Next-generation Sequencing For Detection Research Articles

Use of next generation sequencing for detection of hereditary cancer genes: A shared experience from the UAE national reference laboratory

Use of next generation sequencing for detection of hereditary cancer genes: A shared experience from the UAE national reference laboratory

Developmental validation of the ForenSeq® Kintelligence kit, MiSeq FGx® sequencing system and ForenSeq Universal Analysis Software

Forensic Investigative Genetic Genealogy, a recent sub discipline of forensic genomics, leverages the high throughput and sensitivity of detection of next generation sequencing and established genetic and genealogical approaches to support the identification of human remains from missing persons investigations and investigative lead generation in violent crimes. To facilitate forensic DNA evidence analysis, the ForenSeq® Kintelligence multiplex, consisting of 10,230 SNPs, was developed. Design of the ForenSeq Kintelligence Kit, the MiSeq FGx® Sequencing System and the ForenSeq Universal Analysis Software is described. Developmental validation in accordance with SWGDAM guidelines and forensic quality assurance standards, using single source samples, is reported for the end-to-end workflow from library preparation to data interpretation. Performance metrics support the conclusion that more genetic information can be obtained from challenging samples compared to other commercially available forensic targeted DNA assays developed for capillary electrophoresis (CE) or other current next generation sequencing (NGS) kits due to the higher number of markers, the overall shorter amplicon sizes (97.8% <150 bp), and kit design. Data indicate that the multiplex is robust and fit for purpose for a wide range of quantity and quality samples. The ForenSeq Kintelligence Kit and the Universal Analysis Software allow transfer of the genetic component of forensic investigative genetic genealogy to the operational forensic laboratory.

Mutation landscape in Chinese nodal diffuse large B-cell lymphoma by targeted next generation sequencing and their relationship with clinicopathological characteristics

BackgroundDiffuse large B-cell lymphoma (DLBCL), an aggressive and heterogenic malignant entity, is still a challenging clinical problem, since around one-third of patients are not cured with primary treatment. Next-generation sequencing (NGS) technologies have revealed common genetic mutations in DLBCL. We devised an NGS multi-gene panel to discover genetic features of Chinese nodal DLBCL patients and provide reference information for panel-based NGS detection in clinical laboratories.MethodsA panel of 116 DLBCL genes was designed based on the literature and related databases. We analyzed 96 Chinese nodal DLBCL biopsy specimens through targeted sequencing.ResultsThe most frequently mutated genes were KMT2D (30%), PIM1 (26%), SOCS1 (24%), MYD88 (21%), BTG1 (20%), HIST1H1E (18%), CD79B (18%), SPEN (17%), and KMT2C (16%). SPEN (17%) and DDX3X (6%) mutations were highly prevalent in our study than in Western studies. Thirty-three patients (34%) were assigned as genetic classification by the LymphGen algorithm, including 12 cases MCD, five BN2, seven EZB, seven ST2, and two EZB/ST2 complex. MYD88 L265P mutation, TP53 and BCL2 pathogenic mutations were unfavorable prognostic biomarkers in DLBCL.ConclusionsThis study presents the mutation landscape in Chinese nodal DLBCL, highlights the genetic heterogeneity of DLBCL and shows the role of panel-based NGS to prediction of prognosis and potential molecular targeted therapy in DLBCL. More precise genetic classification needs further investigations.

The Use of Next Generation Sequencing to Detect Early Implant Infections: The Future of Microbiology and Targeted Antibiotic Treatment

Abstract Goals/Purpose The presence of fluid around foreign bodies, particularly breast implants, is a well-established phenomenon; However scant data exists regarding the composition of this fluid. The difficulty in studying this fluid arises from the lack of safe access to aspirate fluid post-breast augmentation, and by the loss of sterility once fluid exits the body via a drain bulb as traditionally placed during breast reconstruction. Our innovative breast reconstruction approach utilizes a drainless technique with a dual chamber tissue expander (TE) in the pre-pectoral plane, offering a unique avenue to sterilely aspirate peri-implant fluid through a percutaneous drainage port. In this study, we employ Next Generation Sequencing (NGS), utilizing polymerase chain reaction technology, to discern the composition of microbial DNA within peri-prosthetic breast implant fluid. NGS, known for its precision and cost-effectiveness, circumvents the biases inherent in traditional culture methods. By integrating this cutting-edge technology with our breast reconstruction methodology, we sought to study peri-prosthetic breast implant fluid to better understand when implant infections arise and if peri-prosthetic fluid could detect infections before clinical symptoms arise. Methods/Technique This is a single surgeon, prospective study of patients undergoing mastectomy, followed by pre-pectoral, drainless, TE reconstruction. Peri-prosthetic fluid was collected through the TE drainage port in the OR immediately after closure, at 1 week post op, and 3 weeks post op. This fluid was sent for traditional culture and NGS. Patients were observed for signs of infection, antibiotics, return to OR, or implant loss. Results/Complications 33 breasts from 20 patients were included. Patients had a mean age of 50 years. The average length of drainage was 21 days post-operative, with a mean of 131 cc of fluid drained per breast per week. Eight breasts had concern for skin necrosis. In 25 breasts (76%), complete sterility was achieved at the end of the case with no NGS detection of microorganisms in the initial peri-implant fluid sample. However, NGS detected microorganisms in the initial sample in 8 breasts (24%). In 5 of these 8 breasts, microorganisms were cleared by the 1-week sample and another 2 cleared by 3 weeks. The remaining 1 breast continued to have positive NGS and developed clinical signs of infections by week 2, which were not picked up by traditional culture until week 3. One breast with skin necrosis developed a positive NGS at week 3 and clinical signs of infection at week 4. No other clinical infections were seen throughout the study. Conclusion We are one of the first groups to demonstrate the ability to achieve peri-implant sterility at the time of implant insertion in the operating room. This could have significant implications for understanding the necessity of peri-operative antibiotics following breast implant placement. Our investigation into the microbial composition of ostensibly "sterile" peri-prosthetic breast implant fluid provides insight into when microorganisms arise and when they become clinical infections. These promising preliminary results suggests that peri-prosthetic fluid monitoring through Next-Generation Sequencing (NGS) holds significant potential in preemptively detecting implant infections before the onset of clinical symptoms, thereby reducing infection rates. Furthermore, our findings open avenues for targeted antibiotic therapy, contributing to the imperative goal of antibiotic stewardship. Ongoing work is beginning to explore the impact of microorganisms within the fluid on breast capsule formation, as we strive to enhance our understanding of breast implants and infections.

Prevalence and detection methodology for preliminary exploration of NTRK fusion in gastric cancer from a single-center retrospective cohort

The fusion of neurotrophic tyrosine receptor kinase (NTRK) is a novel target for cancer therapy and offers hope for patients with gastric cancer (GC). However, there are few studies on the prevalence and detection methods of NTRK fusions in GC. In this study, we used immunohistochemistry (IHC) as a screening method to select cases for molecular testing and evaluated the effectiveness of IHC, fluorescence in situ hybridization (FISH), and next-generation sequencing (NGS). We retrospectively collected 1970 patients with GC. Pan-TRK IHC was conducted in all cases, and three cases were positive: one with strong and diffuse cytoplasmic staining, while two with weak cytoplasmic staining. All three cases were validated using NTRK1/2/3 FISH. FISH results revealed a single 3′ signal of NTRK1 in 95% of the tumor cells in the first case, while the remaining two cases were negative. NGS confirmed LMNA-NTRK1 fusion in the first case, with no gene fusion detected in the other two cases. Out of 46 negative controls, one had a non-functional fusion of IGR-NTRK1, and four had point mutations. The case with LMNA-NTRK1 fusion were negative for pMMR, EBV, HER2, and AFP. The pan-TRK IHC showed a 33.33% (1/3) concordance rate with RNA-based NGS. If the criterion for positivity was 3+ cytoplasmic staining, the agreement between IHC and RNA-based NGS was 100% (1/1). In conclusion, the incidence of NTRK fusion in GC is extremely low (0.05%). If the criteria are strict, pan-TRK IHC is highly effective for screening NTRK fusions. FISH could complement NGS detection, particularly when NTRK fusion is detected by DNA sequencing. NTRK fusion in GC may not be limited to specific subtypes.

Application Gene Variation Detection before and after Hematopoietic Stem Cell Transplantation to Predict the Relapse of Hematopoietic Neoplasm Patients after Transplantation: Retrospective Data from a Single Center in China

Objective: To evaluate the changes of gene variation in patients with hematological neoplasms at the time of diagnosis and after allogeneic hematopoietic stem cell transplantation (allo-HSCT), and the relationship between gene mutation and recurrence after transplantation. Methods: 76 patients with hematological neoplasms were analyzed retrospectively, including 69 acute myeloid leukemia, 21 acute lymphoblastic leukemia, 5 myelodysplastic syndrome, 1 mixed acute leukemia and 1 chronic myelomonocytic leukemia. The median age was 37 (7-63) years, with 52 males and 45 females. Among 97 patients, 21 were matched sibling donor HSCT (MDS-HSCT), 14 were matched unrelated donor HSCT (MUS-HSCT), and 62 were haploidentical HSCT (haplo-HSCT). There were 88 cases of myeloablative conditioning regimen and 9 cases of non-myeloablative conditioning regimen. All patients underwent next generation sequencing (NGS) of hematological neoplasms variation genes at the time of diagnosis and 28 days after transplantation. Results: Primary and Secondary variations were detected in 89 of 97 patients during diagnosis. Common variations include CEBPA (18/97), NRAS (13/97), FLT3 (12/97), DNMT3A (11/97), TP53 (9/97), etc. After transplantation, only 29 patients had primary and Secondary variations, including ABCB1 (30/97), CYP2C19 (13/97); The number of other variations did not exceed 3 cases. The above NGS detection results suggest that the number of primary and Secondary variations after transplantation is significantly reduced (p=0.019), and ABCB1 and CYP2C19 are the main variation forms. The membrane related protein encoded by ABCB1 is a member of the ATP binding cassette (ABC) transporter superfamily, and CYP2C19 is a drug metabolizing enzyme, both of which are involved in drug metabolism. Single Nucleotide Polymorphisms (SNP) variations were detected in 52 of 97 patients at diagnosis, and GATA2 (27/97) and TET2 (25/97) variations were the most common variation forms. SNP variations of GATA2 and TET2 increased significantly after transplantation, 59/97 and 54/97 respectively. The increase of GATA2/TET2 double variation was more significant after transplantation (19/97 vs. 46/97, P&amp;lt;0.001). The increased variation frequency of GATA2 and TET2 after transplantation may be related to the mixed chimerism at the early stage of transplantation, which needs to be observed for a long time. Furthermore, we analyzed the relationship between gene variation and recurrence of 97 patients with hematological neoplasms at the time of diagnosis and after allo-HSCT. Eleven of 97 patients recurred after transplantation. The single factor competitive risk model showed that the presence of primary and Secondary variations after transplantation was an independent risk factor for recurrence (P=0.017) (Figure 1). The primary and secondary variations before transplantation, SNP variations before and after transplantation had no effect on recurrence after transplantation. The schemes of transplantation in 29 patients with primary and secondary variations after transplantation were analyzed, including 9 cases of MSD-HSCT, 14 cases of haplo-HSCT, and 6 cases of MUD-HSCT. It suggests that relative donors are more likely to have primary and secondary variations related to diseases. We used Fisher's Exact Test to analyze the relationship between ASXL1, TP53, GATA2, RUNX1 and other high-risk variations at the time of diagnosis/after transplantation and recurrence, and did not find that the presence of these gene variations would increase the risk of recurrence after transplantation. Conclusion: The NGS gene variation detection before and after transplantation can predict the recurrence after transplantation. Large samples and long-term monitoring are also needed to find a more accurate variation pattern to predict the recurrence of hematological neoplasms after transplantation.

Application Value of Next-Generation Sequencing of Bronchial Alveolar Lavage Fluid in Emergency Patients with Infection.

This study was performed to explore the application value of next-generation sequencing (NGS) of bronchial alveolar lavage fluid (BALF) in emergency patients with infection. In this regard, a total of 52 patients with infection who were diagnosed and treated in the emergency department of our hospital from September 2019 to September 2021 were selected as the research objects. The BALF of the patients was analyzed by NGS, and the results were compared with the pathogen detection results of traditional microbial culture of the patients to analyze the diagnostic value of NGS in patients with infection. The results showed that among the 52 patients, 47 were positive by NGS and 13 were positive by traditional microbial culture. The pathogen detection rate of NGS was higher than that of traditional microbial culture[90.4% (47/52) VS 25% (13/52), χ2=45.539, P<0.001], and was able to detect viruses, fungi and other special pathogens that were difficult to be detected by traditional microbial culture, such as Chlamydia psittaci. A total of 129 pathogens were detected in the NGS test results of 47 of these patients, including bacteria, fungi, viruses and mycoplasma/chlamydia. 14 pathogens were detected in the conventional microbiological cultures of 13 patients, including bacteria and fungi. Overall, compared with traditional microbial culture methods, NGS detection has higher accuracy and can effectively detect pathogens that cannot be detected by traditional microbial culture. It has a high application value in the diagnosis of pathogens and can provide clinical guidance for the diagnosis of pathogens in patients with emergency infections.

Heterogeneous expression of mismatch repair proteins and interpretation of immunohistochemical results in colorectal cancer and endometrial cancer

To investigate the heterogeneous expression patterns of four mismatch repair (MMR) proteins in colorectal cancer (CRC) and endometrial cancer (EC), and their effects on the interpretation of immunohistochemical (IHC) results. A total of 1636 CRC and EC specimens were collected from two hospitals. Seventy-eight cases had heterogeneous expression of MMR proteins, including 49 CRC and 29 EC cases. Polymerase chain reaction-capillary electrophoresis (PCR-CE) was then performed to detect the microsatellite instability (MSI) status, and 44 cases were further verified by targeted next-generation sequencing (NGS). Heterogeneous expression of MMR proteins was observed in 66 cases (66/78, 84.6%) of proficient MMR (pMMR) and 12 cases (12/78, 15.4%) of deficient MMR (dMMR). The proportion of heterogeneous MMR protein expression in EC (12.0%) was higher than that in CRC (3.5%). The heterogeneous expression patterns were divided into focal clonal heterogeneity (6/78, 7.7%) and glandular mosaic heterogeneity (72/78, 92.3%). Surprisingly, three pMMR CRC cases showed isolated small focal clonal heterogeneity of mutS homologue 6 (MSH6), with < 15% positive tumour cells, which was validated as high MSI (MSI-H) with PCR-CE and NGS. However, the other three EC pMMR cases with > 50% focal clonal heterogeneity of MMR proteins were verified as microsatellite stable (MSS) or low MSI (MSI-L). Fifteen EC cases with glandular mosaic heterogeneous expression of MMR proteins included two MSI-H cases, which were validated using PCR-CE and NGS. Among the dMMR cases, only two EC cases with mutL homologue 1 (MLH1)/PMS1 homologue 2, mismatch repair system component (PMS2) loss and MSH2/MSH6 mosaic heterogeneous expression were confirmed as MSS using PCR-CE and NGS, which may be related to the mechanism of MLH1 promoter methylation. Thus, in CRC, only cases with small focal clonal heterogeneous expression of MSH6 have a high likelihood of MSI-H, and further PCR-CE or NGS testing is recommended. The possibility of MSI-H cannot be ruled out in EC cases with glandular mosaic heterogeneous expression of MMR proteins; PCR-CE or NGS detection is therefore necessary.

Postoperative prognosis in patients with NSCLC with different EGFR mutation sites.

e20528 Background: Lung cancer can be divided into small cell lung cancer and non-small cell lung cancer. At the same time, non-small cell lung cancer can be divided into non-small cell lung cancer with different molecular mutations. Among them, the EGFR mutation accounts for about 50% of the patients with non-small cell lung cancer in China, which is a large group of patients. The EGFR mutation has many sites and many forms of mutational subtypes, including common mutations such as L858R, and rare mutations. Although the proportion of rare mutations is small, it is still different from common mutations in patients with TKI treatment and immunity. Methods: This study collected and analyzed the data from Formalin-Fixed Paraffin-Embedded (FFPE) tissues of 330 NSCLC patients which received surgery and underwent a targeted next-generation sequencing (NGS) assay performed by 3DMed Clinical Laboratory Inc., a College of American Pathologists (CAP) certified and Clinical Laboratory Improvement Amendments (CLIA) certified laboratory of 3D Medicines Inc. between January, 2019 and June, 2022 in China, to obtain a comprehensive molecular profile of EGFR mutations. genomic alterations, tumor mutational burden (TMB) and PD-L1 expression were analyzed. Gene mutation and TMB level were analyzed by NGS, detected PD-L1 expression by using Dako PD-L1 IHC 22C3 pharmDx, Tumor Proportion Score (TPS) was used to determine expression of PD-L1. Finally, the postoperative follow-up data were also collected and analyzed in this study. Results: Therefore, we retrospectively analyzed the tissue samples of patients with non-small cell lung cancer who received surgical treatment, and carried out NGS detection and analysis, as well as postoperative follow-up. Among them, patients with EGFR mutations accounted for 51.2%. We further analyzed the TMB and PD-L1 expression levels of different EGFR mutation subtypes. First, we found that different EGFR mutation sites, whether common mutations such as L859R, 19del or unusual mutations such as S768I, There is no difference in the prognosis after operation, but it is very significant that our results show that there are two or more EGFR mutations at the same time. Conclusions: NSCLC with EGFR mutation is a very large group of patients, which should be further subdivided into different subtypes according to different EGFR mutation sites, and should perform individualized management. This study found that patients with two or more EGFR mutations and patients with EGFR single site mutations are different types of NSCLC, with different postoperative prognosis, and it may be related to the differences in the expression levels of TMB and PD-L1, which is a direction worthy of further exploration in the future.

Effects of concomitant chronic obstructive pulmonary disease on immune microenvironment in non-small cell lung cancer patients.

e20522 Background: Chronic obstructive pulmonary disease (COPD) as a progressive deterioration of lung function, has caused significant morbidity and mortality, especially in developing countries. Persistent lung inflammation and potential lung stroma remodeling in COPD may be important causes of lung cancer. Previous studies have shown that non-small cell lung cancer (NSCLC) patients with COPD have a worse prognosis than those without, but the reasons have not been determined. Due to the increasing application of ICIs in the NSCLC, the characteristics of the immune microenvironment in the NSCLC patients concomitant COPD are worth exploring. Methods: Immune microenvironment and the next generation sequencing (NGS) data of twenty NSCLC patients from Sun Yat-sen Memorial Hospital were analyzed. Formalin-fixed paraffin-embedded biopsy tissue before first-line treatment to prepare tumor tissue slides. Samples were stained using an Opal automation mIF Detection Kit (Akoya) for immune cells. The immune cell infiltration were reported as percentages and density from each individual cell subpopulation in the tumor or stromal area. The landscape of gene mutantions was obtained by NGS detection of 733-gene panel, sequencing depth was 35000x. Results: There were 8 (40%) patients concomitant COPD, and the median age was 65.5-year-old. Analysis the immune cell infiltration in all patients, including T cells, B cells, macrophages and NK cells through 13 markers, showed no difference between COPD and non-COPD groups. However, comparing the differences between tumor and stromal immune microenvironment, it was found that there was a significant difference in the percentages of M1 macrophages between tumor and stromal in COPD group (P = 0.031), but no difference in non-COPD group (P = 0.47). Further analysis of the difference in cell density between tumor and stroma showed that the stromal M1 macrophages in the COPD group was significantly higher than that in the non-COPD group (P = 0.017), as well as PD-1+CD8+ T cells (P = 0.05). Additionally, a preliminary analysis of the gene mutation characteristics had done. In COPD patients with somatic mutations, the most frequently mutated gene was EGFR (60%), followed by TP53 (50%) and GLI2 (50%), which were similar as non-COPD patients. The most common variant of COPD patients are the missense mutation of EGFR L858R, but it was EGFR 19del in non-COPD. Conclusions: The results showed that the COPD was correlated with the infiltration status of M1 macrophages and PD-1+CD8+ T cell, and poor immune cell infiltration in tumor of COPD patients may be the potential influencing factors for the worse prognosis. The characteristics of immune microenvironment suggest that promoting the entry of immune cells from stroma into tumor may be a potential mechanism for the benefit of immunotherapy in NSCLC patients with COPD.

Tumour purity as an underlying key factor in tumour mutation detection in colorectal cancer

Tumour purity as an underlying key factor in tumour mutation detection in colorectal cancer

A CRISPR-enhanced metagenomic NGS test to improve pandemic preparedness

The lack of preparedness for detecting and responding to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogen (i.e., COVID-19) has caused enormous harm to public health and the economy. Testing strategies deployed on a population scale at day zero, i.e., the time of the first reported case, would be of significant value. Next-generation sequencing (NGS) has such capabilities; however, it has limited detection sensitivity for low-copy-number pathogens. Here, we leverage the CRISPR-Cas9 system to effectively remove abundant sequences not contributing to pathogen detection and show that NGS detection sensitivity of SARS-CoV-2 approaches that of RT-qPCR. The resulting sequence data can also be used for variant strain typing, co-infection detection, and individual human host response assessment, all in a single molecular and analysis workflow. This NGS work flow is pathogen agnostic and, therefore, has the potential to transform how large-scale pandemic response and focused clinical infectious disease testing are pursued in the future.

Abstract 2133: Next-generation sequencing-based MET focal amplification can predict treatment efficacy of MET inhibitors in lung cancer

Abstract Background: MET amplification (METamp) occurs in 1-5% of treatment-naïve or 5-20% of epidermal growth factor receptor tyrosine kinase inhibitors-treated lung cancers (LC). It is a promising therapeutic target in LC. Although fluorescence in situ hybridization (FISH), next-generation sequencing (NGS), and immunohistochemical staining (IHC) are available for METamp detection, but the most appropriate detection method and criteria for defining METamp are uncertain. Here, we explored the concordance of these three methods in informing METamp status and identified patients likely to benefit from MET inhibitors. Methods: Tissue samples from LC patients who underwent MET detection by FISH, NGS, and IHC tests at Shanghai Chest Hospital were retrospectively collected. MET focal amplification and polysomy by FISH were determined as MET/CEP7 ratio ≥ 2, and &amp;lt; 2 with the average MET signals per cell ≥ 5, respectively. NGS-based MET focal amplification and polysomy were determined with in-house scripts (Burning Rock Biotech). Focal amplification and polysomy were defined as METamp-positive. IHC-based MET positive was determined with H-score ≥ 200. FISH was used as a reference. Results: A total of 231 patients with a median age of 65 years were enrolled. With FISH as reference testing, NGS detection for METamp showed 66.9% (87/130) sensitivity and 95.1% sensitivity (96/101), and IHC had 71.5% (93/130) sensitivity and 91.1% (92/101) specificity. Furthermore, NGS detection for MET focal amplification achieved 87.2% (75/86) sensitivity and 96.6% (140/145) specificity, while MET polysomy detection showed 20.5% (9/44) sensitivity and 98.4% (184/187) specificity. Of 153 treatment-naïve patients, patients with focal amplification (FISH: 16.7%; NGS: 16.3%) had a significantly lower frequency of classic LC oncogene alterations than those with polysomy (FISH: 51.6%, p = 0.001; NGS: 58.3%, p = 0.006) and METamp-negative patients (METneg; FISH: 72.1%, p &amp;lt; 0.001; NGS: 64.1%, p &amp;lt; 0.001). A total of 27 patients had follow-up data. The disease control rate (DCR) to MET inhibitors was 73.1% (19/26) for overall METamp, 81.0% (17/21) for focal amplification, and 40.0% (2/5) for polysomy using FISH results. In contrast, corresponding DCRs using NGS results were 84.2% (16/19), 88.9% (16/18), and 0% (0/1), respectively. Moreover, patients with NGS-based focal amplification had a significantly longer progression-free survival (PFS) than those with polysomy or METneg. Of note, 1 patient with METneg by FISH was determined to harbor focal amplification by NGS and achieved partial response to crizotinib with a PFS of 314 days. Conclusion: Our results suggest that MET focal amplification leads to oncogenic MET addiction and is associated with promising efficacy of MET inhibitors. MET focal amplification by NGS tests might be a feasible and powerful biomarker to identify LC patients who benefit from MET inhibitors. Citation Format: Chan Xiang, Lianying Guo, Ruiying Zhao, Haohua Teng, Zhou Zhang, Ting Kuang, Xinze Lv, Ting Hou, Chenglin Liu, Wenjie Sun, Haiwei Du, Yuchen Han. Next-generation sequencing-based MET focal amplification can predict treatment efficacy of MET inhibitors in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2133.

525. A Universal ‘Day Zero’ Infectious Disease Testing Strategy Exploiting Crispr-Based Sample Depletion And Metagenomic Sequencing

Abstract Background The lack of preparedness for detecting the highly infectious SARS-CoV-2 pathogen — the pathogen responsible for the COVID-19 disease — caused enormous harm to the public health, the economy and society as a whole. It took ∼60 days for the first RT-PCR tests for SARS-CoV-2 infection developed by the United States Centers for Disease Control (CDC) to be made available. It then took &amp;gt;270 days to deploy 800,000 of these tests at a time when the estimated actual testing needs required over 6 million tests per day. Testing was therefore limited to only individuals with symptoms or individuals in close contact with confirmed positive cases. Testing strategies that can be deployed on a population scale at ‘day zero’ (i.e., at the time of the first reported case) are needed. Next Generation Sequencing (NGS) has such day zero capabilities with the potential to enable feasible and broad large-scale testing strategies, however it has limited detection sensitivity for low copy numbers of pathogens which may be present. Here we demonstrate that using CRISPR-Cas9 to remove abundant sequences that do not contribute to pathogen detection, NGS detection sensitivity is equivalent to RT-PCR. In addition, we show that this assay can be used for variant strain typing, co-infection detection, and individual human host response assessment – all in a single workflow using existing open-source analysis pipelines. This NGS workflow is pathogen agnostic, and therefore has the potential to radically transform how both very large-scale pandemic response and focused clinical infectious disease testing are pursued in the future. microbial composition at two test sites Detection Sensitivity Methods Covid positive samples with RT-PCR Ct values from 16-39 were processed through the CRISRP enhanced mNGS pipeline. Data processing workflow Results Sn/Sp compared to RT-PCR was 97%/100%. Strain calling concordance compared to amplicon sequencing was 100%. Co-infections from Covid positive samples were identified with high confidence. Host response signatures match the published literature. Conclusion Applying CRISPR enhanced metagenomic NGS at Day Zero of the next pandemic can mitigate the time gap in developing approved diagnostics at population scale and potentially save lives. Disclosures All Authors: No reported disclosures.

Comparing Data Generated Using Multiparametric Flow Cytometry, Capillary Electrophoresis, and Next-Generation Sequencing in a Cohort of B-Cell Lymphoproliferative Disorder Samples

Background: Determining whether suspected lymphoid proliferation disorder (LPD) samples are benign or malignant remains a significant challenge for the hematopathologist. For the vast majority of LPDs, the current gold standard includes assessment of clinical characteristics, cytomorphology, and immunophenotyping by flow cytometry; however, 10-15% of LPDs require further resolution by molecular methods, such as PCR-based capillary electrophoresis or next-generation sequencing, to rule out clonality based on V-D-J rearrangements in the immunoglobulin heavy-chain (IGH) gene. Invivoscribe (San Diego, CA) has developed multiple complementary methodologies to evaluate LPDs; including multiparametric flow cytometry (MFC), capillary electrophoresis (CE), and next-generation sequencing (NGS). To compare performance between these three methods, a cohort of 21 clinical samples were tested in a pilot study. Method: The CAP/CLIA Hematolymphoid Screening assay, a 10-color flow panel, was used to identify all major types of hematopoietic neoplasia according to the 2006 Bethesda Consensus and 2017 WHO Classification. For molecular testing, DNA was extracted from peripheral blood (PB), then PCR-based assays were used to determine presence or absence of clonality. The Research Use Only (RUO) molecular assays were designed for CE (ABI 3500xL) and NGS (MiSeqTM) detection, utilizing framework multiplex PCR master mixes targeting the conserved framework regions FR1, FR2, or FR3 within the VH and the JH regions of the IGH gene. Twenty-one (21) de-identified PB samples were collected from informed consensus donors (LabPMM, San Diego, CA) or from residual samples (Lahey Hospital, Burlington, MA) and tested under an IRB approved protocol. An aliquot of each PB sample was used for MFC evaluation, then the remaining DNA was extracted and tested using the CE and NGS methods. Test results were reported as "present abnormal" or "normal" for MFC and "clonal" or "non-clonal" for molecular assays based on pre-defined criteria. Results: The MFC assay identified 12 out of 21 (57%) samples as "present abnormal" and 9 out of 21 (43%) samples as "normal." Both molecular assays (CE and NGS) reported 12 out of 21 (57%) samples as "clonal" and 9 out of 21 (43%) samples as "non-clonal." The results between CE and NGS assays were 100% in agreement for clonality detection. Comparisons between the CE and MFC assays again resulted in 100% agreement; all 12 "present abnormal" samples by MFC were reported as "clonal" by CE, while all 9 "normal" samples by MFC were "non-clonal" by CE. In addition to presence of clonality, the NGS method was able to detect the prevalence of a specific clonal V-D-J rearrangement sequence (expressed as % reads), which ranged from 10% to 99% in the 12 clonal samples. This pilot study demonstrated excellent concordance (21/21) between these 3 methods (MFC, CE and NGS). Accordingly, MFC, CE and NGS are all suitable methods for the evaluation and stratification of B-cell LPD samples. The combined use of all 3 methods provides a comprehensive assessment of clonality: including surface markers identified by MFC, DNA rearrangement lengths from CE, and the specific DNA rearrangement sequences from NGS. In addition, the NGS method allows for tracking of these clonal sequences in relation to disease status. Conclusion: This pilot study demonstrated 100% concordance between 3 different methods (MFC, CE, and NGS) when evaluating samples from LPD subjects. Depending on laboratory capabilities and the type of information desired, any one of these technologies can provide appropriate results when studying clonality. The alignment between MFC and NGS allows the combined use of both methods to provide rapid diagnosis via the MFC service and identification of specific biomarkers for subsequent tracking of these clonal populations in clinical studies.

Differences in Variants in the Structural Domain of BCR-ABL1 Kinase between Chinese Han and Minority Patients with Chronic Myeloid Leukemia by Sanger Sequencing and Next-Generation Sequencing

This study aimed to detect differences in BCR-ABL1 kinase domain (KD) variants in patients with chronic myeloid leukemia (CML) who have been warned and failed in tyrosine kinase inhibitor (TKI) treatment among Chinese Han and ethnic minorities through Sanger sequencing (SS) and next-generation sequencing (NGS), and analyze the difference between SS and NGS detection. Peripheral blood samples from 51 CML patients with warning and failure of TKI therapy were analyzed using SS and NGS, and the detection differences between both sequencing types were compared. BCR-ABL1 KD variants were found in 23.53% of the cohort, including 7 Han Chinese (58.33%) and 5 ethnic minority cases (41.67%). Y253H, F317L, M244V, D276G, F359I, L387F, E459K, E255K, T315I, M351V, and heterozygous insertional mutated genes (ABL1 c.1068_1070dup) were detected. Comparison of the two sequencing assays revealed that NGS could detect compound variants and low frequency variants that were not detected by SS. More compound variants were detected in Han patients than in ethnic minority patients. In conclusion, there is no significant difference in BCR-ABL1 KD mutations between Han and ethnic minority patients. NGS has a higher mutation detection rate than SS, and can detect compound variants and genes with lower mutation frequency that are not detected by SS.

Cell block-based RNA next generation sequencing for detection of gene fusions in lung adenocarcinoma: An institutional experience.

Targeted therapy is an important part of the treatment of lung adenocarcinoma. Tests for EGFR mutation, ALK, ROS1, RET and NTRK gene fusions are needed to make a treatment decision. These gene fusions are traditionally detected by fluorescence in situ hybridisation (FISH) or immunohistochemistry. In this study, we investigated whether gene fusions in pulmonary adenocarcinoma could be accurately detected by RNA next-generation sequencing (RNA-NGS) and whether cytology cell blocks could be used effectively for this test. Archived cytological specimens of lung adenocarcinoma submitted for RNA sequencing between 2019 and 2022 at Fox Chase Cancer Center were retrospectively retrieved. Hybrid capture-based targeted RNA next generation sequencing was used, which covers 507 fusion genes, including ALK, ROS1, RET and NTRKs, irrespective of their partner genes. DNA NGS, FISH and chromosomal microarray analysis were used to confirm the results of the RNA-NGS. A total of 129 lung adenocarcinoma cytology specimens were submitted for molecular testing. Eight of 129 (6.2%) cases were excluded from RNA sequencing as their cell blocks contained inadequate numbers of tumour cells. One case (0.8%) failed to yield adequate RNA. The overall success rate was 93% (120/129). Ten of 120 (8.3%) cytology cases were positive for gene fusions, including 7 ALK, 2 ROS1 fusion genes, and 1 RET fusion gene. Twenty-two cell block cases were also tested for ALK fusion genes using FISH. However, 11 of 22 (50%) failed the testing due to inadequate material. Cytology cell blocks can be used as the main source of material for molecular testing for lung cancer. Detection of gene fusions by RNA-based NGS on cell blocks is convenient and reliable in daily practice.

A comparison of DP-TOF Mass Spectroscopy (MS) and Next Generation Sequencing (NGS) methods for detecting molecular mutations in thyroid nodules fine needle aspiration biopsies.

Mutations in the B-Raf proto-oncogene, serine/threonine kinase (BRAF), have been linked to a variety of solid tumors such as papillary thyroid carcinoma. The purpose of this study was to compare the DP-TOF, a DNA mass spectroscopy (MS) platform, and next-generation sequencing (NGS) methods for detecting multiple-gene mutations (including BRAFV600E) in thyroid nodule fine-needle aspiration fluid. In this study, we collected samples from 93 patients who had previously undergone NGS detection and had sufficient DNA samples remaining. The MS method was used to detect multiple-gene mutations (including BRAFV600E) in DNA remaining samples. NGS detection method was used as the standard. The MS method’s overall sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were 95.8%, 100%, 100%, and 88%, respectively in BRAFV600E gene mutation detection. With a kappa-value of 0.92 (95%CI 0.82–0.99), the level of agreement between these methods was incredibly high. Furthermore, when compared to NGS in multiple-gene detection, the MS method demonstrated higher sensitivity and specificity, 82.9% and 100%, respectively. In addition, we collected the postoperative pathological findings of 50 patients. When the postoperative pathological findings were used as the standard, the MS method demonstrated higher sensitivity and specificity, at 80% and 80%, respectively. Our findings show that the MS method can be used as an inexpensive, accurate, and dependable initial screening method to detect genes mutations and as an adjunct to clinical diagnosis.

OTHR-25 Germline Mutations in Pediatric Brain Tumor

Abstract BACKGROUND: The exploration of germline mutations in pediatric brain tumor is helpful to improve the understanding of tumor genesis, guide the treatment, and provide genetic counseling for patients and their families. METHODS: In this study, children with primary brain tumor aged ≤18 years old in Guangdong Sanjiu Brain Hospital were enrolled．Tumor tissue or cerebrospinal fluid (CSF) next-generation sequencing (NGS) tests were required for all included patients. The genetic mutations in these children were analyzed and patients with germline mutations were screened. RESULTS: 135 pediatric patients with NGS detection were included in this patients. 63 patients were medulloblastoma, 54 were glioma. The rest were 5 ependymoma, 2other embryonal tumors, 2 pineoblastoma, 2 germ cell tumors, 1 pituitary adenoma, 1 neuroepithelial tumor, 1 Schwannoma, 1 neuronal tumor, 1 craniopharyngioma and 1 Rosai-Dorfman disease. NGS was performed on tissues of 122 patients and CSF of 13 patients. 7 patients underwent whole exome sequencing and the remaining 115 patients underwent panel detection. 6.67% (9/135) patients took germline mutations, which included 4 gliomas, 3 medulloblastomas, 1 schwannoma and 1 ependymoma. The rate of germline mutations were 7.41% (4/54) and 4.76% (3/63) in gliomas and medulloblastoma, respectively. The germinal mutations included TP53 missense mutation, BLM nonsense mutation, MSH2 nonsense mutation, MSH2 frameshift mutation, SUFU frameshift mutation, FANCI frameshift mutation, SBDS splicing site mutation, NF2 nonsense mutation and DIS3L2 nonsense mutation. These mutations are closely related to with Ligemeni syndrome, Bloom syndrome, Lynch syndrome, nevus basal cell carcinoma syndrome, Fanconi anemia, Shwachman-Diamond syndrome, neurofibromatosis type II and Perlman syndrome, respectively. 2 glioma patients had a family history of cancer. CONCLUSION: Pediatric patients with glioma, medulloblastoma, schwannoma and ependymoma are easily harboring germline mutations. For these patients, a detailed genetic history of family members is required and genetic counseling is recommended.

LGG-36. Analysis of BRAF-related mutations in pediatric low-grade glioma

BACKGROUND: Most pediatric low-grade gliomas (pLGGs) are driven by a single genetic event resulting in up-regulation of the RAS/MAPK pathway. BRAF-related mutations are the most freauent molecular alteration in the pathway. To explore BRAF-related mutations in pediatric low-grade glioma is helpful for clinical practice. METHODS: In this study, patients with low-grade glioma aged ≤18 years in Guangdong Sanjiu Brain Hospital were enrolled. All patients accepted the tests of BRAF-related mutations with tumor tissue by next-generation sequencing (NGS). Results: A total of 26 patients diagnosed low-grade glioma and underwent NGS detection were included in this study. The male to female ratio was 6:7, and the median age was 9.5 years. 8 patients had tumors located in the cerebral hemisphere, 6 in the third or fourth ventricle, 5 in the cerebellum, 4 in the optic pathway, and 3 in the brain stem. A total of 14 patients took BRAF-related mutations, such as BRAF-KIAA1549 fusion, BRAF p.V600E mutation and other fusion. BRAF-KIAA1549 fusion was detected in 7 patients with pilocytic astrocytoma or pilomyxoid astrocytoma. BRAF p.V600E mutation was detected in 6 patients, two of whom were pleomorphic xanthoastrocytoma. A rare genetic fusion, BCAS1-BRAF fusion, was detected in 1 patient who had brain stem ganglioglioma. Among the 26 patients, 2 patients without BRAF-related mutations had typical multiple cafe-au-lait macules and were diagnosed as NF1-pLGG. These patients were treated with surgery, radiation, chemotherapy and targeted therapy. Only 2 patients received targeted therapy by Trametinib, Vimofinib and Everolimus after progression of the tumor. However, due to the severity of the disease, they eventually died. CONCLUSIONS: More than half of pLGG patients have BRAF-related mutations, which have the opportunity for targeted therapy. However, the optimal timing of targeted therapy still needs further exploration.