Complete Concordance Research Articles

Morphology combined with HER2 D-DISH ploidy analysis to diagnose partial hydatidiform mole: an evaluation audit using molecular genotyping

AimsA hydatidiform mole (HM) is classified as complete (CHM) or partial (PHM) based on its morphology and genomic composition. Ancillary techniques are often required to confirm a morphologically suspected PHM...

Novel scoring system provides high separation of diploidy and triploidy to aid partial hydatidiform mole diagnosis: an adaption of HER2 D-DISH for ploidy analysis

AimsDiagnosis of hydatidiform mole or molar pregnancy based on morphology alone can be challenging, particularly in early gestation, necessitating the use of ancillary techniques for accurate diagnosis. We sought to...

The application of targeted RNA sequencing for the analysis of fusion genes, gene mutations, IKZF1 intragenic deletion, and CRLF2 overexpression in acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is characterized by highly genetic heterogeneity, owing to recurrent fusion genes, gene mutations, intragenic deletion, and gene overexpression, which poses significant challenges in clinical detection. RNA sequencing (RNA-seq) is a powerful tool for detecting multiple genetic abnormalities, especially cryptic gene rearrangements, in a single test. Sixty samples (B-ALL, n = 49; T-ALL, n = 9; mixed phenotype acute leukemia (MPAL), n = 2) and 20 controls were analyzed by targeted RNA-seq panel of 507 genes developed by our lab. Of these, 16 patients were simultaneously analyzed for gene mutations at the DNA level using a next-generation sequencing panel of 51 genes. Fusion genes, CRLF2 expression, and IKZF1 intragenic deletion were also detected by reverse transcription-polymerase chain reaction (RT-PCR). Karyotype analysis was performed using the R-banding and G-banding technique on bone marrow cells after 24 hours of culture. Partial fusion genes were analyzed using fluorescence in situ hybridization (FISH). Compared with the results of Karyotype analysis, FISH, and RT-PCR, the detection rate of fusion genes by targeted RNA-seq increased from 48.3% to 58.3%, and six unexpected fusion genes were discovered, along with one rare isoform of IKZF1 intragenic deletion (IK10). The DNA sequencing analysis of 16 ALL patients revealed that 96.2% (25/26) of gene mutations identified at the DNA level were also detectable at the RNA level, except for one mutation with a low variant allele fraction. The detection of CRLF2 overexpression exhibited complete concordance between RT-PCR and RNA-seq. The utilization of RNA-seq enables the identification of clinically significant genetic abnormalities that may go undetected through conventional detection methods. Its robust analytical performance might bring great application value for clinical diagnosis, prognosis, and therapy in ALL.

Comprehensive Analysis of Clinically Relevant Copy Number Alterations (CNAs) Using a 523-Gene Next-Generation Sequencing Panel and NxClinical Software in Solid Tumors

Copy number alterations (CNAs) are significant in tumor initiation and progression. Identifying these aberrations is crucial for targeted therapies and personalized cancer diagnostics. Next-generation sequencing (NGS) methods present advantages in scalability and cost-effectiveness, surpassing limitations associated with reference assemblies and probe capacities in traditional laboratory approaches. This retrospective study evaluated CNAs in 50 FFPE tumor samples (breast cancer, ovarian carcinoma, pancreatic cancer, melanoma, and prostate carcinoma) using Illumina’s TruSight Oncology 500 (TSO500) and the Affymetrix Oncoscan Molecular Inversion Probe (OS-MIP) (ThermoFisher Scientific, Waltham, MA, USA). NGS analysis with the NxClinical 6.2 software demonstrated a high sensitivity and specificity (100%) for CNA detection, with a complete concordance rate as compared to the OS-MIP. All 54 known CNAs were identified by NGS, with gains being the most prevalent (63%). Notable CNAs were observed in MYC (18%), TP53 (12%), BRAF (8%), PIK3CA, EGFR, and FGFR1 (6%) genes. The diagnostic parameters exhibited high accuracy, including a positive predictive value, negative predictive value, and overall diagnostic accuracy. This study underscores NxClinical as a reliable software for identifying clinically relevant gene alterations using NGS TSO500, offering valuable insights for personalized cancer treatment strategies based on CNA analysis.

Abstract 4239: Pancreatic cancer organoids from the Human Cancer Models Initiative biobank reflect disease genotypes, capture patient heterogeneity, and are amenable to therapeutic screening

Abstract Pancreatic cancer is the fourth leading cause of cancer-related death in the United States (~3% of all cancer cases) and has one of the lowest 5-year relative survival rates. There is a lack of clinically representative, easily available, validated, in vitro pancreatic cancer models that reflect the genomic and phenotypic diversity of the disease. The Human Cancer Models Initiative (HCMI) is an international collaborative project devoted to the development and distribution of approximately 1,000 novel human primary tissue-derived tumor models supported with clinical and molecular annotation. These models were manufactured, characterized, and validated at a not-for-profit biobank and made available for academic and commercial use in basic research, compound screening, and drug development. Available pancreatic cancer organoids (PCOs) from the HCMI biobank (n=46 unique models, n=41 unique donors) were acquired and subjected to a targeted next-generation sequencing hybridization capture, pan-cancer panel of 663 genes. Data were compared to publicly available clinical and genotyping results from the patient’s tumor (n=42) that the model was derived from as well as cancer datasets such as The Cancer Genome Atlas (TCGA). A subset of models was cultured following standard procedures and tested for response to chemotherapeutics (cisplatin, nab-paclitaxel), KRAS-targeting inhibitors (MRTX1133, KR-pep-2D), or PARP inhibitors (PJ34, olaparib) at three time points. A luminescent ATP viability assay was used to assess response. PCOs from the HCMI were predominantly derived from primary tumors (75%), had an adenocarcinoma ductal histological type, and were from female patients (56%) with an average age of 65 years at diagnosis. Models had mutations in key disease-relevant driver genes including KRAS, TP53, SMAD4, and/or CDKN2A at frequencies similar to the TGCA cohort. Sequencing revealed mutations in genes less commonly associated with pancreatic cancer, including BRCA, APC, BRAF, TGFBR2, and ARID1A, albeit at lower frequencies. Most models had mutations in KRAS including G12D, G12V, G12C, and G12R variants. Significant but not complete concordance was seen in comparison with the mutation status of the patient’s respective tumor, indicating potential genotypic drift had occurred. Response to acute treatment varied by model, genotype, timepoint, and drug. Chemotherapeutics like Cisplatin were broadly cytotoxic while MRTX1133 exhibited greater toxicity in models with KRAS G12D variants as compared with KRAS wild type. Patient-derived cancer organoids from the HCMI recapitulate genotypes seen in patient populations and respond to therapeutic treatment in vitro. Citation Format: Stephen Friend, Rachel Demerath, Matthew Graziano, James Clinton. Pancreatic cancer organoids from the Human Cancer Models Initiative biobank reflect disease genotypes, capture patient heterogeneity, and are amenable to therapeutic screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4239.

Bile Culture May Guide Antibiotic Stewardship in Acute Bacterial Cholangitis.

Bile cultures are often sent with blood cultures in patients with acute bacterial cholangitis. To assess the yield of blood and bile cultures in patients with cholangitis and the clinical utility of bile cultures in guiding therapy. All patients diagnosed with cholangitis, based on the Tokyo 2013/2018 guidelines were recruited retrospectively over ten years. The clinical and investigation details were recorded. The results of bile and blood cultures including antibiotic sensitivity patterns were noted. The concordance of microorganisms grown in blood and bile cultures and their sensitivity pattern were assessed. A total of 1063 patients with cholangitis were included. Their mean age was 52.7 ± 14years and 65.4% were males. Blood cultures were positive in 372 (35%) patients. Bile culture was performed in 384 patients with 84.4% being positive, which was significantly higher than the yield of blood culture (p < 0.001). Polymicrobial growth was more in bile (59.3%) than in blood cultures (13.5%, p < 0.001). E.coli, Klebsiella, Enterococcus and Pseudomonas were the four most common organisms isolated from both blood and bile. Extended spectrum betalactamase producing organisms were isolated in 57.7% and 58.8% of positive blood and bile cultures, respectively. Among 127 patients with both blood and bile cultures positive, complete or partial concordance of organisms was noted in about 90%. Bile and blood cultures have a similar microbial profile in most patients with cholangitis. As bile cultures have a significantly higher yield than blood cultures, they could effectively guide antimicrobial therapy, especially in those with negative blood cultures.

Concordance in Molecular Tumor Board Case Reviews in the ASCO TAPUR Study.

With the advent of precision medicine, molecular tumor boards (MTBs) were established to interpret genomic results and guide decision making for targeted therapy in oncology patients. There are currently no universal guidelines for how MTBs should operate and thus variance can be seen depending on which MTB is reviewing the case. This study assesses the concordance of MTB recommendations when a participant case is reviewed by two different MTBs, establishes potential reasons for discordance, and advocates for the establishment of standard MTB operating guidelines. Participants with advanced cancer, who had exhausted all standard treatment options were screened for the Targeted Agent and Profiling Utilization Registry (TAPUR) Study. Cases were submitted for MTB review if the treatment proposal was outside the protocol genomic matching rules, or if multiple treatment options were identified. Of the 306 cases submitted for review by the TAPUR MTB from 2016 to 2018, 107 were randomly selected for secondary review by a different MTB group. Recommendations from the original review were not disclosed. Concordance between MTB group recommendations was assessed. Concordance was defined as agreement between MTB reviews on the genomic alteration and study drug match proposed by the clinical site. Thematic qualitative analysis was conducted for the discordant cases to assess reasons for discordance. Complete or partial concordance was observed in 79% of cases (95% CI, 70 to 86; one-sided P = .25). Most discordant analyses were due to disagreements on the strength of evidence regarding efficacy of the proposed treatment (32%). When presented with identical participant cases, different MTB review groups make the same or similar treatment recommendations approximately 80% of the time.

Diagnostic utility of intraoperative squash smear cytology of Intra-cranial lesions in a resource limited setup of central India

IntroductionIntraoperative squash smear cytology is a useful diagnostic tool in case of CNS lesions. In resource poor countries where frozen section is unavailable, cytology is the mainstay method in giving a rapid intraoperative diagnosis which helps Surgeons regarding the extent of excision. The current study aims to assess the feasibility and accuracy of intraoperative squash cytology in evaluation of CNS tumors. Definitive diagnosis is confirmed by histopathological examination. Materials and methodsThis retrospective observational study was carried out at Department of Neuropathology. All patients diagnosed with space occupying lesion (SOL) in CNS were enrolled in the study. Intra-operative cytological diagnosis was compared to histopathological diagnosis. WHO classification of CNS tumors 2021 was used to classify tumors. ResultsTotal seventy patients with intracranial SOLs were enrolled in this study who were operated for the same and their specimens were examined. Two samples were found to be inadequate and not included in final analysis. Mean age was 38.8 ± 17.85 (SD). Male–female ratio was 1.5:1 with 41 (60.3%) males and 27 (39.7%) females. 58 had neoplastic and rest 10 had non-neoplastic lesions. Complete concordance was achieved in 61 cases (Diagnostic Accuracy-88.2%). 3 (4.4%) cases showed partial concordance and 4 (5.9%) were discordant. For detection of malignant lesions through squash smear cytology overall Sensitivity was 73.9%, Specificity-97.8%, Positive Predictive Value-94.4% and Negative Predictive Value-88%. ConclusionsSquash smear cytology is a simple, rapid and cost-effective method relying solely on the expertise of pathologist. High diagnostic accuracy can be achieved with squash cytology by taking clinical and radiological findings into consideration.

Establishing guidelines for sentinel lymph node ultrastaging in endometrial cancer

BackgroundMany sentinel lymph node (SLN) ultrastaging protocols for endometrial cancer exist, but there is no consensus method.ObjectiveThis study aims to develop guidelines for size criteria in SLN evaluation for endometrial...

Plasma Microbial Cell-Free DNA Sequencing for Pathogen Detection and Quantification in Children With Musculoskeletal Infections.

Nearly half of all pediatric musculoskeletal infections (MSKIs) are culture negative. Plasma microbial cell-free DNA (mcfDNA) sequencing is noninvasive and not prone to the barriers of culture. We evaluated the performance of plasma mcfDNA sequencing in identifying a pathogen, and examined the duration of pathogen detection in children with MSKIs. We conducted a prospective study of children, aged 6 months to 18 years, hospitalized from July 2019 to May 2022 with MSKIs, in whom we obtained serial plasma mcfDNA sequencing samples and compared the results with cultures. A pathogen was recovered by culture in 23 of 34 (68%) participants, and by initial mcfDNA sequencing in 25 of 31 (81%) participants. Multiple pathogens were detected in the majority (56%) of positive initial samples. Complete concordance with culture (all organisms accounted for by both methods) was 32%, partial concordance (at least one of the same organism(s) identified by both methods) was 36%, and discordance was 32%. mcfDNA sequencing was more likely to show concordance (complete or partial) if obtained prior to a surgical procedure (82%), compared with after (20%), (RR 4.12 [95% CI 1.25, 22.93], p = .02). There was no difference in concordance based on timing of antibiotics (presample antibiotics 60% vs no antibiotics 75%, RR 0.8 [95% CI 0.40, 1.46], p = .65]). mcfDNA sequencing was positive in 67% of culture-negative infections and detected a pathogen for a longer interval than blood culture (median 2 days [IQR 1, 6 days] vs 1 day [1, 1 day], p < .01). Plasma mcfDNA sequencing may be useful in culture-negative pediatric MSKIs if the sample is obtained prior to surgery. However, results must be interpreted in the appropriate clinical context as multiple pathogens are frequently detected supporting the need for diagnostic stewardship.

Comparative performance of two different detection chemistries of qPCR for their implementation in a same-day detection method to determine the presence of Shiga toxin–producing Escherichia coli in ready-to-eat salads

Shiga toxin-producing E. coli (STEC), are among the most frequently reported foodborne pathogens worldwide. They produce two powerful cytotoxins, called Shiga toxins (Stx1 and Stx2). Most DNA-based methods target these genes, and rely on lengthy enrichment protocols which delay early identification of potential threats. Vegetable samples are particularly problematic, as they are rich in components that may inhibit these techniques. In the present study, a short enrichment and improved matrix lysis protocols were combined and tested with qPCR implementing two different detection chemistries, a hydrolysis probe, and an intercalating dye-based one taking advantage of SYBR Green, to determine which performed best. The methodology presented a turnaround time of ∼6 h including the enrichment, sample treatment followed by DNA extraction, and STEC detection with either detection chemistry. Minor differences were identified among both detection chemistries, as the LOD95 were 6.9 and 8.6 CFU/25 g for the probe-based and intercalating dye respectively; both got relative sensitivities, specificities, and accuracies of 100 % and a Cohen's kappa, of 1.00, being this an “almost complete concordance” with the expected result. Overall, same-day detection of STEC in ready-to-eat salad samples, was achieved, and both qPCR detection chemistries demonstrated suitable for the intended application.

A novel targeted NGS panel identifies numerous homologous recombination deficiency (HRD)-associated gene mutations in addition to known BRCA mutations

Deleterious mutations in the BRCA1 and BRCA2 genes have significant therapeutic relevance in clinical settings regarding personalized therapy approaches. BRCA1 and BRCA2 play a pivotal role in homologous recombination (HR) and thus are sensitive for PARP inhibitors (PARPi). Beyond the narrow scope of evaluating only the BRCA mutation status, PARPi can be beneficial for HR deficient (HRD) patients, who harbor mutations in other HR-associated genes. In the present retrospective study, a novel targeted HRD gene panel was validated and implemented for use with FFPE tissue. Samples of patients with ovarian, breast, pancreatic and prostate cancer were included. Variants were robustly detected with various DNA input amounts and the use of test samples showed complete concordance between previously known mutations and HRD panel results. From all the 90 samples included in this cohort, TP53 was the most frequently altered gene (73%). Deleterious BRCA1/2 mutations were found in 20 (22%) of all samples. New pathogenic or likely pathogenic mutations in additional HR-associated genes were identified in 22 (24%) patients. Taken together, the present study proves the feasibility of a new HRD gene panel with reliable panel performance and offers the possibility to easily screen for resistance mutations acquired over treatment time.Mutations in HR-associated genes, besides BRCA1/2, might represent promising potential targets for synthetic lethality approaches. Thus, a substantial number of patients may benefit from expanding the scope of therapeutic agents like PARPi.

Comparative analysis of Streptococcus agalactiae S03 and S07 isolated from Schizothorax spp. with different antibiotic resistance and virulence

Streptococcus agalactiae (GBS) is an important Gram-positive pathogen of fish aquaculture worldwide. In this study, we performed a comparative analysis of GBS S03 (serotype III) and S07 (serotype Ia) isolated from Schizothorax spp.. We explored the association between phenotypic antibiotic resistance, virulence, and genomic characteristics. Antimicrobial sensitivity tests on 12 common antibiotics using the disc diffusion method revealed that the GBS S03 resisted seven antibiotics, while GBS S07 showed sensitivity to eleven antibiotics. Pathogenicity analysis demonstrated greater virulence of GBS S07 than S03. Then, the occurrence of antibiotic resistance and virulence genes were identified using the whole-genome sequence (WGS) of GBS S03 and GBS S07. Besides just GBS S03 had gyrA and parC gene mutations, there were mefE, tetO, lnuB, lsaE, APH3’, and sat-4 resistance genes present only in GBS S03 genome. In addition to 51 virulence genes in both GBS S03 and S07 genomes, GBS S07 carried virulence genes associated with invasion, such as SAN_1519, rfbA and cylE genes. There was complete concordance between genotypic evidence and phenotypic characteristics. Virulence factors and phylogenetic analysis showed that GBS S03 and human sources shared an extremely close evolutionary relationship. Our findings also provide important proof for using WGS as an effective tool for phenotypic predictions of GBS.

Intraoperative pathologic diagnosis of central nervous system lymphomas: A comparison of frozen and permanent section diagnoses, and the significance of preoperative imaging

BackgroundCentral nervous system (CNS) lymphomas, either primary or secondary in origin, are rare malignant tumors affecting the brain, spinal cord, or leptomeninges. Diagnosis of CNS lymphomas is complicated by their diverse clinical presentations, radiological features, and histopathological characteristics. Although frozen section (FS) analysis is commonly employed for various CNS tumors, its role and accuracy in CNS lymphoma diagnosis are less explored. In this study, we conducted a comparative analysis to assess the impact of knowledge of preoperative imaging on enhancing the accuracy of FS diagnosis in CNS lymphomas. MethodsData collection involved a retrospective review of CNS lymphoma patients from January 2009 to August 2021. Patients who underwent intraoperative consultation were included, excluding those with prior cortisone treatment. The dataset incorporated patient demographics, classification as primary or secondary lymphoma, radiological preliminary diagnoses, FS diagnosis, and permanent section diagnosis. We employed various archived materials, including FSs, touch imprint slides, crush cytology slides, H&E-stained sections, and immunohistochemical stains, and re-evaluated all slides for diagnostic validation. ResultsOur study included 25 patients, of whom 60 % were female and had a mean age of 56.5 years. Preoperative radiology data were available for 80 % of cases, with preliminary diagnoses commonly including lymphoma and/or metastasis. Intraoperative consultation results indicated lymphoma in 18 (72 %) patients, with discordance observed in 28 % of cases when compared to permanent section diagnoses. Most permanent section diagnoses were diffuse large B-cell lymphomas (92 %), with the remainder being T-cell non-Hodgkin lymphoma (4 %) and follicular lymphoma (4 %). Intraoperative misdiagnoses were significantly associated with the absence of knowledge of preoperative imaging. ConclusionOur study demonstrates the reliability of FS diagnosis for CNS lymphomas during surgery, with a favorable complete concordance rate of 72 % when compared to permanent diagnoses. Importantly, lack of knowledge of preoperative imaging significantly impaired diagnostic accuracy in FS, emphasizing the need for close collaboration between pathologists and radiologists.

Reporting of paediatric osteoporotic vertebral fractures in Duchenne muscular dystrophy and potential impact on clinical management: the need for standardised and structured reporting

BackgroundIn boys with Duchenne muscular dystrophy (DMD), initiation of bisphosphonate is recommended upon identification of moderate or severe vertebral fractures, even if asymptomatic. Clear radiological reporting is important for consistency of clinical interpretation and management.ObjectivesTo audit radiology reports of spine imaging for vertebral fracture assessment in DMD, and assess potential impact on diagnosis and management.Materials and MethodsLateral thoracolumbar spine imaging (71 lateral spine radiographs and 13 lateral dual energy absorptiometry spine image) in 84 boys with DMD performed across two centres. Anonymised radiology reports by paediatric radiologists were circulated to two neuromuscular clinicians and two endocrinologists. Clinicians determined if there was vertebral fracture, no vertebral fracture, or unclear interpretation. Endocrinologists also determined if bisphosphonate was indicated. A single observer (a clinician with expertise in vertebral fracture assessment) performed vertebral fracture assessment in 37 images and re-reported using a structured format. Structured reports were re-circulated to the four clinicians to re-evaluate the degree of concordance in clinical diagnosis of vertebral fracture and treatment decisions with bisphosphonate.ResultsThe term “fracture” was used in 25/84 (30%) radiology reports and only in 8/43 (19%) with description of vertebral body abnormalities. Fracture grading was included in 7/43 (16%) radiology reports. Diagnostic concordance by the clinicians was noted in 36/84 (43%). Unclear interpretation was noted in 22% to 51% based on radiology reports. No unclear interpretation was noted with structured reports. Complete diagnostic (37/37, 100%) and treatment (37/37, 100%) concordance was noted with the structured reports, whereas complete diagnostic and treatment concordance was noted in only 16/37 (43%) and 17/37 (46%) of the radiology reports, respectively.ConclusionOnly a third of radiology reports of spine imaging in DMD explicitly used the terminology “fracture”. Grading was only noted in a small percentage. Variability in diagnostic interpretation by clinicians may lead to differing management plans. As identification of vertebral fracture is a trigger for treatment, developing reporting guidelines for paediatric vertebral fracture assessment will improve care. A structured template should be introduced for radiological reporting of paediatric vertebral fracture assessment.

Evaluation of routine proximal bone analysis for microbiology and histopathology in diabetes-related foot infections and osteomyelitis.

The value of proximal bone analysis for surgical clearance of infection remains debated. Real-world practice traditionally utilized proximal bone microbiology rather than histopathology to diagnose residual diabetes-related osteomyelitis of the foot (DFO) post-amputation. We assessed the concordance between proximal bone microbiology and histopathology in determining residual infection and their predictability for revision operation in DFO and diabetes-related foot infection (DFI). A single-centre retrospective study was conducted between June and December 2020 at a tertiary institution. We recruited patients with diabetes mellitus who had minor amputations for DFO and DFI and analyzed their proximal bone microbiology, histopathology and outcomes at 6 months. Eighty-four patients were recruited; 64 (76.2%) were male. The mean age was 69.3 years. The mean HbA1c was 8.6%. Seventy-seven operations were performed for DFO and 17 for DFI. Negative microbiology showed complete concordance with histopathology; and none had revision operation (P = 0.99). Positive microbiology had 9.8% concordance with histopathology (P = 0.99). Positive histopathology was associated with a higher rate of revision operation (80% vs. 12.5%; P = 0.01). High preoperative C-reactive protein was associated with residual DFO (P = 0.02) and revision operation (P = 0.01). Positive histopathology was more reliable for determining significant residual DFO and predicting revision operation. Positive microbiology was valuable for guiding antibiotic selection. We suggest routine proximal bone analysis for both histopathology and microbiology to optimize the treatment of DFO and DFI.

Deciphering the Structural Variants in B Cell Acute Lymphoblastic Leukemia By Optical Genome Mapping

Introduction Structural variants (SVs) of the genome play a key role in the leukemogenesis and development of B cell lymphoblastic leukemia (B-ALL), including gene deletion, amplification, translocation, and other forms. Methods for analyzing SVs and CNVs include karyotype analysis, fluorescence in situ hybridization, gene chips, and high-throughput gene sequencing technologies. Optical genome mapping (OGM) is a novel ultra-long single-molecule DNA detection based high throughput SVs analysis technology, which has the characteristics of whole genome coverage, high detection incidence and sensitivity. There were few publications presenting technical concordance of SVs detection beween karyotyping and OGM, and there are no systematic studies comparing the concordance of OGM and RNA-seq in detecting gene fusion. This study aimed to compare the concordance of OGM and RNA-seq in the detection of fusion genes in B-ALL cases, and the additional findings using OGM technology. Methods Bone marrow aspirates of 70 patients with newly diagnosed or relapsed/refractory B-ALL were recruited into the study. All samples were conducted with both OGM and transcriptome sequencing (RNA-seq). Among these cases, 42 males and 28 females, 30 pediatric (&amp;lt; 18 years) and 40 adults (≥ 18 years) cases, ages ranged 4-60 years (median 16 years). Results Totally 50 gene fusions were reported in 48 cases, with 2 cases each carrying two fusion genes ( BCR::ABL1/ PAX5::ELK3 and P2RY8::CRLF2/ PAX5::GSE1, respectively) (Figure 1A). OGM and RNA-seq have complete concordance in reporting gene fusions, and OGM provides further insight into the genomic events that lead to gene fusion. A subtype of B-ALL characterized by UBTF microdeletion leading to UBTF::ATXN7L3 fusion and PAN3 downstream deletion-induced CDX2 aberrant expression has been reported recently, and has been accepted by the International Consensus Classification as a novel molecular subtype of UBTF/CDX2 of B-ALL. We performed the OGM investigation on 6 UBTF/CDX2 cases which were identified by RNA-seq. OGM identified both microdeletion events in all 6 cases, and further reported 1q amplification in 3 of them (Figure 1B, C). B-lymphocyte differentiation-associated transcription factor gene defects are common and important genetic abnormalities in B-ALL, often manifested as complete or partial deletion of genes. We focused on a panel including 30 genes in the OGM data and determined IKZF1 deletion in 35 patients, CDKN2A/B deletion in 32 patients, and PAX5 deletion in 25 patients (Figure 1 A). Follow-up data showed that patients with CDKN2A/B deletion had a worse prognosis than those without CDKN2A/B deletion ( P = 0.025). Conclusion This is the first B-ALL cohort to systematically compare OGM and RNA-seq in gene fusion detection, and the result showed complete consistency. RNA-seq has significant advantages in analyzing gene fusion transcripts, while OGM has technical advantages in analyzing SVs at the genome level. Combining the two technologies can accurately analyze gene fusion and other SVs in B-ALL, significantly improve the detection rate of SVs with important pathological/diagnostic classification significance, and may bring new discoveries on SVs and pathogenic mechanisms.

Analytical and Clinical Validation of Duoseq, a Novel Assay for Rapid, on-Site Clinical DNA and RNA Sequencing of Hematologic Malignancies

Introduction: Next generation sequencing (NGS) has become a critical component of the workup of malignancies. NGS can provide important diagnostic information including mutations, chromosomal copy number alterations and translocations (from DNAseq) as well as gene expression and fusions (from RNAseq). Incorporation of NGS in the clinic remains difficult. DNAseq and RNAseq remain distinct assays with challenging bioinformatics and turnaround times (TAT) that can last several weeks. Duoseq is a novel assay for combined DNA- and RNAseq in a single workflow that detects single nucleotide variants (SNVs), insertions/deletions (indels), splice site variants (SSVs), structural variants (SVs) and Epstein-Barr virus (EBV) status. Initially implemented for hematologic malignancies, Duoseq assesses nearly all known gene drivers (N=475) and is compatible with both fresh-frozen and formalin-fixed, paraffin-embedded (FFPE) tissues. It is available as a reagent kit and includes cloud-based bioinformatics, enabling any clinical lab with an Illumina sequencer to perform NGS in-house with a TAT of 2-3 days. Methods/Results: Duoseq was validated for concordance with commercial DNA- and RNAseq kits (New England BioLabs (NEB)). Sequencing libraries were prepared on DNA alone, RNA alone and total nucleic acid (TNA) from cell lines in accordance with kit protocols and captured using the Duoseq panel. Results were equivalent between Duoseq and individual (NEB) library preparations with nearly complete concordance in DNA variants (99.4%, P&amp;lt;10 -6) and RNA expression (R 2=0.97, P&amp;lt;10 -6). Separately, copy number alteration calls across 42 cases had high concordance between Duoseq and Affymetrix array approaches (P&amp;lt;10 -6). Analytical validation was performed at two CLIA laboratories to establish assay precision and reproducibility, sensitivity (LoD), and specificity. Analytical validation was performed on well characterized and control samples that were selected to harbor an array of clinically important variants at known allele frequencies. Precision and reproducibility testing confirmed Duoseq's ability to produce accurate results independent of external variables. We tested inter-lab reproducibility, intra-day and inter-day repeatability and technician-to-technician reproducibility. In each case, Duoseq achieved &amp;gt;98% precision and reproducibility for each variant class (SNVs, indels/SSVs and SVs). We confirmed the LoD using TNA from the same samples diluted in normal TNA to create 3-point curves that spanned our expected LoD. We confirmed the assay to be 100% accurate down to ≥5% mutant allele fraction (MAF) for SNVs, ≥10% MAF for indels/SSVs, and ≥20% tumor purity for SVs. Specificity was additionally assessed by including normal cell lines in each run to determine the incidence of cross-contamination indicated by false positives. Analytical specificity was 100%, indicating no instances of sample cross-contamination. We assessed the clinical accuracy of Duoseq using FFPE myeloid and lymphoid specimens (N=153) collected at both laboratories and characterized via orthogonal methods including Foundation Medicine (SNVs and indels/SSVs), fluorescent in situ hybridization (translocations) and in situ hybridization (EBV status). Duoseq achieved a positive predictive agreement of 98% for SNVs (N=139), 96% for indels/SSVs (N=55), 95% for translocations (N=282), and 100% for EBV status (N=58). Cell of origin (COO) for diffuse large B cell lymphoma (n=42) using Hans criteria and had high concordance (90%) with the COO calls rendered from Duoseq expression data. Given Duoseq's array of measurements and short TAT, we plan a potential application in “match” clinical trials where patients are enrolled in treatment arms based on molecular profiling. In ongoing work to test a potential trial workflow, we enrolled four institutions that will provide cases to a central site for pathology review and molecular profiling enabling trial arm assignment. Specimen enrollment is anticipated to be completed to allow for results to be presented at the meeting. Conclusions: Duoseq is comparable to current methods and provides a cost and time effective option for library preparation.The large gene panel enables testing a broad range of clinical features in a single assay, including genes required for diagnosis and prognostication in heme malignancies. These results support clinical use of Duoseq.

Basic ctDNA Panel Promises Affordable Clinical Validity in Colon Cancer Patients but Not in Pancreas Cancer Patients.

The potential of circulating tumor DNA (ctDNA) as a biomarker to assess the progression of various solid tumors has been explored extensively. In this study, we investigated the feasibility of utilizing a ctDNA sequencing panel specifically designed to target the most frequently mutated genomic regions in colon and pancreas cancers. Through somatic analysis of colon and pancreas tumors, we targeted 27 regions within eight genes. By employing PCR amplification and Illumina NGS, we ensured that each region was adequately covered with a minimum of 5000 reads (with an average of 12,000 reads). Our method exhibited reproducibility with repetition and dilutions. The positive detection threshold for ctDNA was set at a cutoff value of 0.5% ctDNA of the total reads using IGV. Among the samples analyzed, 71% of colon cancer cases displayed somatic mutations covered by the targeted regions. Within this group, detectable ctDNA was observed in 34% of the cases. Conversely, in pancreatic cancer, 55% of mutations were covered by the panel's regions, but only 13% of these cases exhibited detectable ctDNA. In follow-ups with the patients, changes in ctDNA percentages demonstrated complete concordance with changes in the clinical condition in 88% of the cases. Our findings suggest that employing a basic ctDNA-targeted panel can serve as a cost-effective and reliable approach for repeated monitoring of the efficacy of colon cancer therapy. However, in the case of pancreatic cancer, ctDNA showed limited utility, and alternative biomarkers may offer superior diagnostic value. Additionally, we found that a negative ctDNA test is not a guarantee for a relapse-free recovery; thus, we recommend a continuous follow-up with the patient on a long-term basis.

PATH-64. COMPREHENSIVE CNS TUMOUR MOLECULAR DIAGNOSTICS USING THIRD GENERATION SEQUENCING

Abstract The WHO classification of CNS tumours 2021 recommends reporting a wide range of molecular alterations for WHO-compatible diagnoses. Conventional workflows warrant considerable investment and long turnaround times limited by batching. Nanopore sequencing enables long read sequencing, real-time targeting and simultaneous base modification detection with compact devices. Rapid-CNS2- a rapid, comprehensive adaptive sampling based sequencing pipeline with a turnaround time of 5 days was previously described. The ad-hoc methylation classification model employed by Rapid-CNS2 is limited to MNP v11 classes. This study presents an optimised Rapid-CNS2 and validation cohort that successfully reports hard-to-detect variants. We also present a whole genome sequencing workflow that affords direct upload to the MNP website for MNP v12 classification. We sequenced and analysed 156 samples from the Department of Neuropathology, University Hospital Heidelberg using Rapid-CNS2. RESULTS: were compared to NGS panel sequencing and methylation array analyses. Rapid-CNS2 showed improved resolution over NGS panel sequencing for CNAs. High accuracy was obtained for detection of SNV/Indels. Long reads spanning the breakpoint identified clinically relevant fusions in 10/10 cases. Subclonal SVs like EGFR vIII missed by NGS panel sequencing were detected with high confidence. Complete concordance was achieved for MGMT promoter methylation status and methylation classification for MNP v11 classes. The optimized Rapid-CNS2 workflow shortened the turnaround time to 30 hours from sample receipt and minimum input DNA required was reduced to 500ng. For WGS, samples were sequenced for 72h on a Promethion device. WGS enabled accurate reporting of SNVs, CNAs, SVs, MGMT promoter status in addition to MNP v12 classification directly from the website eliminating the need for ad-hoc training of methylation classification models. The presented nanopore sequencing based workflows provide feasible single assay alternatives to conventional molecular diagnostic approaches with the aim to make such analyses more accessible.