Generation Sequencing-based Assay Research Articles

Identification of EWSR1 rearrangements in patients with immature hematopoietic neoplasms: A case series and review of literature

Rearrangement of the EWSR1 gene (22q12.2) is a well-recognized genetic lesion in bone and soft tissue tumors. However, few reports have suggested that EWSR1 rearrangements may also occur in the setting of hematopoietic tumors. We herein describe two cases of immature hematopoietic neoplasms presenting with EWSR1 rearrangements. The first occurred in a 41-year-old female diagnosed with mixed-phenotype acute leukemia, B/T/myeloid, in which conventional chromosome analysis revealed a t(2;22)(q35;q12). Further analysis with whole genome sequencing revealed that this rearrangement led to an EWSR1::FEV gene fusion. The second case was identified in an 18-year-old male with a high-grade B-cell lineage malignant neoplasm with immature features in which conventional chromosome analysis revealed a t(17;22)(q25;q12). Mate-pair sequencing, a next generation sequencing-based assay, was performed and revealed three in-frame chimeric gene fusions involving the EWSR1, TEF and STRADA gene regions. This report further expands the repertoire of hematopoietic neoplasms with EWSR1 fusions and partner genes involved in these rearrangements.

Prognostic Roles of BRAF, KIT, NRAS, IGF2R and SF3B1 Mutations in Mucosal Melanomas.

Background: The prognostic value of commonly recurrent mutations remains unclear in mucosal melanomas. Methods: Clinicopathologic parameters of 214 cases of mucosal melanomas diagnosed in 1989–2020 in several clinical institutions were analyzed. NRAS, KIT, BRAF, IGF2R and SF3B1 mutational analyses by Sanger sequencing and next generation sequencing-based assay were performed in a subset of cases. Results: Of the triple (BRAF, NRAS, NF1)-negative cases, APC, KIT and KRAS are detected mainly in sinonasal, vulvovaginal and anorectal melanomas, respectively. NRAS, KIT, BRAF, IGF2R and SF3B1 mutations are detected in 19% (37/198), 22% (44/197), 12% (25/201), 16% (22/138) and 15% (20/133) of cases, respectively. In univariate analyses, advanced stage (p = 0.016), 65 years or older (p = 0.048) and presence of ulceration (p = 0.027) are significantly correlated with worse overall survival (OS), respectively. NRAS mutation significantly correlates with worse OS (p = 0.028) and worse melanoma-specific survival (MSS) (p = 0.03) for all cases of mucosal melanomas. In multivariate analyses, NRAS mutation remains as an independent predictor of worse OS (p = 0.036) and worse MSS (p = 0.024). Conclusion: NRAS mutation is a predictor of worse survival, independent of stage in mucosal melanomas. The significance of frequently mutated IGF2R in mucosal melanomas remains unclear.

Screening of Bacteriophage Encoded Toxic Proteins with a Next Generation Sequencing-Based Assay.

Bacteriophage vB_EcoM_fHy-Eco03 (fHy-Eco03 for short) was isolated from a sewage sample based on its ability to infect an Escherichia coli clinical blood culture isolate. Altogether, 32 genes encoding hypothetical proteins of unknown function (HPUFs) were identified from the genomic sequence of fHy-Eco03. The HPUFs were screened for toxic properties (toxHPUFs) with a novel, Next Generation Sequencing (NGS)-based approach. This approach identifies toxHPUF-encoding genes through comparison of gene-specific read coverages in DNA from pooled ligation mixtures before electroporation and pooled transformants after electroporation. The performance and reliability of the NGS screening assay was compared with a plating efficiency-based method, and both methods identified the fHy-Eco03 gene g05 product as toxic. While the outcomes of the two screenings were highly similar, the NGS screening assay outperformed the plating efficiency assay in both reliability and efficiency. The NGS screening assay can be used as a high throughput method in the search for new phage-inspired antimicrobial molecules.

Next generation sequencing of a set of ancestry-informative SNPs: ancestry assignment of three continental populations and estimating ancestry composition for Mongolians.

When traditional short tandem repeat profiling fails to provide valuable information to arrest the criminal, forensic ancestry inference of the biological samples left at the crime scene will probably offer investigative leads and facilitate the investigation process of the case. That is why there are consistent efforts in developing panels for ancestry inference in forensic science. Presently, a 30-plex next generation sequencing-based assay was exploited in this study by assembling well-differentiated single nucleotide polymorphisms for ancestry assignment of unknown individuals from three continental populations (African, European and East Asian). And meanwhile, relatively balanced population-specific differentiation values were maintained to avoid the over-estimation or under-estimation of co-ancestry proportions in individuals with admixed ancestry. The principal component analysis and STRUCTURE analysis of reference populations, test populations and the studied Mongolian group indicated that the novel assay was efficient enough to determine the ancestry origin of an unknown individual from the three continental populations. Besides, ancestry membership proportion estimations for the Mongolian group revealed that a large fraction of the ancestry was contributed by East Asian genetic component (approximately 83.9%), followed by European (approximately 12.6%) and African genetic components (approximately 3.5%), respectively. And next generation sequencing technology applied in this study offers possibility to incorporate more single nucleotide polymorphisms forindividual identification and phenotype prediction into the same assay to provide as many as possible investigative cluesin the future.

Abstract CT053: Preliminary safety, efficacy, and PK/PD characterization from GARNET, a phase 1 clinical trial of the anti-PD-1 monoclonal antibody, TSR-042, in patients with recurrent or advanced NSCLC and MSI-H endometrial cancer

Abstract Introduction: TSR-042 is a humanized monoclonal antibody targeting programmed death (PD)-1, effectively blocking interaction with its ligands PD-L1 and PD-L2. TSR-042 is being evaluated in patients (pts) with advanced solid tumors in the ongoing phase 1 GARNET trial (NCT02715284). Weight based dose escalation (Part 1) and fixed-dose safety studies (Part 2A) have been completed,1 and the study is currently enrolling pts with specific tumor types into expansion cohorts. Here, we present safety and efficacy data from the microsatellite instability-high (MSI-H) endometrial cancer (EC) and non-small cell lung cancer (NSCLC) cohorts, as well as pharmacokinetic (PK) and receptor occupancy (RO) characterization at the recommended phase 2 dose (RP2D). Methods/Procedures: 30 NSCLC pts and 19 MSI-H EC pts with previously treated recurrent or advanced disease were enrolled; median number of prior lines of therapy for metastatic disease was 1.0 for both cohorts. MSI status for EC pts was determined centrally using a next generation sequencing-based assay. Key exclusion criteria included prior therapy with agents targeting anti-PD-1, PD-L1, or PD-L2, and uncontrolled CNS metastases. Pts were treated at the R2PD of TSR-042: 500 mg Q3W for the first 4 cycles and 1000 mg Q6W thereafter. Serum and PBMCs were collected for PK and RO analyses, respectively. Antitumor activity was assessed by investigators using immune-related Response Evaluation Criteria in Solid Tumors (irRECIST). RO was assessed using a CD3+ binding assay (direct receptor occupancy).1 Results: Among the 30 NSCLC and 19 MSI-H EC pts, 42 (85.7%) pts reported ≥1 adverse event (AE), with grade ≥3 AEs reported in 13/49 (26.5%) pts. The most common AEs were diarrhea and nausea (11 pts each), arthralgia and fatigue (9 pts each), and cough, decreased appetite, and dyspnea (7 pts each). 25/49 (51.0%) pts reported treatment-related AEs; 11/49 (22.4%) pts had serious AEs; 1 case of grade 3 fatigue and 1 case of grade 3 leukopenia and grade 3 neutropenia were deemed treatment-related. 21 NSCLC and 11 MSI-H EC pts had at least 1 tumor assessment. Among NSCLC pts, 7/21 (33.3%) had a partial response (irPR; confirmed and unconfirmed), and 6/21 (28.6%) had stable disease (irSD); among MSI-H EC pts, 4/11 (36.4%) had irPR, and 2/11 (18.2%) had irSD. TSR-042 demonstrated dose-proportional PK. The maximal achievable RO was observed in pts treated at the RP2D, consistent with the results reported for Parts 1 and 2A.1 Conclusions: These results indicate clinical benefit of TSR-042 in previously treated NSCLC and MSI-H EC patients, with a safety profile similar to other PD-1 inhibitors. PK results were consistent across all patients evaluated and show that maximal achievable receptor occupancy was attained at the RP2D. 1. Sachdev JC et al. Ann Oncol. 2017(suppl 5):28:420;1185P Citation Format: Victor Moreno, Maria-Pilar Barretina-Ginesta, Wei Guo, Sharon Lu, David Jenkins, Kristen McEachern, Vienna Reichert, Steven Dunlap, Ellie Im, Lucy Gilbert, Ana Oaknin, Charles Leath, III, Janakiraman Subramanian. Preliminary safety, efficacy, and PK/PD characterization from GARNET, a phase 1 clinical trial of the anti-PD-1 monoclonal antibody, TSR-042, in patients with recurrent or advanced NSCLC and MSI-H endometrial cancer [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 CT053.

A Comprehensive Clinical Next Generation Sequencing-Based Assay Can Impact Hematopathologic Diagnosis in a Significant Subset of Patients with Hematologic Malignancies

High throughput genomic studies have identified novel recurrent somatic alterations with prognostic or therapeutic relevance in hematological malignancies. By contrast, few studies have investigated the impact on pathologic assessment, and clinicopathologic diagnosis. We therefore assessed the impact of a CLIA-certified CAP-accredited comprehensive clinical grade next-generation sequencing-based assay, FoundationOne Heme (FOH), on hematopathologic assessment of patients at our institution.The FOH assay targets 405 cancer-related genes and 31 frequently rearranged genes by DNA-capture and sequencing, and 265 frequently-rearranged genes by RNA-capture and sequencing. We prospectively tested 92 cases as part of routine clinical practice, including malignant lymphoid neoplasms (n=51) and suspected myeloid malignancies (n=41). (Table 1) The samples submitted included 30 blood, 38 bone marrow and 24 FFPE specimens.We were able to obtain genomic profiling data in90 of the 92 submitted cases (98%) including all FFPE specimens. A total of 282 genomic abnormalities (range 0-11, average 3.2 abnormalities/case) including substitutions, insertions/deletions and gene fusions were detected. There was excellent concordance between conventional cytogenetic or molecular genetic assays and FOH assay. Only 10 cases lacked any genomic abnormality. Six of these were morphologically normal bone marrows submitted to rule out myeloid neoplasia. Of the 4 remaining cases without recurrent somatic alterations, 2 cases were derived from myeloma patients where the sample analyzed had less than 20% plasma cells and 2 cases were from patients with myelodysplasia with likely low tumor content. In 84 cases with a diagnosis of hematologic malignancy, we identified genomic abnormalities with diagnostic relevance in 42 cases (50%). Most importantly, in 12 cases (14% of cohort), the presence of specific genomic alterations led to a change or refinement of the diagnosis. (Table 2) This included two cases in which a diagnosis of T-LGL was confirmed based on the presence of STAT3 mutations, three cases of lymphoma/myeloma in which a specific diagnosis was reached based on identification of pathogenic fusions/rearrangements, and 4 cases of MPN/MDS which could be confirmed based on the presence of known MPN/MDS disease alleles. In addition, we identified genomic alterations with prognostic relevance in 54 cases (64%), and with potential therapeutic impact in 64 cases (76%).Our data demonstrate that the FOH assay can be performed with a very high success rate (98%) in a routine clinical setting and that comprehensive genomic profiling can substantively impact pathologic assessment and diagnosis of a wide spectrum of hematologic malignancies. Genomic testing provided critical diagnostic information in half of the cases, in some instances refining or changing the conventional pathological diagnosis. These findings suggest that comprehensive targeted genomic testing has an important role to play not only in identifying prognostic and therapeutic targets but also in hematopathology diagnosis, and should be considered as a first line testing platform in hematologic malignancies. Table 1:Clinical characteristicsTotal number of casesN=92Median age58 (18-82)SexMale58 (63%)Female34 (37%)Myeloid neoplasms33 (36%)AML14MDS11MPN6CMML1CML1Lymphoid neoplasms51 (55%)DLBCL17B-ALL6TLPD6MCL5Myeloma5CLL3MZL3FL3BCL-NOS2CHL1Normal marrow6 (7%)Sample failed2 (2%)Genomic abnormalities in hematologic malignancies80/84 (93%)Diagnostic42/84 (50%)Prognostic54/84 (64%)Potential therapeutic64/84 (76%)Table 2:Genomic changes which led to improved diagnosis and classification of the hematologic malignancyCaseDiagnosis/ProblemGenomic alterationFinal/refined diagnosis1Neutropenia, T-LGL?STAT3 (N647I)T-LGL2Neutropenia, T-LGL?STAT3 (D661Y)T-LGL3T-LPD?JAK3 (M511I)T-PLL4T-LPD?TET2 (Q1523*), TP53 (R175G)PTCL, NOS5Transformed FLCIITA-DSCAML1 (Fusion)PMBL6CHL vs ALCLIGH-BCL2 (Rearrang.)DLBCL7BCL-NOSIGL-MYC (Rearrang.)BCL between DLBCL/BL8MyelomaIGH-MAF8 (Rearrang.)High risk myeloma9MDS?STK11 (F354L) RUNX1-MECOM (Fusion)MDS10MDS?MLL (ITD)MDS11MDS?KDM6AMDS12MPNMPL (R514_W515>KK)ET DisclosuresMoskowitz:Seattle Genetics, Inc.: Consultancy, Research Funding; Genentech: Research Funding; Merck: Research Funding. Horwitz:Research: Celgene, Millennium, Infinity, Kiowa-Kirin, Seattle Genetics, Spectrum-Consulting: Amgen, Bristol-Myers Squibb, Celgene, Jannsen, Millennium, seattle genetics: Consultancy, Honoraria, Research Funding. Stein:Janssen Pharmaceuticals: Consultancy. He:Foundation Medicine: Employment. Stephens:Foundation Medicine, Inc. : Employment, Equity Ownership. Miller:Foundation Medicine: Employment. Younes:Novartis: Research Funding; J & J: Research Funding; Curis: Research Funding; Bayer; Bristol Meyer Squibb; Celgene; Incyte; Janssen R & D; Sanofi; Seattle Genetics; Takeda Millenium: Honoraria. Dogan:Foundation Medicine: Consultancy.

Abstract 3570: Development and validation of a clinical next generation sequencing-based assay for hematologic malignancies

Abstract Background: Next-generation sequencing (NGS) is rapidly becoming an indispensable cancer diagnostic, as it can detect most genomic alterations in a single assay from limited tissue. We developed a novel, NGS-based assay designed to provide targeted assessment of the genomic landscape of hematologic malignancies from archived FFPE, blood and bone marrow aspirate samples, sequencing both DNA and RNA to improve sensitivity for driver fusion events which are common in these tumors. Methods: The high accuracy of the assay for detection of substitutions, indels and CNAs was previously demonstrated by extensive validation studies achieving 95-99% across all alteration types with high specificity (PPV>99%) [Frampton et al, Nat Biotech, 2013]. To validate assay performance in detecting gene fusions we created reference samples by mixing 21 cell-lines with previously characterized fusions in 39 combinations representing 167 fusion events in 10-50% tumor cell fractions. In addition, we confirmed accuracy in 76 clinical hematologic FFPE and bone-marrow samples profiled for 212 substitutions, indels and fusions in 11 genes by Sanger sequencing, PCR, fragment sizing and FISH. DNA and RNA were extracted from all samples; adaptor ligated sequencing libraries were captured by solution hybridization using custom baits targeting 405 cancer related genes by DNA-seq, and 265 frequently rearranged genes by RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq) in a CLIA-certified laboratory (Foundation Medicine), averaging 467x for DNA and 6M unique pairs for RNA. Results: On reference samples, sensitivity for detection of fusions events reached >99% for tumor cell fractions of 20-50%, and 97% for tumor fraction of 10%, all with high specificity (PPV>95%). Robust performance translated to the clinical samples: we observed a concordance rate of 98.6% relative to prior calls, with only 3/212 differing calls (2+, 1-) by NGS. 129 additional known oncogenic alterations in 57 different genes were detected in these samples, for a total of 3.1 alterations per sample. Analysis of 290 additional leukemia, lymphoma and myeloma patient samples revealed known and novel gene fusions in 15% of cases, more than half of which were identifiable only by RNA-seq. Conclusions: We describe the analytic validation of a sensitive, high throughput assay to detect somatic alterations in hundreds of genes known to be deregulated in hematologic malignancies, which can be used to identify a spectrum of somatic alterations from blood, bone marrow and paraffin embedded patient samples. We demonstrate that targeted DNA and RNA sequencing can be used to identify all classes of genomic alterations, including gene fusions, with high accuracy. This approach offers the opportunity to streamline the characterization of genomic alterations in hematologic malignancies and to expand targeted treatment options for patients with liquid tumors. Citation Format: Doron Lipson, Michelle K. Nahas, Geoff A. Otto, Jie He, Kai Wang, Kristina M. Knapp, Kristina W. Brennan, Amy L. Donahue, Lauren E. Young, Geneva Young, Alex Fichtenholtz, Jeffrey S. Ross, Roman Yelensky, Philip J. Stephens, Vincent A. Miller, Ross Levine. Development and validation of a clinical next generation sequencing-based assay for hematologic malignancies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3570. doi:10.1158/1538-7445.AM2014-3570