Targeted Next-generation Sequencing Analysis Research Articles

Side-by-side comparison of next-generation sequencing, cytology, and histology in diagnosing locally advanced pancreatic adenocarcinoma

EUS-guided biopsy sampling is the method of choice for obtaining pancreatic tissue. Next-generation sequencing (NGS) has been applied to EUS-guided biopsy sampling and may classify patients based on specific molecular profiles. Our study aimed to compare side-by-side the diagnostic yield achievable by genetic identification of somatic mutations detected with NGS versus histologic and cytologic typing in locally advanced pancreatic carcinoma (LAPC) in samples acquired under EUS guidance. We conducted a prospective comparative pilot study at Humanitas Research Hospital. The study included 33 patients referred for LAPC who underwent EUS-guided tissue acquisition using a 22-gauge Franseen needle. Material was obtained for both pathologic diagnosis and DNA extraction and targeted NGS analysis with the AmpliSeq Comprehensive Panel v3 (Illumina Inc, San Diego, Calif, USA). Twenty-one genes were prioritized for somatic mutation detection. The final diagnosis was pancreatic ductal adenocarcinoma (PDAC) in all patients (100%). A macroscopic core was obtained in 30 patients (91%). In 3 lesions no cores adequate for histologic analysis were obtained, but cytologic analysis revealed tumoral cells from PDAC. DNA was extracted from 32 of 33 samples (97%), most of which (27/32) carried at least 2 clearly pathogenic mutations in different genes. Detection of K-ras mutation allowed for molecular diagnosis of PDAC in most of the patients (30/32). In our study we demonstrated that proper tissue specimens obtained under EUS guidance allowed DNA sample extraction and subsequent NGS analysis in 97% of cases. These results support the potential role of NGS as a complementary diagnostic test to be implemented in association with standard diagnostic modalities. (Clinical trial registration number: NCT03578939.).

Genomic and Immunophenotypic Landscape of Aggressive NK-Cell Leukemia.

Aggressive natural killer-cell leukemia (ANKL) is a rare, lethal disease with pathologic features that are underdescribed in the literature, particularly in Western nations. In addition, although data on the molecular pathogenesis of ANKL has been reported, evaluation of such data in a clinicopathologic context remains limited. Patients diagnosed with ANKL were identified retrospectively. Detailed demographic and clinicopathologic data were analyzed. We assessed novel markers by immunohistochemistry and performed targeted next-generation sequencing analysis. The study group included 9 men and 3 women with a median age at diagnosis of 47.5 years (range, 20 to 75 y). Two distinct patterns of bone marrow involvement were identified: interstitial and sinusoidal. The neoplastic cells were positive for CD56 and CD94, and negative for surface CD3, CD5, and CD57 in all cases assessed. They were also positive for CD2 (10/12), c-MYC (6/8), BCL2 (6/8), CD16 (5/7), EBER (9/12), CD7 (6/11), pSTAT3 (3/8), CD8 (2/6), PD-L1 (2/8), CD4 (2/11), CD8 (2/6), and CD158 (1/5). Aberrant p53 expression was identified in most (7/8) cases; p53 was strongly expressed in 4 cases. Conventional cytogenetic analysis showed clonal abnormalities in 5 of 12 cases. TP53 mutations were detected in 3 of 6 cases, whereas ASXL1 and TET2 mutations were each detected in 2 of 6 cases. Patients had very poor outcomes despite intensive chemotherapy, with a median survival of 2 months. ANKL exhibits 2 distinct patterns of tissue involvement. Neoplastic cells in ANKL are commonly positive for c-MYC and EBER, and they have a high frequency of p53 overexpression, frequently with corresponding TP53 mutations.

Personalized and targeted mutational analysis of multiple second primary melanomas under kinase inhibitors.

Second primary melanomas (SPMs) are new developed primary melanomas occurring in a subset of patients affected by BRAF-mutated metastatic melanoma during treatment with BRAF-inhibitors. A drug-induced paradoxical activation of mitogen-activated protein kinase (MAPK) signaling pathway in BRAF-wild type/RAS-mutated cells have been proposed as a possible molecular mechanism but data on the mutational status of SPMs are lacking. In order to better understand genetic alterations affecting the biological mechanism of SPMs, we performed a personalized and targeted next-generation sequencing analysis of a patient affected by metastatic melanoma who developed multiple SPMs during treatment with encorafenib (LGX818). Using a cancer panel of 50 genes for solid tumors enriched with a custom panel of 10 genes specifically involved in melanoma pathogenesis, we analyzed the primary melanoma, two SPMs, one benign compound nevus and the normal DNA extracted from blood lymphocytes of the patient. We identified HRAS Q61 somatic mutation in one SPM developed in a pre-existing nevus. In the primary melanoma, besides the BRAF mutation, we identified the clinically actionable IDH1 R132C somatic mutation. Both SPMs were BRAF wild type. The patient harbors the recently recognized pathogenetic germline variant KDR Q472. We observed that mutations detected in tumor samples involving genes related to melanoma pathogenesis (TP53, PIK3CA, FGFR3, ATF1, KIT, HRAS and MAP2K2) were present in heterozygosis in the germline status of the patient. Our results support the paradoxical mechanism of MAPK pathway for SPMs under BRAF inhibitors. Moreover, they suggest that targeted mutational assessment based on matching somatic and germline analysis represent a promising approach to detect the neoplastic landscape of the tumor and to identify most accurate treatment in metastatic melanoma patient.

NPM1 mutations Using Deep Amplicon Sequencing and Broad Next Generation Sequencing at the Time of Complete Remission Is Informative to Predicting Risk of Relapse Following Intensive Chemotherapy

Introduction: NPM1 gene mutations are a common molecular aberration in acute myeloid leukemia (AML). In the absence of concurrent high FLT3-ITD ratio mutations (>0.5), NPM1 mutations typically associate with higher complete remission (CR) rates following intensive induction chemotherapy. NPM1 mutations have been shown to be stable markers of persistent disease or impending relapse during CR or complete remission with incomplete count recovery (CRi). Given the clinical implications that persistent NPM1 mutations can have during CR/CRi, we used Deep Amplicon sequencing on CR/CRi bone marrow (BM) samples collected from adult de novoNPM1-mutated AML patients to determine the ability of NPM1 mutations at both a high and lower sensitivity next generation sequencing methods and also the presence of additional clonal abnormalities on relapse risk. Methods: We performed targeted next generation sequencing (NGS) analysis in addition to NPM1 Deep Amplicon sequencing on paired BM or blood samples collected from 38 newly diagnosed NPM1-mutated AML patients during CR/CRi after successful induction (1-2 courses of 7 + 3) and, if available, at relapse. NPM1 mutated NGS libraries were prepared using a KAPA HyperPlus Kit (Roche, Pleasanton, CA) and xGen Lockdown Probes (IDT, Coralville, IA). Libraries were sequenced using the Illumina HiSeq 4000 (Illumina, San Diego, CA). GATK's MuTect2 was used to perform variant calling. Variant allele frequency (VAF) cut-off for the NGS panel was 0.05 (5%) with the exception of hotspot variants in IDH1 (R132) and IDH2 (R140) where variants detected to a level of 0.01 (1%) were included. The VAF cut off used for NPM1 Deep Amplicon sequencing was 0.00012 (0.012%). Results: Targeted NGS analysis and NPM1 Deep Amplicon sequencing had exceptional concordance at the level of detection of VAF= 0.05 (Figure 1). Of 38 patients, 23 patients had undetectable NPM1 mutations as analyzed through NPM1 Deep Amplicon sequencing of whom 9 (38.1%) relapsed. In contrast, 15 patients were positive by NPM1 Deep Amplicon sequencing and 9 (60%) relapsed. Only 4 patients had detectable persistent NPM1 mutations after induction according to both detection techniques and two of these relapsed. We next examined the potential impact of clearing both NPM1 mutation and co-occurring mutations together on relapses (Figure 2). A total of 15 patients cleared all of their clonal abnormalities and 5 (27%) relapsed. In contrast, of the 23 patients who did not clear the NPM1 mutation and/or another co-occurring mutation at remission, 14 (61%) have relapsed. Eleven of the relapsed patients had relapse samples available of whom all had persistent NPM1 mutation at this time. Paired CR/CRi and relapsed samples showed acquisition or recurrence of several other mutations, most notably FLT3-ITD, IDH1, and IDH2 which are all targetable with small molecule therapeutics. Conclusions: The use of Deep Amplicon sequencing to identify NPM1 mutations at a lower detection threshold compared to standard NGS techniques was more sensitive, but did not appear to fully inform relapse rates in NPM1-mutated AML patients after receipt of induction therapy. The appearance of other AML-associated mutations, identified together with NPM1 at time of remission, was more frequent among patients relapsing. These pilot data provide support for concurrent assessment of Deep Amplicon sequencing together with a broad standard NGS AML mutational assay to further enhance risk stratification of NPM1-mutated patients. Additionally, while NPM1 clones are present in all patients examined at the time of relapse, persistence or development of targetable clones justifies repeat broad NGS sequencing at this time. Figure Disclosures Bhatnagar: Novartis and Astellas: Consultancy, Honoraria; Cell Therapeutics, Inc.: Other: Research support; Karyopharm Therapeutics: Other: Research support. Mims:Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Astellas Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; PTC Therapeutics: Membership on an entity's Board of Directors or advisory committees. Behbehani:Fluidigm corporation: Other: Travel funding. Byrd:Novartis: Other: Travel Expenses, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; BeiGene: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S.

Technical advance in targeted NGS analysis enables identification of lung cancer risk-associated low frequency TP53, PIK3CA, and BRAF mutations in airway epithelial cells

BackgroundStandardized Nucleic Acid Quantification for SEQuencing (SNAQ-SEQ) is a novel method that utilizes synthetic DNA internal standards spiked into each sample prior to next generation sequencing (NGS) library preparation. This method was applied to analysis of normal appearing airway epithelial cells (AEC) obtained by bronchoscopy in an effort to define a somatic mutation field effect associated with lung cancer risk. There is a need for biomarkers that reliably detect those at highest lung cancer risk, thereby enabling more effective screening by annual low dose CT. The purpose of this study was to test the hypothesis that lung cancer risk is characterized by increased prevalence of low variant allele frequency (VAF) somatic mutations in lung cancer driver genes in AEC.MethodsSynthetic DNA internal standards (IS) were prepared for 11 lung cancer driver genes and mixed with each AEC genomic (g) DNA specimen prior to competitive multiplex PCR amplicon NGS library preparation. A custom Perl script was developed to separate IS reads and respective specimen gDNA reads from each target into separate files for parallel variant frequency analysis. This approach identified nucleotide-specific sequencing error and enabled reliable detection of specimen mutations with VAF as low as 5 × 10− 4 (0.05%). This method was applied in a retrospective case-control study of AEC specimens collected by bronchoscopic brush biopsy from the normal airways of 19 subjects, including eleven lung cancer cases and eight non-cancer controls, and the association of lung cancer risk with AEC driver gene mutations was tested.ResultsTP53 mutations with 0.05–1.0% VAF were more prevalent (p < 0.05) and also enriched for tobacco smoke and age-associated mutation signatures in normal AEC from lung cancer cases compared to non-cancer controls matched for smoking and age. Further, PIK3CA and BRAF mutations in this VAF range were identified in AEC from cases but not controls.ConclusionsApplication of SNAQ-SEQ to measure mutations in the 0.05–1.0% VAF range enabled identification of an AEC somatic mutation field of injury associated with lung cancer risk. A biomarker comprising TP53, PIK3CA, and BRAF somatic mutations may better stratify individuals for optimal lung cancer screening and prevention outcomes.

Pediatric myeloid sarcoma: a single institution clinicopathologic and molecular analysis

Myeloid sarcoma (MS) is a neoplastic condition composed of immature myeloid cells involving an extramedullary site. We investigated underlying chromosomal and molecular alterations to assess potential molecular markers of prognosis and outcome in this rare pediatric disease. We conducted a retrospective review of clinicopathologic and cytogenetic data from 33 pediatric patients with MS (ages 1 month–18 years) at our institution over a 32 year period (1984–2016). Tissue-based cancer microarray and targeted next-generation sequencing analysis were performed on six cases. The median age at diagnosis was 2.8 years with a male-to-female ratio of 2.6:1. MS is commonly presented with concomitant marrow involvement (n = 12, 36.4%) or as a recurrence of acute myeloid leukemia (AML; n = 14, 42.4%). The skin (n = 18, 54.5%) and soft tissue (n = 9, 27.3%) were the most common sites of involvement. Twenty-one of 25 samples (84.0%) harbored chromosomal aberrations; KMT2A alterations (n = 10, 40.0%) or complex cytogenetics (n = 7, 28.0%) were most frequent. Mutations in RAS, tyrosine kinase, cell signaling, and chromatin remodeling genes were detected. When compared to pediatric patients with AML without extramedullary involvement (EMI), inferior overall survival (OS) was observed (18.8 months vs. 89.3 months, p = .008). Pediatric patients with MS with non-favorable cytogenetics [abnormalities other than t(8;21), inv(16)/t(16;16), or t(15;17)] had a significantly lower OS compared to patients with AML with non-favorable cytogenetics and no extramedullary involvement (8.0 months vs. 28.1 months, p < .001). Pediatric MS is a rare disease with diverse clinical presentations. Non-favorable cytogenetics may be a poor prognostic marker for pediatric patients with MS and molecular diagnostics can assist with risk stratification and identify potentially actionable targets.

Morule-like features in pulmonary adenocarcinoma associated with epidermal growth factor receptor mutations: two case reports with targeted next-generation sequencing analysis.

Morules, or morule-like features, can be identified in benign and malignant lesions in various organs. Morular features are unusual in pulmonary adenocarcinoma cases with only 26 cases reported to date. Here, we describe two cases of pulmonary adenocarcinoma with morule-like features in Korean women. One patient had a non-mucinous-type adenocarcinoma in situ and the other had an acinarpredominant adenocarcinoma with a micropapillary component. Both patients showed multiple intra-alveolar, nodular, whorled proliferative foci composed of atypical spindle cells with eosinophilic cytoplasm. Targeted next-generation sequencing was performed on DNA extracted from formalin-fixed paraffin-embedded samples of the tumors. Results showed unusual epidermal growth factor receptor (EGFR) mutations, which are associated with drug resistance to EGFR tyrosine kinase inhibitors, revealing the importance of identifying morule-like features in pulmonary adenocarcinoma and the need for additional study, since there are few reported cases.

P2.17-25 Genomic Insight into Stage I Pulmonary Adenocarcinoma for Recurrence by Targeted Next-Generation Sequencing Analysis

P2.17-25 Genomic Insight into Stage I Pulmonary Adenocarcinoma for Recurrence by Targeted Next-Generation Sequencing Analysis

Cutaneous metastasis as a primary presentation of a pulmonary enteric adenocarcinoma

Primary pulmonary enteric adenocarcinoma (PEAC) is a rare non-small cell lung cancer subtype sharing morphologic and immunohistochemical features with colorectal adenocarcinoma. Given the frequency of lung metastases in colorectal cancer, the differential diagnosis of PEAC according to routine morphological and immunohistochemical findings may be difficult. Genome sequence by next-generation sequencing has recently introduced new perspectives to better define the diagnosis and tumor sensitivity to treatments, while the rarity of this subtype of cancer still limits the current knowledge of its molecular features and provides no information to address patients to tailored therapies. We diagnosed a rare case of subcutaneous metastasis as a first symptom of a PEAC. Formalin-fixed paraffin-embedded samples of the primary tumor and subcutaneous metastases were examined by immunohistochemistry, and subsequently by targeted next-generation sequencing analysis. Morphological and immunohistochemical findings suggested a rare case of metastatic pulmonary adenocarcinoma with enteric aspects. Next-generation sequencing analysis performed on both the primary tumor sample and the cutaneous lesion identified two pathogenic variants on CDKN2A and KRAS in both of them. However, the metastasis showed two additional pathogenic mutations located in SMAD4 and FLT3 genes. We describe for the first time an extensive molecular analysis on a rare case of PEAC with an unusual cutaneous metastasis. Our observation suggests that a specific pattern of mutations is harbored in this neoplasm, and that additional molecular studies may provide further information to identify prognostic and hopefully predictive genes of response to treatment.

Small lung tumor biopsy samples are feasible for high quality targeted next generation sequencing.

Next‐generation sequencing (NGS) has been implemented in clinical oncology to analyze multiple genes and to guide therapy. In patients with advanced lung cancer, small biopsies such as computed tomography‐guided needle biopsy (CTNB), endobronchial ultrasound‐guided transbronchial needle aspiration (EBUS‐TBNA) and transbronchial biopsy (TBB) are less invasive and are preferable to resection to make a pathological diagnosis. However, the quality of DNA/RNA and NGS from small lung tumor biopsy samples is unknown. Between April 2017 and March 2018, 107 consecutive samples were obtained from thoracic tumors or metastatic sites for targeted NGS analysis. Fifteen samples were obtained through CTNB, 11 through EBUS‐TBNA, 11 through TBB and 70 through surgical resection. All samples were formalin‐fixed and paraffin‐embedded. DNA and RNA quality was measured using the ddCq method and the percentage of RNA fragments above 200 nucleotides (DV200), respectively. Our custommade probes were designed to capture exon sequences of 464 cancer‐related genes and transcripts of 463 genes. DNA and RNA yield from the 3 biopsy methods were similar, and less than the yield obtained from resected samples. The quality of DNA and RNA was similar across all methods. Overall, 12 of 15 CTNB samples (80%), all 11 EBUS‐TBNA samples, and 9 of 11 TBB samples (82%) underwent successful NGS assays from DNA. NGS analysis from RNA was successful in all 12 CTNB samples, 9 of 11 EBUS‐TBNA samples (82%), and 8 of 11 TBB samples (73%). CTNB, EBUS‐TBNA and TBB mostly resulted in adequate DNA and RNA quality and enabled high‐quality targeted NGS analysis.

Identification of causative variants in patients with non-syndromic hearing loss in the Minnan region, China by targeted next-generation sequencing

Background: Due to extreme genetic heterogeneity, targeted next-generation sequencing (NGS) can be an efficient tool for rapid genetic diagnosis of hereditary non-syndromic hearing loss (NSHL).Aims/Objectives: This study was aiming to identify causative variants in NSHL patients from the Minnan region through targeted NGS.Material and methods: Genomic DNA samples from 90 NSHL subjects were extracted and subjected to SureSelect target enrichment system to capture the entire coding exons and flanking intronic regions of gene GJB2, SLC26A4, and MT-RNR1. The captured DNA was then sequenced by Illumina HiSeq2500. The sequence data was processed and quality-checked using Burrows-Wheeler Alignment, Picard, and GATK, and annotated by SnpEff, SIFT, and GERP++.Results: Twenty-six candidate variants in GJB2, SLC26A4, and MT-RNR1 were detected in 57 of 90 NSHL patients. GJB2 c.109G > A was the most frequent variant, followed by GJB2 c.608T > C and c.235delC. Genetic diagnosis was available for 22 NSHL patients, including 19 patients associated with autosomal recessive NSHL, one patients with autosomal dominant NSHL, and two individuals with mitochondrial disorders.Conclusions and significance: Our targeted NGS analysis added supports for the application of NGS in routine diagnosis and provided an overview of genetic variants with allele frequencies in the deaf population from the Minnan region.

Molecular genetic analysis using targeted NGS analysis of 677 individuals with retinal dystrophy

Inherited retinal diseases (IRDs) are a common cause of visual impairment. IRD covers a set of genetically highly heterogeneous disorders with more than 150 genes associated with one or more clinical forms of IRD. Molecular genetic diagnosis has become increasingly important especially due to expanding number of gene therapy strategies under development. Next generation sequencing (NGS) of gene panels has proven a valuable diagnostic tool in IRD. We present the molecular findings of 677 individuals, residing in Denmark, with IRD and report 806 variants of which 187 are novel. We found that deletions and duplications spanning one or more exons can explain 3% of the cases, and thus copy number variation (CNV) analysis is important in molecular genetic diagnostics of IRD. Seven percent of the individuals have variants classified as pathogenic or likely-pathogenic in more than one gene. Possible Danish founder variants in EYS and RP1 are reported. A significant number of variants were classified as variants with unknown significance; reporting of these will hopefully contribute to the elucidation of the actual clinical consequence making the classification less troublesome in the future. In conclusion, this study underlines the relevance of performing targeted sequencing of IRD including CNV analysis as well as the importance of interaction with clinical diagnoses.

GLIS Rearrangement is a Genomic Hallmark of Hyalinizing Trabecular Tumor of the Thyroid Gland.

Hyalinizing trabecular tumor (HTT) is a rare thyroid neoplasm with a characteristic trabecular growth pattern and hyalinization. This lesion has been the subject of long-term controversy surrounding its genetic mechanisms, relationship to papillary thyroid carcinoma (PTC), and malignant potential. Due to the presence of nuclear features shared with PTC, HTT frequently contributes to a false-positive cytology, which hampers patient management. The goal of this study was to apply genome-wide sequencing analyses to elucidate the genetic mechanisms of HTT and its relationship to PTC. Whole-exome, RNA-Seq, and targeted next-generation sequencing analyses were performed to discover and characterize driver mutations in HTT. RNA-Seq results were used for pathway analysis. Tissue expression of GLIS3 and other proteins was detected by immunohistochemistry. The prevalence of GLIS fusions was studied in 17 tumors initially diagnosed as HTT, 220 PTC, and 10,165 thyroid fine-needle aspiration samples. Using whole-exome and RNA-Seq analyses of the initial three HTT, no known thyroid tumor mutations were identified, while in-frame gene fusion between PAX8 exon 2 and GLIS3 exon 3 was detected in all tumors. Further analysis identified PAX8-GLIS3 in 13/14 (93%) and PAX8-GLIS1 in 1/14 (7%) of HTT confirmed after blind pathology review. The fusions were validated by Sanger sequencing and FISH. The fusions resulted in overexpression of the 3'-portion of GLIS3 and GLIS1 mRNA containing intact DNA-binding domains of these transcription factors and upregulation of extracellular matrix genes including collagen IV. Immunohistochemistry confirmed upregulation and deposition of collagen IV and pan-collagen in HTT. The analysis of 220 PTC revealed no PAX8-GLIS3 and one PAX8-GLIS1 fusion. PAX8-GLIS3 was prospectively identified in 8/10,165 (0.1%) indeterminate cytology fine-needle aspiration samples; 5/5 resected fusion-positive nodules were HTT on surgical pathology. This study demonstrates that GLIS rearrangements, particularly PAX8-GLIS3, are highly prevalent in HTT but not in PTC. The fusions lead to overexpression of GLIS, upregulation of extracellular matrix genes, and deposition of collagens, which is a characteristic histopathologic feature of HTT. Due to unique genetic mechanisms and an indolent behavior, it is proposed to rename this tumor as "GLIS-rearranged hyalinizing trabecular adenoma."

Common origin of sequential cutaneous CD30+ lymphoproliferations with nodal involvement evidenced by genome‐wide clonal evolution

This study sought to clarify the molecular pathways underlying the putative evolution from lymphomatoid papulosis (LyP) to cutaneous anaplastic large-cell lymphoma (c-ALCL) and lymph node invasion (LNI). We analysed nine sequential tumours from the same patient presenting with parallel evolution of LyP (n=3) and c-ALCL (n=1) with LNI (n=1), combined with systemic diffuse large B-cell lymphoma (DLBCL) (n=4). Clonality analysis showed a common clonal T-cell origin in the five CD30+ lesions, and a common clonal B-cell origin in the four DLBCL relapses. Array-comparative genomic hybridisation and targeted next-generation sequencing analysis demonstrated relative genomic stability of LyP lesions as compared with clonally related anaplastic large-cell lymphoma (ALCL) tumours, which showed 4q and 22q13 deletions involving the PRDM8 and TIMP3 tumour suppressor genes, respectively. The three analysed CD30+ lesions showed mostly private (specific to each sample) genetic alterations, suggesting early divergence from a common precursor. In contrast, DLBCL tumours showed progressive accumulation of private alterations, indicating late divergence. Sequential cutaneous and nodal CD30+ tumours were clonally related. This suggests that LyP, c-ALCL and LNI represent a continuous spectrum of clonal evolution emerging from a common precursor of cutaneous CD30+ lymphoproliferations. Therefore, nodal ALCL tumours in the context of LyP should be considered as a form of transformation rather than composite lymphoma.

Targeted next-generation sequencing reveals recurrence-associated genomic alterations in early-stage non-small cell lung cancer.

PurposeThe identification of genomic alterations related to recurrence in early-stage non-small cell lung cancer (NSCLC) patients may help better stratify high-risk individuals and guide treatment strategies. This study aimed to identify the molecular biomarkers of recurrence in early-stage NSCLC.ResultsOf the 42 tumors evaluable for genomic alterations, TP53 and EGFR were the most frequent alterations with population frequency 52.4% and 50.0%, respectively. Fusion genes were detected in four patients, which had lower mutational burden and relatively better genomic stability. EGFR mutation and fusion gene were mutually exclusive in this study. CDKN2A, FAS, SUFU and SMARCA4 genomic alterations were only observed in the relapsed patients. Increased copy number alteration index was observed in early relapsed patients. Among these genomic alterations, early-stage NSCLCs harboring CDKN2A, FAS, SUFU and SMARCA4 genomic alterations were found to be significantly associated with recurrence. Some of these new findings were validated using The Cancer Genome Atlas (TCGA) dataset.ConclusionsThe genomic alterations of CDKN2A, FAS, SUFU and SMARCA4 in early-stage NSCLC are found to be associated with recurrence, but confirmation in a larger independent cohort is required to define the clinical impact.Materials and MethodsPaired primary tumor and normal lung tissue samples were collected for targeted next-generation sequencing analysis. A panel targets exons for 440 genes was used to assess the mutational and copy number status of selected genes in three clinically relevant groups of stage I/II NSCLC patients: 1) Early relapse; 2) Late relapse; and 3) No relapse.

Archival May-Grünwald-Giemsa-Stained Bone Marrow Smears Are an Eligible Source for Molecular DNA Research.

Biobanking is increasingly important in studying complex heterogeneous diseases. Therefore, it is essential to ensure the sample quality after long-term storage for reliable downstream analyses. The Clinical Biobank of the Jessa Hospital and the University Biobank Limburg (UBiLim) hold a continuously growing collection of hematological samples, including May-Grünwald-Giemsa (MGG)- and Perls' Prussian Blue (PPB)-stained bone marrow (BM) smears, stored at room temperature (RT) for up to 20 years. In this study, we investigated the effect of short- and long-term storage on the quality of DNA and RNA extracted from these BM smears to assess their fitness-for-purpose in downstream molecular applications, including agarose gel electrophoresis, bio-analyzer analysis, quantitative polymerase chain reaction (qPCR), and targeted next-generation sequencing (NGS). The RNA quality was very low for all samples, independent of storage time or staining method. The DNA from PPB-stained BM smears was already degraded after 1 year of storage and correspondingly could not be used for reliable downstream molecular analysis. In contrast, DNA extracted from MGG-stained BM smears stored for up to 10 years was able to generate high-quality data in qPCR and targeted NGS analyses. Longer storage periods (>15 years) of these samples revealed a high degree of degradation and a significant amount of DNA transitions and transversions. In conclusion, the DNA extracted from archival MGG-stained BM smears with a storage time up to at least 10 years was qualitatively good and fit for downstream analysis, including targeted NGS. This indicates that these samples are an eligible source for molecular DNA research and for studying complex diseases.

Results of next-generation sequencing gene panel diagnostics including copy-number variation analysis in 810 patients suspected of heritable thoracic aortic disorders.

Simultaneous analysis of multiple genes using next‐generation sequencing (NGS) technology has become widely available. Copy‐number variations (CNVs) in disease‐associated genes have emerged as a cause for several hereditary disorders. CNVs are, however, not routinely detected using NGS analysis. The aim of this study was to assess the diagnostic yield and the prevalence of CNVs using our panel of Hereditary Thoracic Aortic Disease (H‐TAD)‐associated genes. Eight hundred ten patients suspected of H‐TAD were analyzed by targeted NGS analysis of 21 H‐TAD associated genes. In addition, the eXome hidden Markov model (XHMM; an algorithm to identify CNVs in targeted NGS data) was used to detect CNVs in these genes. A pathogenic or likely pathogenic variant was found in 66 of 810 patients (8.1%). Of these 66 pathogenic or likely pathogenic variants, six (9.1%) were CNVs not detectable by routine NGS analysis. These CNVs were four intragenic (multi‐)exon deletions in MYLK, TGFB2, SMAD3, and PRKG1, respectively. In addition, a large duplication including NOTCH1 and a large deletion encompassing SCARF2 were detected. As confirmed by additional analyses, both CNVs indicated larger chromosomal abnormalities, which could explain the phenotype in both patients. Given the clinical relevance of the identification of a genetic cause, CNV analysis using a method such as XHMM should be incorporated into the clinical diagnostic care for H‐TAD patients.

Abstract 1286: Analytical validation of an integrated next-generation sequencing pan-cancer liquid biopsy approach for detection of microsatellite instability

Abstract Failure of the DNA mismatch repair (MMR) pathway during DNA replication in cancer leads to the increased accumulation of somatic mutations. The molecular hallmark of MMR deficiency is microsatellite instability (MSI), which presents as polymorphism of tandem nucleotide repeat lengths ubiquitously distributed throughout the genome. Furthermore, the presence of MMR-deficiency or MSI across solid tumors has been recently demonstrated to be a robust biomarker for immunotherapy response with checkpoint inhibition. We have previously developed technology to detect MSI in tumor tissue through hybrid-capture based targeted next-generation sequencing analyses of a 125 gene panel, with probes capturing mononucleotide repeat markers. Using a set of 32 MSI and 27 microsatellite stable (MSS) formalin-fixed paraffin-embedded tumor tissue specimens, we obtained 100% sensitivity and 100% specificity with a limit of detection of 20% tumor purity. The clinical importance of MSI status now requires a fast, objective, highly sensitive screening method, particularly in late-stage patients where tumor material may not be readily obtained. However, to extend this approach to a liquid biopsy panel requires technological advances to overcome the inherent challenges associated with low circulating tumor DNA (ctDNA) levels that are compounded by polymerase slippage in mononucleotide repeat regions during PCR amplification. To overcome these limitations, we applied a multifactorial error correction approach together with a novel peak finding algorithm to more accurately identify the specific mononucleotide sequences in cell-free DNA (cfDNA) analyses. By eliminating a significant majority of sequencing errors and polymerase slippage artifacts, we were able to reduce background error rates by &amp;gt; 90%. Combined with implementation of a distribution modeling and a peak finding algorithm, we were able to accurately sequence the mononucleotide tracts to minimize false discovery rates for cfDNA analyses. To evaluate the performance of this approach in clinical samples, for a cohort of plasma samples obtained from 13 late-stage clinical cases with matched tumor tissue data available (9 MSI and 4 MSS) together with 66 healthy donor plasma samples, we obtained 89% sensitivity and 100% specificity, with a lowest reported mutant allele fraction of 0.4%. Taken together, this integrated next-generation sequencing liquid biopsy approach is valuable for the determination of MSI status to inform immunotherapy treatment. Citation Format: Andrew Georgiadis, Derrick Wood, Derek Murphy, Sonya Parpart-Li, David Riley, Naomi Sengamalay, Marian Novak, Sian Jones, Samuel A. Angiuoli, Mark Sausen. Analytical validation of an integrated next-generation sequencing pan-cancer liquid biopsy approach for detection of microsatellite instability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1286.

A novel homozygous variant of GPR98 causes usher syndrome type IIC in a consanguineous Chinese family by next generation sequencing

BackgroundUsher syndrome (USH) is a common heterogeneous retinopathy and a hearing loss (HL) syndrome. However, the gene causing Usher syndrome type IIC (USH2C) in a consanguineous Chinese pedigree is unknown.MethodsWe performed targeted next-generation sequencing analysis and Sanger sequencing to explore the GPR98 mutations in a USH2C pedigree that included a 32-year-old male patient from a consanguineous marriage family. Western blot verified the nonsense mutation.ResultsTo identify disease-causing gene variants in a consanguineous Chinese pedigree with USH2C, DNA from proband was analyzed using targeted next generation sequencing (NGS). The patient was clinically documented as a possible USH2 by a comprehensive auditory and ophthalmology evaluation. We succeeded in identifying the deleterious, novel, and homologous variant, c.6912dupG (p.Leu2305Valfs*4), in the GPR98 gene (NM_032119.3) that contributes to the progression of USH2C. Variant detected by targeted NGS was then confirmed and co-segregation was conducted by direct Sanger sequencing. Western blot verified losing almost two-thirds of its amino acid residues, including partial Calx-beta, whole EPTP and 7TM-GPCRs at the C-terminus of GPR98. Furthermore, our results highlighted that this p.Leu2305Valfs*4 variant is most likely pathogenic due to a large deletion at the seven-transmembrane G protein-coupled receptors (7TM-GPCRs) domain in GPR98 protein, leading to significantly decreased functionality and complex stability.ConclusionsThese findings characterized the novel disease causativeness variant in GPR98 and broaden mutation spectrums, which could predict the pathogenic progression of patient with USH2C, guide diagnosis and treatment of this disease; and provide genetic counseling and family planning for consanguineous marriage pedigree in developing countries, including China.

Molecular dissection of engraftment in a xenograft model of myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are oligoclonal disorders of the hematopoietic stem cells (HSC). Recurrent gene mutations are involved in the MDS physiopathology along with the medullar microenvironment. To better study the heterogeneity of MDS, it is necessary to create patient derived xenograft (PDX).We have reproduced a PDX model by xenografting HSC (CD34+) and mesenchymal stromal cells (MSC) in NOD/SCID/IL2rγ-/- mice with primary samples from one RAEB2, two RAEB1 and one RARS patients harboring karyotype abnormalities and gene mutations. The average human chimerisms ranged from 59.7% to 0.0175% for the 4 patients. Secondary grafts (G2) were only performed for mice derived from the RAEB2 patient and the average human chimerism was 53.33%. G1 mice 1 and 2, and their derived G2 mice showed less than 20% of medullar blasts whereas mouse 3 and the resulting G2 mice transformed to AML. Clonal architecture was dissected in the different hematopoietic progenitors (HP) harvested from G1 and G2 mice. By direct Sanger sequencing, we found the 4 initial mutations in each HP subpopulation and those mutations had the same variant allele frequency in the CD34+ CD38- HSC from G1 and G2 mice by next generation sequencing (NGS). Targeted NGS analysis done in HSC of mouse 3 did not show any additional driver gene mutations explaining the transformation to AML.To conclude, we have generated a PDX mouse model that perfectly reproduces the MDS founder clone which is stable over time, allowing us to consider this system as a powerful tool to test therapeutic approaches.