognosis. CIC fusions activate PEA3 family genes, ETV1/4/5, leading to tumorigenesis and progression. We report three pediatric sarcomas with novel fusions and similar gene-expression/methylation patterns without CIC rearrangement. One disseminated tumor of unknown origin and two high-grade CNS tumors of unclear histological diagnosis were received for genomic evaluation. All three patients were infants with aggressive diseases, and two experienced rapid disease deterioration and death. A series of IHC staining and comprehensive NGS panel analyses for mutations, copy number alterations, and fusions were not diagnostic. Whole-transcriptome sequencing identified an ATXN1L-NUTM2A fusion in case 1 and ATXN1-NUTM2A in the two CNS tumors. ETV1/4/5 and WT1 overexpression were observed in all three cases. Methylation analyses predicted CIC-rearranged sarcoma for all cases. Retrospective IHC staining on case 2 demonstrated ETV4 and WT1 overexpression. An ATXN1-DUX4 fusion was recently reported in a CNS sarcoma that showed a gene expression pattern similar to that of CIC-rearranged sarcomas. ATXN1 and ATXN1L interact with CIC forming a transcription repressor complex. We propose that ATXN1/ATXN1L-associated fusions disrupt their interaction with CIC and decrease the transcription repressor complex, leading to downstream gene overexpression. These three cases, including a non-CNS tumor, with novel ATXN1/ATXN1L-associated fusions and features of CIC-rearranged sarcomas further expand the scope of 'CIC-rearranged' sarcomas without CIC rearrangement. Additional cases are needed to demonstrate if ATXN1/ATXN1L-NUTM2A fusions are associated with younger age and more aggressive diseases.

THE ROLE OF LABORATORY EVALUATION IN THE DIAGNOSIS AND MANAGEMENT OF NLRP12- ASSOCIATED AUTOINFLAMMATORY DISEASE

<h3>Introduction</h3> This case highlights the relevance of including autoinflammatory diseases in the differential diagnosis of adults with recurrent fever or episodes of shock. The role of genetic testing and confirmatory functional testing in the diagnosis and therapeutic management of autoinflammatory diseases is also discussed. <h3>Case Description</h3> A 67-year-old man presented with a 2-year history of episodes of shock of unknown etiology. Symptoms included hypotension, fever and abdominal pain requiring vasopressor support and antibiotics. Extensive infectious, autoimmune and rheumatological work-up was negative. Bone marrow biopsy showed chronic myelomonocytic leukemia (CMML) that Oncology determined was non-contributory and did not require treatment. Immunological investigations demonstrated elevated IgE, CD8+ T cell lymphocytosis during episodes of shock and increased sCD163. A targeted NGS panel revealed a heterozygous VUS in the <i>NLRP12</i> gene. Functional studies were performed offline to evaluate inflammasome activity. PBMCs stimulated with LPS for 24h revealed substantially elevated levels of IL-1β, IL-6 and TNF-α compared to healthy controls suggesting inflammasome activation and NFκB dysregulation. This prompted initiation of IL-1β blockade with canakinumab, with symptom resolution. <h3>Discussion</h3> In adults presenting with a hyperinflammatory state of unknown etiology, immunological investigations should be pursued for a possible autoinflammatory syndrome. In this case, genetic testing identified a VUS in <i>NLRP12</i>. NLRP12 is an inflammasome in which LOF and GOF variants have been reported with disease manifestations, including cold-induced autoinflammatory syndrome (FCAS). NLRP12 also regulates NFκB, which may explain some of the observed hyperinflammation. This patient had complete response to IL-1β blockade. Functional evaluation of VUS may provide an opportunity for therapeutic intervention.

Specificities of the DMD Gene Mutation Spectrum in Russian Patients.

Duchenne/Becker muscular dystrophy (DMD/BMD) is the most common form of muscular dystrophy, accounting for over 50% of all cases. In this regard, in Russia we carry out a program of selective screening for DMD/BMD, which mainly involves male patients. The main inclusion criteria are an increase in the level of creatine phosphokinase (&gt;2000 U/L) or an established clinical diagnosis. At the first stage of screening, patients are scanned for extended deletions and duplications in the DMD gene using multiplex ligase-dependent probe amplification (MLPA SALSA P034 and P035 DMD probemix, MRC-Holland). The second stage is the search for small mutations using a custom NGS panel, which includes 31 genes responsible for various forms of limb-girdle muscular dystrophy. In a screening of 1025 families with a referral Duchenne/Becker diagnosis, pathogenic and likely pathogenic variants in the DMD gene were found in 788 families (in 76.9% of cases). In the current study, we analyzed the mutation spectrum of the DMD gene in Russian patients and noted certain differences between the examined cohort and the multi-ethnic cohort. The analysis of the DMD gene mutation spectrum is essential for patients with DMD/BMD because the exact mutation type determines the application of a specific therapeutic method.

Improving Time-to-Treatment for Advanced Non-Small Cell Lung Cancer Patients through Faster Single Gene EGFR Testing Using the Idylla™ EGFR Testing Platform.

Introduction: Patients with advanced-stage non-small cell lung cancer (NSCLC) may benefit from a short time-to-treatment (TTT). Predictive biomarker testing is performed prior to treatment, as recommended by various international expert consensus bodies. Genetic testing is more time-intensive than immunohistochemistry (IHC) and commonly contributes to prolonged TTT. For epidermal growth factor receptor-positive patients (EGFR+), further genetic testing may not be required due to the mutual exclusivity of actionable mutations. Methods: The trial cohort (N = 238) received both BC Cancer NGS panel (Oncopanel) and Idylla EGFR testing. Data were also collected for a control cohort (N = 220) that received Oncopanel testing. For each patient, the time that the lab received the sample, the time taken to report the NGS and Idylla tests, the time of first treatment, and the final treatment regimen were recorded. Results: A concordance frequency of 98.7% (232/235) was observed between the Idylla and NGS panel. The lab turnaround time (TAT) was faster for the Idylla test by an average of 12.4 days (N = 235, p < 0.01). Overall, the average TTT in the trial cohort (N = 114) was 10.1 days faster (p < 0.05) than the control (N = 114), leading to a 25% reduction in TTT. For patients treated based on EGFR positivity, the mean TTT was 16.8 days faster (p < 0.05) in the trial cohort (N = 33) than the control cohort (N = 28), leading to a 48% reduction in TTT. Conclusion: Using the Idylla EGFR test as part of the molecular testing repertoire in advanced-stage NSCLC patients could significantly reduce TTT.

004 Systemic autoinflammatory diseases: a multicenter cohort study

BackgroundSystemic autoinflammatory diseases (SAIDs) are a group of hereditary diseases characterized by inflammatory attacks accompanied by clinical findings such as fever, skin rash, abdominal pain, and musculoskeletal symptoms and with high acute phase response.ObjectivesThis study sought to define the characteristics, to report 24-month follow-up of children with SAIDs and to compare the performances of both the previously validated classification criteria Federici and new Eurofever/PRINTO classification criteria in a multicentre cohort under real-life conditions.MethodThe medical charts of 60 children diagnosed with SAIDs (CAPS, TRAPS and HIDS) who were followed up regularly every 1–3 months between 2010 and 2021 in 6 pediatric rheumatology centers in Turkey were retrospectively analyzed. Demographic data, clinical findings, laboratory results and treatments were recorded. All patients were analyzed by NGS panel containing 16 genes.ResultsA total of 60 patients with SAID were evaluated. Among them 30 (50%) were male and 30 (50%) were female. The median age at disease onset and diagnosis were 22 (0–193) months and 72 (2–213) months, respectively. The current age was median 10.3 (1.7–20.4) years. All patients were follow-up with a median 31 (6–97) months. Among 60 patients, 22 were diagnosed with CAPS, 8 with TRAPS and 30 with HIDS. The frequency of clinical symptoms was 95%, arthralgia 63.3%, gastrointestinal complaints 58.3%, urticarial rash 56.7%, myalgia 48.3%, lymphadenopathy 33.3%, respectively.Urticarial rash (86.4%), cold induced rash (45.5%) and arthritis (50%) were significantly higher in CAPS patients compared with other two groups (p< 0.05). Lymphadenopathy (46.7%) was more common in HIDS patients (p = 0.03).When we applied the diagnostic/classification criteria to our cohort, 71.7% met the Federici criteria, 53.3% fulfilled the Eurofever/PRINTO classification criteria. When performance of two diagnostic criteria (Federici and Eurofever/PRINTO criteria) was compared, the proportion of patients fulfilling the Eurofever/PRINTO criteria higher in TRAPS patients compared with other groups (p = 0.008).Statistically significant decreases were found in AIDAI scores, CRP, ESR levels from the 1st month and in the leucocyte counts from the 6th month of the treatment in CAPS patients (p = <0.001, p = 0.035, p = 0.03 and p = 0.006, respectively). In addition, AIDAI scores were correlated with CRP values (p = 0.047).In HIDS patients, there was a statistically significant decrease in AIDAI scores and ESR levels from the 1st month, CRP levels from the 3rd month, and leucocyte counts from the 12th month of treatment (p = <0.001, p = 0.003, p = <0.001 and p = 0.034, respectively). In addition, ESR values were correlated with CRP (p = 0.006). In TRAPS patients, on the other hand, there was a statistically significant decrease in AIDAI scores from the 1st month of treatment (p = 0.018).At the last visit, 93.3% of the patients were receiving anti-IL-1, 5% anti-IL-6, and 1.7% were receiving colchicine. Ninety percent of the patients were in complete remission.ConclusionWe showed that the diagnostic/classification criteria may not always be sufficient to make the diagnosis, and there is still low agreement between clinical diagnoses and molecular analyzes. Anti-IL-1 treatments have been found to be quite effective in the treatment from the early period.

ANALYTICAL PERFORMANCE OF A DNA/RNA-BASED NEXT-GENERATION SEQUENCING (NGS) PANEL FOR MYELOID NEOPLASMS

Myeloid neoplasms (MNs) are a heterogeneous group of neoplasms including acute myeloid leukemia, myelodysplastic syndrome, myeloproliferative neoplasms and myelodysplastic syndrome/myeloproliferative neoplasms. MNs show a complex genetic basis, and the number of alterations to be analyzed for risk diagnostic, stratification and management of patients increases continuously. Consequently, there is a demand for multiple molecular and cytogenetic techniques with the aim of detecting a wide variety of genetic abnormalities. Since conventional techniques are insufficient to study all such abnormalities, the NGS panel has become a useful tool for broad characterization of the spectrum of mutations in MNs. However, before NGS panels can be introduced in the laboratory routine, their performance should be evaluated. In this context, we describe the analytical performance of the Oncomine™Myeloid Research Assay (OMA) (Thermo Fisher), a DNA/RNA-based amplicon sequencing panel, that evaluated simultaneously 40 genes (17 full genes and 23 hotspots genes) and 29 fusions drivers genes (over 600 partners) known to be recurrently mutated in MNs. Commercial controls Seraseq Myeloid Mutation DNA and the Seraseq Myeloid Fusion RNA were used for sensitivity, specificity, accuracy, repeatability, reproducibility and limit of detection (LoD) testing. Prospective sequencing was performed for samples from 34 patients presenting with suspected or confirmed MN. Libraries was prepared by manual method and sequenced by the Ion S5 Sequencer. Data obtained were analysed on Ion Reporter 5.18 software. The OMA assay showed good performance in terms of depth of coverage, on-target reads, and uniformity. The mean reads lenght ranged from 223 to 231 bp for DNA sample and from 88 to 124 bp for RNA, within the expected standard for the panel. The panel achieved 98,8% and 100% concordance with DNA and RNA controls, reaching analytical sensitivity of 99% and 100% for DNA and RNA, respectively. Regarding the type of DNA mutation: single nucleotide variants (SNP), indels or indels hot spots, the sensitivity found was 100%, 96% and 95%, respectively. The accuracy and analytical specificity was 100% for DNA panel. The LoD was 5% variant allele fraction (VAF) for DNA (SNP and indel) and 1-log reduction from initial number of reads counts for RNA fusion genes. Repeatability and reproducibility were 98% for DNA and 100% for RNA. However, the coefficient of variation (CV) within-run and between-run in VAF was >20% for large indels in CALR, FLT3 (ITD) and SRSF2 . The vertical coverage of each sample was inspected with a coverage threshold of 100X. Some amplicons in the assay failed repeatedly (ASXL1_1.875, BCOR_8.202477, BRAF_18.30633, CEBPA_1.1.86676, PRPF8_12.173121, SH2B3_1.85663). However, the OMA is unable to detect specific variants in ASXL1 (c.1900_1922del23 and c.1934_1935insG) and CEBPA (c.68_69insC). This is a known limitation of the Ion Torrent chemistry for accurate sequencing of large homopolymers and high GC content regions. Of the 34 patient samples, 28 (87.5%) had at least one reportable variant. A total of 58 DNA variants and six gene fusions were detected. Some variants were confirmed by orthogonal techniques. In conclusion, OMA is a highly accurate and reproducible NGS panel for the detection of common genetic abnormalities in MNs.

TERRA R-LOOPS ARE INCREASED IN ACUTE MYELOID LEUKEMIA

Telomeric repeat-containing RNA (TERRA) are long non-coding RNAs transcribed from subtelomeric regions towards telomere ends. TERRA binds to subtelomeric and telomeric regions, forming R-loops, structures composed of DNA-RNA hybrids and displaced single-stranded DNA. TERRA R-loops protect telomeres from DNA damage and promote telomere elongation in short telomeres by enabling telomerase or by activating alternative telomere lengthening. However, high or low TERRA levels are associated with DNA damage and telomere dysfunction. RNaseH1 and RNaseH2 cleave R-loops at telomeres, and regulate R-loops levels. The role of TERRA R-loops in telomere maintenance in human cells is not well understood, since conflicting results derive from experiments using diverse organisms and commercial cell lines, with few studies applying primary human cells. Here we describe for the first time the TERRA physiology in hematopoietic cells of patients with acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), telomeropathies (TEL), and healthy subjects. We initially screened 17 patients with AML, 5 with ALL, 9 with TEL, and 20 healthy subjects. Additionally, 88 AML patients were studied. HeLa, H1, THP1, NB4, and VA13 cell lines were used for functional experiments, and lentivirus vectors containing GFP or RNaseH1-GFP motifs were employed. ATRX, TRF2, TERT, RNaseH1, RNaseH2, TERRA 10q, 15q, 20q, XqYq, XpYp, and 17p expression levels were measured by RT-qPCR. Telomere length was determined by qPCR. R-loops were assessed by immunofluorescence using the S9.6 antibody. Cell line viability was defined by MTT assay. NGS panel was designed to evaluate variants in splicing factors and epigenetic regulators. In the screening population, TERRA expression (10q, 15q, 20q, and XqYq) was significantly increased in AML samples (p < 0.05) in comparison to healthy controls and VA-13 cells. Telomeres were very short in AML samples and TEL patients compared to controls. R-loop formation was significantly increased in AML samples compared to controls (p < 0.01). To examine the TERRA physiology in AML, we further eveluated 88 AML samples. Hierarchical cluster algorithm based on TERRA expression defined two AML groups, one with high and the other with low TERRA expression. Both groups dysplayed short telomeres, but the high TERRA AML group exhibited low TERT and RNaseH2 expression levels (p < 0.05). There was no difference between groups on somatic variants or RNaseH1, TRF2, ATRX, and TRF2 expression levels. AML cell lines were transfected RNaseH1-GFP-containing or empty vector lentiviruses and treated with cytarabine. AML cells with exogenous RNAseH1 expression showed more sensitivity to cytarabine in MTT assays (p < 0.05) than cells transfected with the empty vector. These results show for the first time that TERRA is dysregulated in AML. High TERRA levels are unique to a subset of AML cases with low TERT expression. High TERRA correlated with the accumulation of R-loops, which may be therapeutic targets. TERRA upregulation in AML due to short telomeres is unlikely, given the low TERRA levels in TEL patients. TERRA upregulation did not appear to be directly related to neoplastic differentiation, given low TERRA in ALL. TERRA expression and R-loops are dysregulated in AML. TERRA R-loops inhibition increases chemotherapy-induced apoptosis in AML cell lines and may serve as a therapeutic target.

Association between copy number variations in the OCA2-HERC2 locus and human eye colour

Human eye colour variation is strongly associated with single nucleotide polymorphisms (SNPs) in the OCA2-HERC2 locus, especially rs12913832 that is found in an enhancer element of OCA2. In a previous study we found that 43 out of 166 individuals in a Norwegian population with the brown eye colour genotype HERC2 rs12913832:AA or AG, did not have the expected brown eye colour. To investigate if duplications or deletions in the OCA2-HERC2 locus could explain the blue eye colour in these individuals, we analysed massively parallel sequencing (MPS) data for copy number variations (CNVs) in the OCA2-HERC2 region. The ∼500 kb long OCA2-HERC2 locus was sequenced in 94 individuals with the rs12913832:AG and AA genotypes. Of these, 43 were observed to have blue eye colour and 51 were observed to have brown eye colour. CNVs were analysed using R and the R-package panelcn.MOPS - CNV detection tool for targeted NGS panel data. In rs12913832:AG individuals, CNVs in 32 regions were significantly associated with blue eye colour (Benjamini-Hochberg adjusted p-value ≤ 0.05). In rs12913832:AA individuals, CNVs in 14 regions were associated with blue eye colour using raw p-values (p ≤ 0.05). The functional effects of these CNVs on OCA2 expression are yet to be investigated. However, this study suggests that CNVs in the OCA2-HERC2 locus might explain why some of the rs12913832:AG and AA individuals have unexpectedly blue eyes.

Correlation of plasma cell assessment by phenotypic methods and molecular profiles by NGS in patients with plasma cell dyscrasias

BackgroundNext-generation sequencing (NGS) detects somatic mutations in a high proportion of plasma cell dyscrasias (PCD), but is currently not integrated into diagnostic routine. We correlated NGS data with degree of bone marrow (BM) involvement by cytomorphology (BMC), histopathology (BMH), and multiparameter flow cytometry (MFC) in 90 PCD patients.MethodsOf the 90 patients the diagnoses comprised multiple myeloma (n = 77), MGUS (n = 7), AL-amyloidosis (n = 4) or solitary plasmocytoma (n = 2). The NGS panel included eight genes CCND1, DIS3, EGR1, FAM46C (TENT5C), FGFR3, PRDM1, TP53, TRAF3, and seven hotspots in BRAF, IDH1, IDH2, IRF4, KRAS, NRAS.ResultsMutations were detected in 64/90 (71%) of cases. KRAS (29%), NRAS (16%) and DIS3 (16%) were most frequently mutated. At least one mutation/sample corresponded to a higher degree of BM involvement with a mean of 11% pathologic PC by MFC (range, 0.002–62%), and ~ 50% (3–100%) as defined by both BMC and BMH.ConclusionsThe probability of detecting a mutation by NGS in the BM was highest in samples with > 10% clonal PC by MFC, or > 20% PC by BMC/ BMH. We propose further evaluation of these thresholds as a practical cut-off for processing of samples by NGS at initial PCD diagnosis.

JS04.4.A Beyond β-catenin: Genetic alterations ofTP53 andOTX2 and older age indicate increased risk of relapse in WNT medulloblastomas

Abstract Background This genetic analysis of WNT-activated medulloblastomas (WNT-MBs) aimed to re-evaluate the prognostic impact of age, TP53 mutations and to identify specific chromosomal aberrations as possible prognostic markers. Material and Methods In a cohort of 191 patients with WNT-MBs, mutations in CTNNB1, APC and TP53 were analyzed by Sanger and/or NGS panel sequencing. Chromosomal copy number aberrations were assessed by high-resolution, genome-wide molecular inversion probe technology (MIP), SNP6 array, and/or 850k methylation bead-array hybridization. Association with prognosis was evaluated in 133 patients with follow-up data from the HIT2000 medulloblastoma trial, HIT registries, and the NOA-07 trial. Results CTNNB1 mutations were present in 92.2% of the samples. APC mutations were found in 6.8% (13 samples). One CTNNB1 wildtype tumor gained WNT-activation due to a homozygous deletion of FBXW7. Monosomy 6 was present in 78.6%, and more frequent in children compared to adolescents/adults (≥16 years). Adolescents/adults showed worse overall survival (OS; p=0.009) compared to children, but not worse progression-free survival (PFS; p=0.106). With an age cut-off at 18 years, no survival difference was found. Also adolescents alone (16-20 years) had worse OS (p=0.003) compared to children, whereas in patients ≥21 (n=12 adults with PFS/OS data) no tumor progression/relapse occurred. Only one adult died due to therapy-related complications. WNT-MB patients with tumors harboring TP53 mutations (24/133, 18.1%) showed significant worse PFS (p=0.001), which was also found in children and adolescents individually (p=0.004, resp. p=0.017). Gains of the OTX2 locus on chromosome 14q found in 40.2% (35/87) of samples were independent of TP53 mutations and also associated with poor PFS and OS (p=0.034, resp. p=0.016). Individual analyses of OTX2 gains within age groups showed only worse OS in children (p=0.012). Multivariate Cox regression analysis for PFS identified both genetic alterations, but not age, as independent prognostic markers. For OS, multivariate analysis found OTX2 gains and older age as independent prognostic markers. Conclusion Our data suggest that adolescent patients with WNT-MB and those patients carrying TP53 mutations or OTX2 gains - independent of age - are at higher risk of relapse. Eligibility of these patients for therapy de-escalation trials needs to be debated.

1248P Identification of Epstein-Barr virus (EBV)-associated gastric cancer at RNA-level by evaluating transcriptional status of seven EBV crucial genes

EBV has been an emerging biomarker to inform clinical management of GC, according to the potential mechanisms of EBV infection on GC immune check point blockade (ICB) efficacy. The gold standard for detecting EBV has long been EBV-encoded RNA (EBER) in situ hybridization (ISH). However, with the increasing demand for multi-molecular biomarkers detection recommended by standard treatment guidelines for GC, a single EBV detection has been challenged in clinical practice with limited samples. Herein, an NGS-panel based on RNA for detecting EBV was established and validated in the study. Seven genes including, EBER1, EBER2, and EBNA1, LMP1, LMP2A/B, BZLF1, BARF1 were involved in the NGS panel to evaluate EBV infection status. 42 tumor tissue samples from GC, with half EBV positive and half EBV negative evaluated by ISH were collected, as well as their paired normal tissue sample, for NGS detection. The seven genes expression profile in the enrolled samples were comprehensively analyzed. The raw read counts of the five genes EBNA1, LMP1, LMP2A/B, BZLF1, BARF1 based on RNA sequencing in normal tissue samples were detected as zero. Only 12 normal tissue samples were detected EBER1and/or EBER2, of which the raw read counts did not exceed 10. In ISH-EBV negative tumor samples, raw read counts of the five genes EBNA1, LMP1, LMP2A/B, BZLF1, BARF1 based on RNA were also zero. And, expression signals were detected for EBER1 in 7 samples and EBER2 in 3 samples, and the raw RNA counts did not exceed 17 and 5, respectively. However, in ISH-EBV positive tumor samples, the average number of RNA raw read counts of EBER1 was 7424, with the minimum value was 458; the average number of RNA raw read counts of EBER2 was 3730, with the minimum value was 137. Besides, RNA expression signals of three gene, EBNA1, LMP2A/B, and BARF1, were also been detected with lower raw read counts. Due to the significantly high expression characteristics of EBER1/EBER2 in EBV infected positive samples, the detection of EBV based on RNA by NGS panel is feasible, which provides a basis for the subsequent design of NGS panel including EBV and other molecular biomarkers for GC.

558P Molecular subtyping endometrial carcinoma in Chinese population applying an NGS-based classifier and its association with prognosis

Molecular subtypes of EC is widely recognized and applied in clinics. While, it is still less recognized the distribution and prognostic value of molecular subtypes of endometrial carcinoma in Chinese population. An NGS-based classification strategy with reference to these two classifiers was developed and applied for the first time to explore the prognostic correlation of EC patients in China. Formalin-Fixed Paraffin Embedded (FFPE) tissue of 233 EC patients were retrospectively collected from Qilu Hospital, China. Genomic DNA was extracted and subjected to an all-in-one simplified NGS panel covering POLE, TP53 as well as MSI for subtyping. The information for ProMisE classification were also collected. With the median follow-up was 66 (range 7-122) months, overall survival (OS) and disease-specific survival (DSS) were also been analyzed across the subtypes. Base on NGS panel classification, the fractions of POLE, MSI, CNH and CNL group were 8.15%, 18.88%, 11.59% and 61.37%, respectively. 61patients with ProMisE classification available, the fractions of the former subtypes were11.48%, 36.07%, 14.75%, and 37.70% respectively. The OS and DSS was (P=0.046 for OS, P=0.024 for DSS) significantly in the four subtypes based on NGS testing in 131 followed patients. There was no significant difference in OS (P = 0.91) and DSS (P = 0.95) across the four subtypes by ProMisE in 50 followed patients (Table).Table: 558PSurvival analysis results based on the two classifiersNGS-based classifier（N=131）POLEMSICNHCNLP8 years -OS rate100%80.00%50.00%92.26%0.0468 years -DSS rate100%88.89%66.67%100%0.024ProMisE classifier (N=50)8 years -OS rate83.33%89.47%100%80.00%0.918 years -DSS rate100%94.73%100%92.31%0.95 Open table in a new tab The NGS-based strategy was confirmed to effectively classify four prognostically significant subgroups in endometrial cancer patients. Further analysis showed that the clinicopathological features of each molecular subtype were significantly different. All the results confirmed the feasibility of molecular subtyping of EC in clinical practice by a simplified NGS approach.