Next-generation Exome Sequencing Research Articles

Decoding the Dynamics of Circulating Tumor DNA in Liquid Biopsies.

Circulating tumor DNA (ctDNA), a fragment of tumor DNA found in the bloodstream, has emerged as a revolutionary tool in cancer management. This review delves into the biology of ctDNA, examining release mechanisms, including necrosis, apoptosis, and active secretion, all of which offer information about the state and nature of the tumor. Comprehensive DNA profiling has been enabled by methods such as whole genome sequencing and methylation analysis. The low abundance of the ctDNA fraction makes alternative techniques, such as digital PCR and targeted next-generation exome sequencing, more valuable and accurate for mutation profiling and detection. There are numerous clinical applications for ctDNA analysis, including non-invasive liquid biopsies for minimal residual disease monitoring to detect cancer recurrence, personalized medicine by mutation profiling for targeted therapy identification, early cancer detection, and real-time evaluation of therapeutic response. Integrating ctDNA analysis into routine clinical practice creates promising avenues for successful and personalized cancer care, from diagnosis to treatment and follow-up.

#817 Genetic analysis in a group of patients with persistent isolated microscopic hematuria

Abstract Background and Aims Autosomal dominant Alport syndrome (ADAS) is a genetic disease caused by pathogenic variants in COL4A3 and COL4A4 genes. ADAS is the main cause of isolated persistent microscopic hematuria (PMH). However, other diseases like IgA nephropathy (IgAN) can present with PMH. Establishing a precise genetic diagnosis in this type of patients would avoid invasive studies like kidney biopsies. In this study we performed a comprehensive genetic test in a cohort of patients with isolated PMH to evaluate the frequency of ADAS or other genetic abnormalities and compare the clinical characteristics of patients with or without ADAS Method Fifty-two patients with PMH were included in the study. PMH was defined by the presence of 3 or more dysmorphic red blood cells per field in at least 5 consecutive urine sediments and a time-averaged proteinuria <1 gr/day. Average proteinuria was determined for every 6 months of follow-up and time-averaged (TA) proteinuria represented an average of the mean proteinuria measurements in each 6-month period. A comprehensive genetic study using whole exome next-generation sequencing was performed. One hundred and one genes related to familial hematuria, focal segmental glomerulosclerosis and cystic kidney diseases were analyzed. Patients with known secondary causes of hematuria were excluded. The baseline clinical characteristics of patients with and without ADAS were compared. Patients with pathogenic variants in COL4A5 gene were excluded of this analysis. Results Thirty-one (60%) patients were diagnosed with ADAS (16 patients with pathogenic variants in COL4A3, 15 in COL4A4), 3 (6%) patients (2 women, 1 man) presented pathogenic variant in COL4A5 gene, and the genetic study was negative in the remaining 18 patients (variants of unknown significance in COL4A3 or COL4A4 were found in 3 of the latter). No other genetic abnormalities were detected in the panel of 101 genes. Table 1 shows the baseline clinical characteristics of patients with and without ADAS. Patients with ADAS presented a higher TA proteinuria [0.23 (0.11-0.6) vs 0.08 (0.05-0.16), p = 0.01], and a more frequent familial history of isolated microscopic hematuria (76% vs 40%, p = 0.01) chronic kidney disease (CKD) (44% vs 0%, p = 0.01) or end stage kidney disease (32% vs 0%, p = 0.02). In the multivariate analysis, only a family history of microscopic hematuria was associated with ADAS: OR 5.62 (1.25-25.49, p = 0.02). Conclusion ADAS is the most common cause of PMH. A genetic study should be the first diagnostic step in order to obtain a precise cause of the disease and avoid invasive procedures. A family history of microscopic hematuria and of CKD increases the likelihood of ADAS prior to the performance of genetic test.

O18 Precision genome editing for targeted correction of pathogenic D50N mutation in keratitis–ichthyosis–deafness syndrome using CRISPR/Cas9 and homology-directed repair

Abstract Introduction and aims Keratitis–ichthyosis–deafness (KID) syndrome is an ectodermal disorder that causes blindness, skin inflammation and deafness. It is caused by autosomal dominant mutations in the gap junction beta 2 (GJB2) gene with 86% being a hotspot mutation on c.148G>A, resulting in the amino acid replacement (D50N) in its coding protein connexin 26 (Cx26). There is currently no curative treatment for KID syndrome. We aim to correct the hotspot mutation D50N using a genome-editing approach. Methods Keratinocytes generated from the patient with KID who had a D50N mutation (KID-KCs) were genetically corrected by homologous-directed repair/CRISPR-Cas9 using an electroporation approach. The correction efficiency was determined and analysed using Sanger sequencing and Synthego Inference of CRISPR Edits analysis. The off-target effects were confirmed by whole-exome sequencing [exome next-generation sequencing (NGS)]. Furthermore, the functional recovery following genome editing was assessed by GJB2 mRNA production, Cx26 protein expression, and trafficking and hemichannel activity. Results The correction efficiencies ranged from 95% to 100% with different doses of CRISPR-Cas9 and 8µg CRISPR-Cas9 was the most optimal concentration with the efficiency of 100% and approximately 0% insertion-deletion (INDEL). Fifty days after gene editing, the INDEL increased to only 2%, indicating a minimum residual CRISPR-Cas9 effect in edited cells. The exome-NGS analysis also showed no off-target effects in the outside target area. Functional studies showed that there was a significant increase in wildtype GJB2 mRNA expression and a reduction of mutant GJB2 expression (n = 5, P < 0.01). Increased Cx26 membranous localization in KID-KCs was also observed (n = 3, P < 0.01). Additionally, Cx26 hemichannel activity assessment using the neurobiotin assay showed a significant decrease in ‘leaky’ hemichannel in gene-edited KID-KCs compared with unedited cells (n = 3, P < 0.05). Conclusions We can genome-correct KID keratinocytes harbouring the mutation D50N with 100% editing efficiency, 0–2% INDEL, and undetectable off-target effects. This result indicates a promising gene therapy for KID syndrome with the hotspot mutation D50N.

"Deciphering the genomic complexity of thyroid cancers: an in-depth exploration through pan-exomic analysis using whole exome next-generation sequencing"

"Deciphering the genomic complexity of thyroid cancers: an in-depth exploration through pan-exomic analysis using whole exome next-generation sequencing"

Malakoplakia with aberrant ALK expression by immunohistochemistry: a case report

BackgroundMalakoplakia is a rare inflammatory disease of the urogenital tract. There have been no reports of malakoplakia expressing anaplastic lymphoma kinase (ALK) to date. Here, we present one case of malakoplakia with aberrant ALK expression by immunohistochemistry and discuss the clinical significance.Case presentationA 65-year-old Chinese woman with a history of diabetes presented with solid masses in the liver and kidney and elevated lesions on the mucosal surface of the colon. Right nephrectomy and partial liver resection were performed. Microscopically, sheets of histiocytes with poor intercellular adhesion were seen, with Michaelis–Gutmann bodies present in both the intracellular and extracellular interstitium. CD10-, CD68-, and CD163-positive cells were present, with Michaelis–Gutmann bodies confirmed by staining with Alcian blue, periodic acid-Schiff (PAS), periodic acid-Schiff with diastase, Von Kossa, and Prussian blue. Aberrant ALK1 and ALK (D5F3) expression was observed in the cytoplasm and nucleus of cells. However, ALK gene mutation was not detected by fluorescence in situ hybridization or whole exome next-generation sequencing. NGS revealed nine individual somatic gene mutations: GOT1L1, GLIS2, SPOUT1, TMEM97, MUC3A, NSD2, SFXN5, ADAD1 and RAD50. The significance of the somatic gene mutations detected in this study is not clear, and the relationship between them and malakoplakia cannot be clarified by existing scientific studies. The pathological diagnosis was malakoplakia with aberrant ALK expression by immunohistochemistry. The antibiotics imipenem and vancomycin were started based on the results of drug sensitivity analysis and the patient was subsequently discharged. She experienced no discomfort during 30 months of follow-up.ConclusionThis is the first reported case of malakoplakia with aberrant ALK expression, it should be differentiated from ALK-positive histiocytosis to avoid misdiagnosis.

Comparative Genomic Analysis of Pancreatic Acinar Cell Carcinoma (PACC) and Pancreatic Ductal Adenocarcinoma (PDAC) Unveils New Actionable Genomic Aberrations in PACC.

Pure pancreatic acinar cell carcinomas (PACC) are rare malignancies with no established treatment. PACC demonstrates significant genetic intertumoral heterogeneity with multiple pathways involved, suggesting using targeted cancer therapeutics to treat this disease. We aggregated one of the largest datasets of pure PACC to examine the genomic variability and explore patient-specific therapeutic targets. PACC specimens (n = 51) underwent next-generation sequencing of DNA (n = 29) or whole exome (n = 22) and RNA (whole transcriptome, n = 29) at a commercial laboratory. We performed comparative analyses of a genomic cohort of pancreatic ductal adenocarcinomas (PDAC; n = 4,205). In parallel, we conducted a retrospective review of patients with PACC treated at Huntsman Cancer Institute (HCI). The real-world dataset included samples from 51 patients with PACC. We found key molecular differences between pure PACC and PDAC, highlighting the unique characteristics of pure PACC. Major differences in PACC include lower MAPK signaling and less stromal cell abundance compared with PDAC. Pure PACC showed genomic loss-of-heterozygosity to largely coincide with mutations in BRCA1, BRCA2, and PALB2. Of the 7 patients treated at HCI, one had a tumor that harbored a BRAF-V600E mutation. Leveraging precision oncology, this patient is being treated with encorafenib plus binimetinib, achieving an exceptionally durable and ongoing complete response of more than 3 years. There are major differences between PACC and PDAC, including downregulation of the MAPK signaling pathway, and less stromal cell abundance. In addition, genomic characterization of pure PACC revealed frequent targetable alterations, which can guide patient treatment.

Rapidly Progressive Aneurysmal Disease Associated with Epstein Barr Virus-Positive Lymphoproliferative Disease in a CVID patient with NFKB-1 Mutation

IntroductionCommon variable immunodeficiency (CVID) is a genetically heterogeneous group characterized by hypogammaglobulinemia and poor immune response to vaccines. CVID patients are at higher risk for autoimmune diseases or malignancy. We demonstrate a rare case of a patient with CVID (NFKB-1 mutation) presenting with features of vasculitis and rapidly progressive aneurysmal disease secondary to an Epstein Barr Virus (EBV)-associated lymphoproliferative disorder. Case descriptionA 36-year-old man with CVID presented with hoarseness and left vocal cord paralysis. Imaging revealed an aortic arch and descending thoracic aorta abnormality concerning for a plaque, vasculitis or focal thrombosed dissection. He later devloped severe back pain and imaging studies demonstrated an abdominal aortic aneurysm, thickening of the common iliac arteries and a focal aneurysm of the right common iliac artery. PET CT scan showed uptake in the upper abdominal aorta extending to the right renal artery and the distal abdominal aorta near the bifurcation extending to the right common iliac artery, concerning for large vessel vasculitis. He was started on high-dose steroids for large vessel vasculitis. One month later, he was hospitalized for disseminated histoplasmosis and disseminated varicella zoster virus (VZV) and started on amphotericin B and acyclovir with steroid taper. Whole exome next-generation sequencing (NGS) revealed NFKB-1 mutation consistent with CVID-12 with autoimmunity. He later developed worsening back pain prompting repeat imaging which showed further progression of his aneurysmal disease. He was also found to have EBV viremia with a titer of 33,700 IU/mL. A left superior gluteal artery biopsy was performed which showed evidence of an EBV+ lymphoproliferative disorder with concern for an EBV-positive fibrin-associated diffuse lymphoproliferative B-cell lymphoma (DLBCL). DiscussionCVID-12 is an autosomal dominant disease resulting from a mutation in NFKB-1. This mutation leads to an impaired immune response associated with recurrent infections and approximately 20% of patients developing lymphomas at some point in their lifetime. However, there are few case reports of CVID associated large vessel aneurysms or vasculitis. Our patient with CVID demonstrated a rare presentation of an EBV-positive lymphoproliferative disorder driving progression of large vessel disease.

Abstract LB288: Biomarker analysis from AMPECT correlating response to nab-sirolimus with TSC1 and TSC2 inactivating alterations

Abstract nab-Sirolimus is an mTOR inhibitor (mTORi) approved in the US for the treatment of adult patients with locally advanced, unresectable, or metastatic malignant perivascular epithelioid cell tumor (PEComa) based on clinical efficacy and safety data from the phase 2, multicenter, open-label AMPECT trial (NCT02494570). TSC1 and TSC2 are tumor suppressor genes and key upstream regulators of mTOR complex 1 (mTORC1); inactivating alterations (loss-of-function mutations or deletions) in these genes lead to mTORC1 hyperactivation, which may contribute to tumor formation. Phosphorylation of S6 ribosomal protein (pS6) is a reliable surrogate for mTORC1 activity. Here we present the results of an exploratory biomarker analysis performed on samples from patients enrolled in the AMPECT study. Targeted exome next-generation sequencing (NGS) using a 500-gene OncoPanel test (Center for Advanced Molecular Diagnostics, Brigham and Women’s Hospital, Boston, MA) assessed mutations, copy number changes, and translocation events. pS6 was assessed by immunohistochemistry (IHC). Twenty-five patients had tissue sufficient for NGS, 56% (14/25) had either TSC1 (N=5, 20%) or TSC2 (N=9, 36%) mutations: TSC1, 1 frameshift, 2 splice site, 1 missense, and 1 nonsense mutation; TSC2, 1 nonsense, 7 frameshift, and 1 homozygous deletion. In this dataset, TSC1 and TSC2 inactivating alterations were mutually exclusive. Patients with TSC1 or TSC2 inactivating alterations achieved a clinically meaningful benefit: 64.3% (9/14) objective overall responses (including complete and partial responses) and median (95% CI) duration of response (DOR) of 45.7 (5.6, not reached [NR]) months, progression-free survival (PFS) of 41.2 (5.5, NR) months, and overall survival (OS) of NR (31.6, NR) months. Mutations in TP53, RB1, and ATRX were also common (48%, 24%, and 20%, respectively). Of tissue samples evaluable for IHC (N=25), responses occurred in 58.8% (10 of 17) of patients with pS6 positive tumors versus 0% (0 of 8) with pS6 negative tumors; absent pS6 staining was negatively associated with response to nab-sirolimus (P=0.008, Fisher’s exact). Median DOR for pS6 positive responders was 39.7 (5.6, NR) months. In pS6 positive vs pS6 negative patients, the median PFS was 24.4 (2.8, 53.1) vs 5.5 (2.8, NR) months, and OS was 53.1 (18.0, NR) vs 37.0 (16.6, NR) months. A variety of pathogenic inactivating alterations were observed in TSC1 and TSC2 genes, though TSC2 mutations were most commonly frameshift mutations; no recurring mutations were observed. A tumor-agnostic study (PRECISION 1: NCT05103358) is now recruiting patients with pathogenic inactivating TSC1 or TSC2 alterations to further examine these biomarker findings. Citation Format: Lee D. Cranmer, Andrew J. Wagner, Vinod Ravi, Richard F. Riedel, Kristen N. Ganjoo, Brian A. Van Tine, Rashmi Chugh, Erlinda M. Gordon, David J. Kwiatkowski, Jason L. Hornick, Heng Du, Li Ding, Anita N. Schmid, Willis H. Navarro, Loretta M. Itri, Mark A. Dickson. Biomarker analysis from AMPECT correlating response to nab-sirolimus with TSC1 and TSC2 inactivating alterations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB288.

Combination Therapy of Eltrombopag and Cyclosporine a Improves Hematopoiesis in Patients with Lower-Risk Myelodysplastic Syndrome

Objective: Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal myeloid disorders characterized by ineffective hematopoiesis and cytopenias, with variable risks of progression to acute myelogenous leukemia (AML). Peripheral blood cytopenias are the major sources of morbidity and mortality for lower-risk MDS (LR-MDS) pts.The strategies guided by risk stratification tools are different for HR- and LR-MDS. The treatment options for cytopenias in IST non-responders, especially thrombocytopenia, are limited. Such pts are prone to a high risk of bleeding. Eltrombopag (EPAG) is an orally administered nonpeptide thrombopoietin (TPO) receptor agonist. The single-agent activity of EPAG was demonstrated in 44-47% of pts with LR-MDS . In this study, we investigated the efficacy and safety of the combination of EPAG and CysA in LR-MDS in a non-randomized phase II investigator-initiated trial. Method(s):Pts with a diagnosis of LR-MDS by WHO 2016 classification and IPSS-R as low to intermediate-risk group with hemoglobin (Hb) <9.0 g/dL, or RBC transfusion-dependence (at least four units of RBC at 8 weeks prior to enrollment); platelet count <20×109/L or platelet transfusion dependence; were enrolled. http://www.chictr.org.cn/ identifier: ChiCTR2000035473. The primary objective of this IIT was to evaluate the response to this regimen based on IWG-MDS 2006 criteria. Results All the 31 pts were evaluated for response at the primary endpoint. Among them, 21 (67.7%) pts achieved treatment response in at least one eligible lineage with an objective response rate (ORR), 4 (12.9%) pts achieved a CR, 5 (16.1%) pts achieved bi-lineage responses (HI-PE R), 11 (24.3%) pts achieved platelet lineage responses (HI-P R), and 1 (3.2%) patient achieved erythroid lineage response (HI-E R). ANC did not increase significantly at the primary endpoint compared to the baseline; however, altered ANC was a response of CR pts. The median time to response was 8 (range, 2-24) weeks, irrespective of whether the pts had a platelet or erythroid response.Of the 21 pts eligible for an erythroid lineage response,10 pts (47.6%) had responded by the endpoint and the platelet response rate was 64.5% (20/31). The median increase in Hb levels and platelets was from 60.4±18.2 g/L to 68.0±21.2 g/L (P=0.015) and from 12.3±11.2×109/L to 32.5±37.3×109/L (P=0.003) (Figure 1). The median DORs of HI-E and HI-P were not achieved, but the response rates of erythroid and platelet lineages in 20 months were 80.0% and 70.6%, respectively (Figure 2). The RBC and platelet transfusion-dependent rate decreased from 42.9% (9/21) to 28.6% (6/21) (P=0.334) and from 58.0% (18/31) to 25.8% (8/31)We also performed targeted next-generation exome sequencing of 80 genes associated with myeloid malignancies in 21 pts. Among them, 18 showed a total 19 somatic mutations (Figure 3). TET2,and ANKRD11 were detected in 3 responding pts,and KMT2D was detected in 3 responding pts. Pts with adverse prognostic genes, such as ASXL1, ARSR2, U2AF1, KIT, and ETV6, did not exhibit a hematological response. Somatic mutations in DHX9 and ROBO1 were associated with BM failure, as detected in responding pts. All 31 pts were evaluated for toxicity, and the treatment was found to be well-tolerated. The most commonly reported adverse events (AEs) of any grade were nausea (6/31,19.4%), elevated liver transaminases (4/31,12.9%), and headache (3/31,9.7%); no severe AEs had been reported until the cutoff date. One MDS-SLD patient who received CR at 3 weeks experienced deep venous thrombosis of the lower extremity after 9 months of medication, thereby requiring dose interruption. After anticoagulant therapy, the platelet count was still 65×109/L. One MDS-U case who received HI-P had increased BM blasts from 3.2% to 15.4% for 4 months. Another MDS-U case received HI-E and HI-P, and the chromosome was changed from normal karyotype to -7/del (20q) chimeric karyotype for 9 months after administration of the drugs. However, the hematological response was maintained for 16 months until the cutoff date for data collection. Conclusions: Our results indicated that the addition of Epag to CysA is well-tolerated and is an effective treatment for LR-MDS. These ORR are better than CysA monotherapy, especially HI-P. Also, the hematological responses are durable, although continued Epag administration might be necessary. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

ERBB2 amplification status in 67 salivary duct carcinomas assessed by immunohistochemistry, fluorescence in situ hybridization, and targeted exome sequencing

AbstractSalivary duct carcinoma (SDC) is an aggressive salivary gland malignancy with poor survival. Approximately 30% SDC harbor HER2 amplification and response to trastuzumab has been reported. However, a systematic approach for HER2 status assessment in this tumor type has not been established. A total of 67 tumor samples were evaluated for HER2 protein overexpression or ERBB2 gene amplification using at least 2 methods: immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and/or targeted exome next-generation sequencing (NGS). NGS assessed ERBB2 copy number fold change (FC) and total copy number (TCN). HER2 status was first determined by IHC/FISH according to the 2018 ASCO/CAP breast cancer guidelines. FISH results, the “gold standard”, were compared with the NGS results. All (15/15) IHC positive, 35% (6/17) equivocal, and no (0/19) IHC negative SDC were HER2 amplified by FISH. HER2 FISH signal/cell showed a good correlation with FC (Spearman correlation: 0.708, R2: 0.501, p < 0.0001) and TCN (Spearman correlation: 0.763, R2: 0.582, p < 0.0001). Receiver operating characteristics curve estimation showed an area under curve (AUC) of 0.975 for ERBB2 FC. FC cutoff of ≥1.8 corresponded to an accuracy of 95.2% for ERBB2 amplification (Youden's index: 0.84, sensitivity: 89.47%, specificity: 100%). FC < 1.3 could be reliably classified as ERBB2 not amplified and FC ≥ 1.3 and <1.8 as equivocal. TCN estimation showed AUC of 0.981. TCN cutoff of >6.0 corresponded to an accuracy of 92% for HER2 amplification (Youden's index: 0.81, sensitivity: 81.2%, specificity: 100%). TCN < 4 could be reliably classified as ERBB2 not amplified and TCN ≥ 4.0 and ≤6.0 as equivocal. FC and TCN were binarized with respective cutoffs of ≥1.8 and ≥6.0 and the proportion of agreement with FISH were 95% and 92%, respectively. The assessment of ERBB2 copy number by NGS is accurate and reliable with FC or TCN nearly equivalent to FISH in identifying HER2 amplified SDC.

Genetic Analysis Algorithm for the Study of Patients with Multiple Congenital Anomalies and Isolated Congenital Heart Disease.

Congenital anomalies (CA) affect 3–5% of newborns, representing the second-leading cause of infant mortality in Argentina. Multiple congenital anomalies (MCA) have a prevalence of 2.26/1000 births in newborns, while congenital heart diseases (CHD) are the most frequent CA with a prevalence of 4.06/1000 births. The aim of this study was to identify the genetic causes in Argentinian patients with MCA and isolated CHD. We recruited 366 patients (172 with MCA and 194 with isolated CHD) born between June 2015 and August 2019 at public hospitals. DNA from peripheral blood was obtained from all patients, while karyotyping was performed in patients with MCA. Samples from patients presenting conotruncal CHD or DiGeorge phenotype (n = 137) were studied using MLPA. Ninety-three samples were studied by array-CGH and 18 by targeted or exome next-generation sequencing (NGS). A total of 240 patients were successfully studied using at least one technique. Cytogenetic abnormalities were observed in 13 patients, while 18 had clinically relevant imbalances detected by array-CGH. After MLPA, 26 patients presented 22q11 deletions or duplications and one presented a TBX1 gene deletion. Following NGS analysis, 12 patients presented pathogenic or likely pathogenic genetic variants, five of them, found in KAT6B, SHH, MYH11, MYH7 and EP300 genes, are novel. Using an algorithm that combines molecular techniques with clinical and genetic assessment, we determined the genetic contribution in 27.5% of the analyzed patients.

Adult-Onset Alexander Disease: New Causal Sequence Variant in the GFAP Gene.

ObjectivesAlexander disease (AD) is a rare disorder of the CNS. Diagnosis is based on clinical symptoms, typical MRI findings, and mutations in the glial fibrillary acid protein (GFAP) gene. In this case study, we describe a new mutation (p.L58P) in GFAP that caused a phenotype of adult-onset AD (AOAD).MethodsIn our outpatient clinic, a patient presented with cerebellar and bulbar symptoms after brain concussion. We used MRI and performed next-generation exome sequencing (NGS) to find mutations in GFAP to diagnose AD. The mutation was then transfected into HeLa cell lines to prove its pathogenicity.ResultsThe brain MRI finding showed typical AD alterations. The NGS found a heterozygous variant of unknown significance in GFAP (c.173T>C; p.L58P). After transfecting HeLa cell lines with this mutation, we showed that GFAP-L58P formed pathogenic clusters of cytoplasmic aggregates.DiscussionWe have found a new mutation that causes AOAD. We recommend that AOAD is included in the diagnostic workup in adult patients with gait ataxia and cerebellar and bulbar symptoms in association with a traumatic head injury.

Testing and extending strategies for identifying genetic disease–related encounters in pediatric patients

PurposeTo better understand health care utilization and develop decision support tools, methods for identifying patients with suspected genetic diseases (GDs) are needed. Previous studies had identified inpatient-relevant International Classification of Diseases (ICD) codes that were possibly, probably, or definitely indicative of GDs. We assessed whether these codes identified GD-related inpatient, outpatient, and emergency department encounters among pediatric patients with suspected GDs from a previous study (the North Carolina Clinical Genomic Evaluation by Next-Generation Exome Sequencing [NCGENES] study). MethodsUsing the electronic medical records of 140 pediatric patients from the NCGENES study, we characterized the presence of ICD codes representing possible, probable, or definite GD-related diagnoses across encounter types. In addition, we examined codes from encounters for which initially no GD-related codes had been found and determined whether these codes were indicative of a GD. ResultsAmong NCGENES patients with visits between 2014 and 2017, 92% of inpatient, 75% of emergency department, and 63% of outpatient encounters included ≥1 GD-related code. Encounters with highly specific (ie, definite) GD codes had fewer low-specificity GD codes than encounters with only low-specificity GD codes. We identified an additional 32 ICD-9 and 56 ICD-10 codes possibly indicative of a GD. ConclusionCode-based strategies can be refined to assess health care utilization among pediatric patients and may contribute to a systematic approach to identify patients with suspected GDs.

Clinicopathologic Implications of Complement Genetic Variants in Kidney Transplantation.

Genetic testing has uncovered rare variants in complement proteins associated with thrombotic microangiopathy (TMA) and C3 glomerulopathy (C3G). Approximately 50% are classified as variants of uncertain significance (VUS). Clinical risk assessment of patients carrying a VUS remains challenging primarily due to a lack of functional information, especially in the context of multiple confounding factors in the setting of kidney transplantation. Our objective was to evaluate the clinicopathologic significance of genetic variants in TMA and C3G in a kidney transplant cohort. We used whole exome next-generation sequencing to analyze complement genes in 76 patients, comprising 60 patients with a TMA and 16 with C3G. Ten variants in complement factor H (CFH) were identified; of these, four were known to be pathogenic, one was likely benign and five were classified as a VUS (I372V, I453L, G918E, T956M, L1207I). Each VUS was subjected to a structural analysis and was recombinantly produced; if expressed, its function was then characterized relative to the wild-type (WT) protein. Our data indicate that I372V, I453L, and G918E were deleterious while T956M and L1207I demonstrated normal functional activity. Four common polymorphisms in CFH (E936D, N1050Y, I1059T, Q1143E) were also characterized. We also assessed a family with a pathogenic variant in membrane cofactor protein (MCP) in addition to CFH with a unique clinical presentation featuring valvular dysfunction. Our analyses helped to determine disease etiology and defined the recurrence risk after kidney transplant, thereby facilitating clinical decision making for our patients. This work further illustrates the limitations of the prediction models and highlights the importance of conducting functional analysis of genetic variants particularly in a complex clinicopathologic scenario such as kidney transplantation.

Patient-Derived Organoids of Cholangiocarcinoma.

Cholangiocarcinoma (CC) is an aggressive malignancy with an inferior prognosis due to limited systemic treatment options. As preclinical models such as CC cell lines are extremely rare, this manuscript reports a protocol of cholangiocarcinoma patient-derived organoid culture as well as a protocol for the transition of 3D organoid lines to 2D cell lines. Tissue samples of non-cancer bile duct and cholangiocarcinoma were obtained during surgical resection. Organoid lines were generated following a standardized protocol. 2D cell lines were generated from established organoid lines following a novel protocol. Subcutaneous and orthotopic patient-derived xenografts were generated from CC organoid lines, histologically examined, and treated using standard CC protocols. Therapeutic responses of organoids and 2D cell lines were examined using standard CC agents. Next-generation exome and RNA sequencing was performed on primary tumors and CC organoid lines. Patient-derived organoids closely recapitulated the original features of the primary tumors on multiple levels. Treatment experiments demonstrated that patient-derived organoids of cholangiocarcinoma and organoid-derived xenografts can be used for the evaluation of novel treatments and may therefore be used in personalized oncology approaches. In summary, this study establishes cholangiocarcinoma organoids and organoid-derived cell lines, thus expanding translational research resources of cholangiocarcinoma.

Whole Exome Sequencing of Plasma Cells Precancerosis MGUS and Malignant AL Amyloidosis Reveals Small Number of Shared Mutated Genes

Background: Immunoglobulin light chain amyloidosis (ALA) is a plasma cell dyscrasia that develops, similarly as multiple myeloma (MM), from clinically indistinguishable precancerous stage, monoclonal gammopathy of undetermined significance (MGUS). The specific mutations causing malignant transformation from MGUS to amyloidogenic immunoglobulin producing ALA are still unknown. The next generation sequencing (NGS) technology is a powerful tool that allows comprehensive description of the whole genome sequence. Previous studies have already shown highly overlapping phenotypic profile between ALA and both MGUS and MM and lack of unifying mutations in ALA patients (Paiva, 2016). Here we present whole exome NGS data of aberrant plasma cells (aPCs) obtained from patients with MGUS (n=10) and ALA (n=9). Further comparison of the exome sequences of the precancerosis and malignant stage will help to identify recurrently occurring mutations, novel oncogenic drivers and tumor suppressor genes. This will offer new prognostic markers to distinguish potential malignant plasma cell clones in MGUS that will ultimately lead to the development of fatal ALA.Aims: Revealing the mutational status of aPCs in MGUS and ALA using the whole exome NGS.Material and methods: aPCs and peripheral blood (to exclude germinal mutations) were collected from 9 ALA and 10 MGUS samples. aPCs were separated using fluorescence activated cell sorting and genomic DNA was isolated using AllPrep DNA/RNA Micro Kit (Qiagen) and amplified with REPLI-g Mini (Qiagen). DNA library was prepared using SureSelect Human All Exon V5 Kit and sequenced on Illumina HiSeq 4000 platform with average coverage 50x. Somatic variants were called against human genome version 38 using VarScan v2.0. Only non-synonymous mutations, indels and splice site alterations were considered for further investigation.Results: Using NGS we describe whole exome profile for 9 ALA and 10 MGUS patients. Total number of mutated genes for ALA was 554 (average 69 SNVs per patient) and for MGUS 558 (average 73 SNVs per patient). The number of shared mutated genes between both groups was only 26. Among these genes, we identified 4 functional groups: A) Killer-Cell Immunoglobulin Like Receptors (KIR2DL1, KIR3DL2), B) Mucins (MUC16, MUC3A, MUC6), C) Poly(A) Binding Cytoplasmic Proteins (PABPC1, PABPC3) and D) Serine Proteases (PRSS1, PRSS2, PRSS3). Genetic profiles in ALA and MGUS did not reveal any already known MM driver mutations (KRAS, NRAS, BRAF, FAM46C, TP53 and DIS3), with the exception of one ALA patient possessing mutated DIS3 gene. Importantly, we have observed a significant heterogeneity between the samples, as the number of mutated genes shared by all MGUS or ALA patients was only 7 and 3 genes, respectively.Conclusions: ALA develops from a precancerous stage MGUS. Whole exome sequencing of the aPCs showed comparable number of mutated genes between both disease stages (554 vs. 558). Nevertheless, the mutated genes in both groups were very distinct. We identified only 26 mutated genes shared in both ALA and MGUS. Interestingly, none of these genes was previously linked to tumorigenesis. Moreover, genetic profiles were very heterogeneous among individual patients. Future analysis of the presented data will include comparison with genomic profiles obtained from MM plasma cells to reveal additional insights into the mechanisms of clonal plasma cells development in monoclonal gammopathies.Acknowledgment: This work was supported by the Ministry of Health (15-29667A; DRO - FNOs/2014, DRO - FNOs/2016) and the Ministry of Education, Youth and Sports (Institutional Development Plan of University of Ostrava-IRP201550). DisclosuresHajek:Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria; Novartis: Consultancy, Research Funding; Pharma MAR: Consultancy, Honoraria; BMS: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Research Funding.

Evaluating the clinical utility of early exome sequencing in diverse pediatric outpatient populations in the North Carolina Clinical Genomic Evaluation of Next-generation Exome Sequencing (NCGENES) 2 study: a randomized controlled trial

BackgroundExome sequencing (ES) has probable utility for shortening the diagnostic odyssey of children with suspected genetic disorders. This report describes the design and methods of a study evaluating the potential of ES as a routine clinical tool for pediatric patients who have suspected genetic conditions and who are in the early stages of the diagnostic odyssey.MethodsThe North Carolina Clinical Genomic Evaluation by Next-generation Exome Sequencing (NCGENES) 2 study is an interdisciplinary, multi-site Phase III randomized controlled trial of two interventions: educational pre-visit preparation (PVP) and offer of first-line ES. In this full-factorial design, parent-child dyads are randomly assigned to one of four study arms (PVP + usual care, ES + usual care, PVP + ES + usual care, or usual care alone) in equal proportions. Participants are recruited from Pediatric Genetics or Neurology outpatient clinics in three North Carolina healthcare facilities. Eligible pediatric participants are < 16 years old and have a first visit to a participating clinic, a suspected genetic condition, and an eligible parent/guardian to attend the clinic visit and complete study measures. The study oversamples participants from underserved and under-represented populations. Participants assigned to the PVP arms receive an educational booklet and question prompt list before clinical interactions. Randomization to offer of first-line ES is revealed after a child’s clinic visit. Parents complete measures at baseline, pre-clinic, post-clinic, and two follow-up timepoints. Study clinicians provide phenotypic data and complete measures after the clinic visit and after returning results. Reportable study-related research ES results are confirmed in a CLIA-certified clinical laboratory. Results are disclosed to the parent by the clinical team. A community consultation team contributed to the development of study materials and study implementation methods and remains engaged in the project.DiscussionNCGENES 2 will contribute valuable knowledge concerning technical, clinical, psychosocial, and health economic issues associated with using early diagnostic ES to shorten the diagnostic odyssey of pediatric patients with likely genetic conditions. Results will inform efforts to engage diverse populations in genomic medicine research and generate evidence that can inform policy, practice, and future research related to the utility of first-line diagnostic ES in health care.Trial registrationClinicalTrials.govNCT03548779. Registered on June 07, 2018.

PATH-03. CLINICAL UTILITY OF NEXT GENERATION SEQUENCING IN IDH-WILDTYPE GLIOBLASTOMA: THE DANA-FARBER CANCER INSTITUTE EXPERIENCE

Abstract BACKGROUND Comprehensive next generation sequencing (NGS) is available through many academic institutions and commercial entities, and is incorporated in practice guidelines for glioblastoma (GBM). We retrospective evaluated the practice patterns and utility of incorporating NGS data into routine care of GBM patients at a clinical trials-focused academic center. METHODS We identified 1,011 consecutive adult patients with histologically confirmed GBM with OncoPanel testing, a targeted exome NGS platform of 447 cancer-associated genes at Dana Farber Cancer Institute (DFCI), from 2013-2019. We selected and retrospectively reviewed clinical records of all IDH-wildtype GBM patients treated at DFCI. RESULTS We identified 557 GBM IDH-wildtype patients, of which 227 were male (40.7%). OncoPanel testing revealed 833 single nucleotide variants and indels in 44 therapeutically relevant genes (Tier 1 or 2 mutations) including PIK3CA (n=51), BRAF (n=9), FGFR1 (n=8), MSH2 (n=4), MSH6 (n=2) and MLH1 (n=1). Copy number analysis revealed 509 alterations in 18 therapeutically relevant genes including EGFR amplification (n= 186), PDGFRA amplification (N=39) and CDKN2A/2B homozygous loss (N=223). Median overall survival was 17.5 months for the whole cohort. Seventy-four therapeutic clinical trials accrued 144 patients in the upfront setting (25.9%) and 203 patients (36.4%) at recurrence. Altogether, NGS data for 107 patients (19.2%) were utilized for clinical trial enrollment or targeted therapy indications. High mutational burden (&amp;gt;17mutations/Mb) was identified in 11/464 samples (2.4%); of whom 3/11 received immune checkpoint blockade. Four patients received compassionate use therapy targeting EGFRvIII (rindopepimut, n=2), CKD4/6 (abemaciclib, n=1) and BRAFV600E (dabrafenib/trametinib, n=1). CONCLUSION While NGS has greatly improved diagnosis and molecular classification, we highlight that NGS remains underutilized in selecting therapy in GBM, even in a setting where clinical trials and off-label therapies are relatively accessible. Continued efforts to develop better targeted therapies and efficient clinical trial design are required to maximize the potential benefits of genomically-stratified data.

HIST1H2BB and MAGI2 Methylation and Somatic Mutations as Precision Medicine Biomarkers for Diagnosis and Prognosis of High-grade Serous Ovarian Cancer.

Molecular alterations that contribute to long-term (LT) and short-term (ST) survival in ovarian high-grade serous carcinoma (HGSC) may be used as precision medicine biomarkers. DNA promoter methylation is an early event in tumorigenesis, which can be detected in blood and urine, making it a feasible companion biomarker to somatic mutations for early detection and targeted treatment workflows. We compared the methylation profile in 12 HGSC tissue samples to 30 fallopian tube epithelium samples, using the Infinium Human Methylation 450K Array. We also used 450K methylation arrays to compare methylation among HGSCs long-term survivors (more than 5 years) and short-term survivors (less than 3 years). We verified the array results using bisulfite sequencing and methylation-specific PCR (qMSP). in another cohort of HGSC patient samples (n = 35). Immunoblot and clonogenic assays after pharmacologic unmasking show that HIST1H2BB and MAGI2 promoter methylation downregulates mRNA expression levels in ovarian cancer cells. We then used qMSP in paired tissue, ascites, plasma/serum, vaginal swabs, and urine from a third cohort of patients with HGSC cancer (n = 85) to test the clinical potential of HIST1H2BB and MAGI2 in precision medicine workflows. We also performed next-generation exome sequencing of 50 frequently mutated in human cancer genes, using the Ion AmpliSeqCancer Hotspot Panel, to show that the somatic mutation profile found in tissue and plasma can be quantified in paired urine samples from patients with HGSC. Our results suggest that HIST1H2BB and MAGI2 have growth-suppressing roles and can be used as HGSC precision medicine biomarkers.

Isolation and next generation sequencing of archival formalin-fixed DNA.

DNA from archived organs is presumed unsuitable for genomic studies because of excessive formalin‐fixation. As next generation sequencing (NGS) requires short DNA fragments, and Uracil‐N‐glycosylase (UNG) can be used to overcome deamination, there has been renewed interest in the possibility of genomic studies using these collections. We describe a novel method of DNA extraction capable of providing PCR amplicons of at least 400 bp length from such excessively formalin‐fixed human tissues. When compared with a leading commercial formalin‐fixed DNA extraction kit, our method produced greater yields of DNA and reduced sequence variations. Analysis of PCR products using bacterial sub‐cloning and Sanger sequencing from UNG‐treated DNA unexpectedly revealed increased sequence variations, compared with untreated samples. Finally, whole exome NGS was performed on a myocardial sample fixed in formalin for 2 years and compared with lymphocyte‐derived DNA (as a gold standard) from the same patient. Despite the reduction in the number and quality of reads in the formalin‐fixed DNA, we were able to show that bioinformatic processing by joint calling and variant quality score recalibration (VQSR) increased the sensitivity four‐fold to 56% and doubled specificity to 68% when compared with a standard hard‐filtering approach. Thus, high‐quality DNA can be extracted from excessively formalin‐fixed tissues and bioinformatic processing can optimise sensitivity and specificity of results. Sequencing of several sub‐cloned amplicons is an important methodological step in assessing DNA quality.