Clinical Biopsy Research Articles

Validation of a single-cell sequencing assay for the detection of circulating tumor cells in metastatic breast cancer.

1053 Background: Blood Biopsy and the evaluation of Circulating Tumor Cells (CTC) has provided valuable clinical information for patients with metastatic breast cancer, specifically where a tissue biopsy is not feasible. A critical problem in the field is that most assays cannot distinguish true CTCs from other types of rare cells. By combining established protein biomarkers associated with circulating epithelial cells with single-cell whole-genome sequencing to assess copy number variations (CNVs, a signature for genomic instability - GI), we differentiate rare cells from true CTCs. Additionally, we can determine the presence or absence of regions that contain gene amplifications (e.g., ERBB2/HER2). Here we report the validation of a clinical blood biopsy CTC assay that can enumerate CTCs, determine if those CTCs have GI and if selected CTCs are expressing HER2 or ER antigens. Methods: Relevant cell line controls of epithelial origin and known expression levels of ER or HER2 were spiked into healthy donor whole blood at varying concentrations. The nucleated cells were deposited on microscope glass slides at high density. Immunofluorescence staining of slides was performed to identify the presence and localization of nuclei (via DAPI), pan-CK, HER2, ER, CD45 and CD31. Nucleated cells were classified as CTC candidates if they were pan-cytokeratin(+) and CD45/CD31(-), and were further characterized for expression of ER and HER2 protein. Defined CTC candidates were imaged and located by specific coordinates allowing single-CTC isolation for whole genome amplification. Single-cell sequencing data was then analyzed with the Epic CTC DNA copy number analysis pipeline to detect GI and amplification at the ERBB2 locus. Results: The limit of detection for CTCs was determined to be 1 CTC in 6 million white blood cells with a linear range of 1- 300 CTC per slide. The sensitivity and specificity for HER2 detection was determined to be 94% and 97%, respectively. ER assay sensitivity and specificity were 91% and 100%, respectively. Overall accuracy was 100% for HER2 and 94% for ER detection. Overall precision across all relevant variables produced %CVs <10% for HER2 and ER. A genome possessed GI if more than 12 large-scale transitions (LSTs) were detected. For detection of 2-fold amplification at the ERBB2 locus, gene amplification was detected with 85% sensitivity and 94% specificity. Tested healthy donor blood samples were absent of detected CTCs and lacked LST/GI or ERBB2 amplification in all (46) isolated WBCs. Conclusions: These results reflect the validation of a novel clinical assay combining CTC detection with single-cell GI and amplification of the ERBB2 locus. This assay better discriminates CTC detection with the addition of an assessment of GI. Additionally, along with protein expression measures of ER and HER2, it provides the ability to assess if HER2 expression is driven by ERBB2 amplification.

A Comprehensive Guide to Artificial Intelligence in Endoscopic Ultrasound.

Endoscopic Ultrasound (EUS) is widely used for the diagnosis of bilio-pancreatic and gastrointestinal (GI) tract diseases, for the evaluation of subepithelial lesions, and for sampling of lymph nodes and solid masses located next to the GI tract. The role of Artificial Intelligence in healthcare in growing. This review aimed to provide an overview of the current state of AI in EUS from imaging to pathological diagnosis and training. AI algorithms can assist in lesion detection and characterization in EUS by analyzing EUS images and identifying suspicious areas that may require further clinical evaluation or biopsy sampling. Deep learning techniques, such as convolutional neural networks (CNNs), have shown great potential for tumor identification and subepithelial lesion (SEL) evaluation by extracting important features from EUS images and using them to classify or segment the images. AI models with new features can increase the accuracy of diagnoses, provide faster diagnoses, identify subtle differences in disease presentation that may be missed by human eyes, and provide more information and insights into disease pathology. The integration of AI in EUS images and biopsies has the potential to improve the diagnostic accuracy, leading to better patient outcomes and to a reduction in repeated procedures in case of non-diagnostic biopsies.

Harmonized Multisite MRI-Based Quantification of Human Liver Fat and Stiffness: A Pilot Study.

Nonalcoholic fatty liver disease (NAFLD) is a leading cause of end-stage liver disease. NAFLD diagnosis and follow-up relies on a combination of clinical data, liver imaging, and/or liver biopsy. However, intersite imaging differences impede diagnostic consistency and reduce the repeatability of the multisite clinical trials necessary to develop effective treatments. The goal of this pilot study was to harmonize commercially available 3 T magnetic resonance imaging (MRI) measurements of liver fat and stiffness in human participants across academic sites and MRI vendors. Cohort. Four community-dwelling adults with obesity. 1.5 and 3 T, multiecho 3D imaging, PRESS, and GRE. Harmonized proton density fat fraction (PDFF) and magnetic resonance spectroscopy (MRS) protocols were used to quantify the FF of synthetic phantoms and human participants with obesity using standard acquisition parameters at four sites that had four different 3 T MRI instruments. In addition, a harmonized magnetic resonance elastography (MRE) protocol was used to quantify liver stiffness among participants at two different sites at 1.5 and 3 T field strengths. Data were sent to a single data coordinating site for postprocessing. Linear regression in MATLAB, ICC analyses using SAS 9.4, one-sided 95% confidence intervals for the ICC. PDFF and MRS FF measurements were highly repeatable among sites in both humans and phantoms. MRE measurements of liver stiffness in three individuals at two sites using one 1.5 T and one 3 T instrument showed repeatability that was high although lower than that of MRS and PDFF. We demonstrated harmonization of PDFF, MRS, and MRE-based quantification of liver fat and stiffness through synthetic phantoms, traveling participants, and standardization of postprocessing analysis. Multisite MRI harmonization could contribute to multisite clinical trials assessing the efficacy of interventions and therapy for NAFLD. 2 TECHNICAL EFFICACY STAGE: 2.

Healthcare resource utilization of patients with mitochondrial disease in an outpatient hospital setting

Background and objectivesMitochondrial diseases present as multi-system disorders requiring a comprehensive multidisciplinary approach. The data on healthcare resource utilization associated with mitochondrial diseases and the clinical drivers of these costs are limited including for the out-patient setting where the majority of the clinical care for mitochondrial disease patients occurs. We performed a cross-sectional retrospective study of out-patient healthcare resource utilization and costs for patients with a confirmed diagnosis of mitochondrial disease.MethodsWe recruited participants from the Mitochondrial Disease Clinic in Sydney and stratified them into three groups: those with mitochondrial DNA (mtDNA) mutations (Group 1), those with nuclear DNA (nDNA) mutations and the predominant phenotype of chronic progressive external ophthalmoplegia (CPEO) or optic atrophy (Group 2) and those without a confirmed genetic diagnosis but clinical criteria and muscle biopsy findings supportive of a diagnosis of mitochondrial disease (Group 3). Data was collected through retrospective chart review and out-patient costs were calculated using the Medicare Benefits Schedule.ResultsWe analyzed the data from 91 participants and found that Group 1 had the greatest average out-patient costs per person per annum ($838.02; SD 809.72). Neurological investigations were the largest driver of outpatient healthcare costs in all groups (average costs per person per annum:—Group 1: $364.11; SD 340.93, Group 2: $247.83; SD 113.86 and Group 3: $239.57; SD 145.69) consistent with the high frequency (94.5%) of neurological symptoms. Gastroenterological and cardiac-related out-patient costs were also major contributors to out-patient healthcare resource utilization in Groups 1 and 3. In Group 2, ophthalmology was the second-most resource intensive specialty ($136.85; SD 173.35). The Group 3 had the greatest average healthcare resource utilization per person over the entire duration of out-patient clinic care ($5815.86; SD 3520.40) most likely due to the lack of a molecular diagnosis and a less customized management approach.ConclusionThe drivers of healthcare resource utilization are dependent on the phenotype–genotype characteristics. Neurological, cardiac, and gastroenterological costs were the top three drivers in the out-patient clinics unless the patient had nDNA mutations with predominant phenotype of CPEO and/or optic atrophy wherein ophthalmological-related costs were the second most resource intensive driver.

Investigating the Role of Ferroptosis-Related Genes in Ovarian Aging and the Potential for Nutritional Intervention.

With advancing age, women experience irreversible deterioration in the quality of their oocytes, resulting in reduced fertility. To gain a deeper understanding of the influence of ferroptosis-related genes on ovarian aging, we employed a comprehensive approach encompassing spatial transcriptomics, single-cell RNA sequencing, human ovarian pathology, and clinical biopsy. This investigation revealed the intricate interactions between ferroptosis and cellular energy metabolism in aging germ cells, shedding light on the underlying mechanisms. Our study involved 75 patients with ovarian senescence insufficiency, and we utilized multi-histological predictions of ferroptosis-related genes. Following a two-month supplementation period with DHEA, Ubiquinol CoQ10, and Cleo-20 T3, we examined the changes in hub genes. Our results showed that TFRC, NCOA4, and SLC3A2 were significantly reduced and GPX4 was increased in the supplement group, confirming our prediction based on multi-omic analysis. Our hypothesis is that supplementation would enhance the mitochondrial tricarboxylic acid cycle (TCA) or electron transport chain (ETC), resulting in increased levels of the antioxidant enzyme GPX4, reduced lipid peroxide accumulation, and reduced ferroptosis. Overall, our results suggest that supplementation interventions have a notable positive impact on in vitro fertilization (IVF) outcomes in aging cells by improving metal ion and energy metabolism, thereby enhancing oocyte quality in older women.

SEBACEOUS CARCINOMA ABOUT A CASE

Sebaceous carcinoma is a rare and aggressive skin cancer that originates in the cells of the sebaceous glands. It mainly affects fair-skinned and chronically sun-exposed people, and is commonly located on areas such as the face, scalp, and neck. Diagnosis is based on clinical evaluation and biopsy. The treatment of choice is Mohs surgery and, in some cases, adjuvant radiotherapy. The prognosis is generally good, but the disease may be recurrent and, in advanced cases, there may be metastases. Long-term follow-up is important for early detection of recurrences and metastases. In this clinical case, the diagnosis and treatment of a patient with sebaceous carcinoma is presented. The importance of early detection, accurate diagnosis and a comprehensive approach to care to achieve positive results in the management of this disease will be highlighted.

Ultrasound-guide prostate biopsy robot and calibration based on dynamic kinematic error model with POE formula

In the transrectal ultrasound-guided transperineal prostate biopsy, the application of robots can effectively reduce the interference of low-quality ultrasound images on the physician to determine the biopsy route and improve the positive detection rate. In this paper, an 8-degree-of-freedom (DOF) prostate biopsy robot for clinical is developed to realize the operation of ultrasonic probe scanning, the inserting point and target lesion point positioning, and the needle insertion. Considering the dynamic errors caused by different motions of robot joints, a dynamic product-of-exponentials (POE) based kinematic model that can reflect non-geometric errors is proposed by integrating the errors into each joint. Moreover, the parameters in the model are identified by linearizing the dynamic POE-based kinematic model. Finally, the experiment shows the maximum errors can be limited to 0.60 and 1.16 mm for inserting point and needle endpoint after compensation, and the maximum inserting angle error is limited to 1.381°. It proves the feasibility of the kinematic model proposed in this paper and indicates that the robot system can meet the requirements of clinical biopsy.

Model for End Stage Liver Disease (MELD) Score: A Tool for Prognosis and Prediction of Mortality in Patients With Decompensated Liver Cirrhosis.

Decompensated liver disease has become a common occurrence inmedical wards. It has become the third most common cause of death in medical wards. This high mortality rate has become a matter of concern. It is important that a reliable scoring system helps to stratify patients with liver cirrhosis who will require liver transplantation. To determine the value of the Model for End-Stage Liver Disease (MELD) score in assessing the mortality of patients with decompensated liver cirrhosis over one month period (30 days). A longitudinal study was conducted. A total of 110 patients diagnosed with decompensated liver cirrhosis wererecruited from the gastroenterology clinic and medical wards of the University of Benin Teaching Hospital (UBTH), Benin City. The patients were recruited consecutively and met the inclusion criteria for the study. Demographic data, history, clinical, biochemical, ultrasonographic, and liver biopsy findings were evaluated in the patients who participated in this study. Results: The mean age of the patients was 57 ± 11.06 years. Out of the 110 study participants, a 2.9:1 male-to-female ratio was appreciated in the patient population, with a total of 82 males and 28 females. Multiple logistic regression analysis identified MELD scores as an independent predictorof mortality in the studied patients. Predictive values of the MELD score for 1-month mortality which was analyzed using the receiver operating characteristic (ROC) curves showed that the MELD score had a sensitivity of 72.2% and positive predictive value of 93.6% with an area under the curve of 0.926 for all-cause mortality among decompensated liver cirrhosis patients. MELD score is a good predictor of mortality among patients with decompensated liver cirrhosis over a 1-month (30 days) period.

Combined Clinical, Molecular, and Muscle Biopsy Approach to Unveil Prevalence and Clinical Features of Rare Neuromuscular and Mitochondrial Diseases in Patients With Cardiomyopathies.

Combined Clinical, Molecular, and Muscle Biopsy Approach to Unveil Prevalence and Clinical Features of Rare Neuromuscular and Mitochondrial Diseases in Patients With Cardiomyopathies.

The Role of Biomarkers in the Diagnosis and Prognosis of Different Stages of Melanoma.

Melanomais a skin cancer arising from melanocytes, the cells responsible for synthesizing melanin pigment, which gives the skin its color. Early diagnosis and treatment of melanoma increase survival rates. Clinical examination and biopsy are the primary tools used to diagnose melanoma. However, distinguishing between pre-malignant melanocytic lesions and early invasive melanoma histopathologically remains challenging. Therefore, additional modalities such as a detailed clinical history, imaging, genetic testing, and biomarkers have been applied to diagnose melanoma. This review discusses the current trends in biomarker advancements over the last 10 years to assist in the early detection and diagnosis of melanoma. Biomarkers such as melanoma-associated antigens (MAAs), S100B, microRNAs (miRNAs), and circulating tumor cells (CTCs) have the potential to aid in the detection, diagnosis, and prognosis of melanoma. However, the application of biomarkers in the diagnosis of melanoma is still evolving.

Anything But a Biopsy: The Quest for Noninvasive Alternatives in Heart Transplantation.

Anything But a Biopsy: The Quest for Noninvasive Alternatives in Heart Transplantation.

Lacrimal gland ultrasonography and elastography as a diagnostic and activity tool for primary Sjögren's syndrome.

To investigate the effectiveness of 2-dimensional shear wave elastography (2D-SWE) in the assessment of lacrimal gland involvement in primary Sjögren's syndrome (pSS) and to determine the association between ultrasonographic findings and clinical activity measures. Forty-six patients who fulfilled the 2016 American College of Rheumatology/European League Against Rheumatism (EULAR) classification criteria of pSS and 23 age and gender-matched healthy control subjects were enrolled. Clinical, laboratory and labial biopsy histopathologic characteristics of patients were recorded. Disease activity of pSS and severity of ocular dryness were evaluated with EULAR Sjögren's Syndrome Patient Reported Index (ESSPRI) and Ocular Surface Disease Index (OSDI), respectively. Parotid and lacrimal gland architectures were assessed by B-mode ultrasound and 2D-SWE techniques. Mean shear wave elastography measurements, reflecting loss of elasticity, were remarkably higher in pSS patients compared to healthy subjects both in the lacrimal and parotid glands (8.99 ± 3.45 vs 3.68 ± 1.76 in lacrimal glands and 14.14 ± 4.39 vs 7.83 ± 1.69 in parotid glands, all P < 0.001). Shear wave elasticity of lacrimal glands was correlated with OSDI and ESSPRI scores (r = 0.69; P = 0.001 and r = 0.58; P = 0.001, respectively). A cut-off value of 4.6 kPa in the lacrimal gland elasticity discriminated pSS patients from healthy subjects with a sensitivity of 94% and specificity of 87%. Results of our study suggest that lacrimal glands lose elasticity in patients with pSS and the assessment of elasticity with 2D-SWE might help to classify patients as having pSS. Further studies are needed to validate the diagnostic utility of lacrimal 2D-SWE by including diseases other than pSS.

Clinical profile of children with acute post-streptococcal glomerulonephritis.

Acute post-streptococcal glomerulonephritis (APSGN) is common in developing countries with a high hospitalization rate. Most patients have acute nephritic syndrome features, although some occasionally present with unusual clinical features. This study aims to describe and analyze clinical features, complications, and laboratory parameters in children diagnosed with APSGN at presentation, 4 and 12weeks later, in a resource-limited setting. This cross-sectional study was conducted among children < 16years with APSGN between January 2015 and July 2022. Hospital medical records and outpatient cards were reviewed for clinical findings, laboratory parameters, and kidney biopsy results. Descriptive analysis of multiple categorical variables was performed using SPSS version 16.0 and presented as frequencies and percentages. The study included 77 patients. Most (94.8%) were older than five years, and age group 5-12years had highest prevalence (72.7%). Boys were affected more frequently than girls (66.2% vs. 33.8%). Edema (93.5%), hypertension (87%), and gross hematuria (67.5%) were the most frequent presenting symptoms, and pulmonary edema (23.4%) was the most common severe complication. Anti-DNase B and anti-streptolysin O titers were positive in 86.9% and 72.7%, respectively, and 96.1% had C3 hypocomplementemia. Most clinical features resolved in three months. However, at 3months, 6.5% of patients had persistent hypertension, impaired kidney function, and proteinuria alone or in combination. Most patients (84.4%) had an uncomplicated course; 12 underwent kidney biopsy, 9 required corticosteroids, and 1 required kidney replacement therapy. There was no mortality during the study period. Generalized swelling, hypertension, and hematuria were most common presenting features. Persistent hypertension, impaired kidney function, and proteinuria persisted in a small proportion who had a significant clinical course and required kidney biopsy. A higher resolution version of the Graphical abstract is available as Supplementary information.

A Case Study: Henoch-Schonlein Purpura in a Geriatric Patient with Gastrointestinal Involvement

IgA-mediated immune vasculitis known as Henoch-Schonlein purpura affects the small blood vessels in the joints, kidneys, GI tract, skin, and, less frequently, the brain and lungs. The present case report describes a 62-year-old female patient with Henoch-Schonlein purpura (HSP). HSP is a type of vasculitis that affects small vessels and is characterized by purpura, arthritis, abdominal pain, and kidney involvement. In this case, the patient presented with a rash with red lesions on the upper and lower limbs, crusted lesions on both lower limbs, and acute onset abdominal pain with hematochezia. Endoscopy revealed diffuse ulcerations in the stomach, duodenum, and right colon(fig.1). Biopsies revealed a leukocytoclastic vasculitis in the skin (fig2) and gastrointestinal tract. The patient had a history of hypertension and smoking, and laboratory investigations revealed leucocytosis, anaemia, and electrolyte imbalances. The diagnosis of HSP was made based on the clinical presentation, laboratory findings, and biopsy results. The patient was treated with a combination of steroids, antibiotics, and supportive care, which led to significant improvement of symptoms and laboratory findings. Steroid therapy was given according to guidelines for the treatment of HSP, and tapering the steroid therapy helped to improve gastrointestinal symptoms. The case report highlights the importance of careful monitoring and appropriate treatment of HSP in geriatric patients, as well as the potential benefits of immunosuppressant therapy.

Clinicopathological features and long-term prognosis of glomerular diseases associated with mercury-containing cosmetics.

The pathological types and long-term prognosis of glomerular diseases related to mercury exposure are unclear. This study retrospectively examined 41 cases of glomerulonephropathy caused by mercury-containing cosmetics. Forty-one subjects with glomerular diseases presumably caused by mercury-containing cosmetics were selected. Clinical features, kidney biopsy, treatment, and follow-up data were collected. All patients were female with an average age of 39.4 ± 6.6years at diagnosis. Median time of exposure to mercury-containing cosmetics was six months, and average urine mercury level was 66.80 ± 38.55ug/(g·Cr). Most patients presented with nephrotic syndrome. Renal histopathology showed membranous nephropathy in 22 patients (53.65%), minimal change disease in 13 patients (31.71%), IgA nephropathy with minimal change disease in 5 patients (12.20%), and focal segmental glomerulosclerosis in 1 patient. Median time of exposure to mercury was longer and the proportion of patients with autoantibodies (mainly antinuclear antibodies) was higher in patients with membranous nephropathy. Both blood phospholipase A2 receptor -Ab and kidney tissue phospholipase A2 receptor were negative. Thirty-six patients received glucocorticosteroids and/or immunosuppressants. Five patients were treated with an angiotensin receptor blocker, and nine patients were treated with chelation therapy. The median follow-up time was 40months (range 27-94). All patients achieved complete remission, and the median time to complete remission was one month. They all successfully discontinued the drugs without relapsing; withdrawal time was 26months. Membranous nephropathy was the most common pathological type in mercury-induced glomerular disease. Patients were sensitive to glucocorticosteroids and immunosuppressants and achieved complete remission quickly. Contrary to primary glomerulonephritides, patients with mercury-induced glomerular diseases had no relapses after withdrawalof the mercury containing cosmetics.

Early breast cancer risk detection: a novel framework leveraging polygenic risk scores and machine learning

BackgroundBreast cancer (BC) is the most common cancer and the second leading cause of cancer death in women; an estimated one in eight women in the USA will develop BC...

A Comparative Approach on Bridge and Elastic Net Regressions

Machine Learning techniques such as Regression have been developed to investigate associations between risk factor and disease in multivariable analysis. However, multicollinearity amongst explanatory variables becomes a problem which makes interpretation more difficult and degrade the predictability of the model. This study compared Bridge and Elastic Net regressions in handling multicollinearity in multivariable analysis. Wisconsin Diagnostic Breast Cancer data was used for comparison for model fit and in handling multicollinearity between the regression techniques. Comparison were made using MSE, RMSE, R^2, VIF, AIC and BIC for efficiency. Scatter plots was employed to show fitted regression models. The results from the study show that, the Bridge regression performed better in solving the problem of multicollinearity with VIF value of 1.182296 when 𝛾 = 2 compared to Elastic Net regression with a VIF value of 1.204298 respectively. In comparison for best model fit, Bridge regression with 𝛾 = 0.5 performed better with MSE of 11.58667, AIC value of 258.9855 and BIC of 277.2217 respectively. Consequently, we can conclude that both the Bridge and Elastic Net Regressions can be used in handling multicollinearity problems that exist in multivariable regression analysis. Information on machine learning such as this, can help those in the medical fields to improve diagnosis, narrow clinical trials and biopsy to proffer effective treatment.

Abstract 5461: Gain early insights from single cell RNA-sequencing of clinical trial needle biopsy cores

Abstract Single cell RNA-sequencing (scRNA-seq) yields valuable insights into the molecular heterogeneity of multiple cell types in normal and cancer tissues. scRNA-seq approach has the potential to not only answer basic research questions but also enhance clinical studies and clinical trials and accelerate drug development and testing. However, a consistent and reproducible method for isolating viable single cells and generating high quality single cell data from needle biopsy cores has been challenging to develop. Based on our experience isolating single cells from a large number of surgical tumor tissues, we set out to optimize a dissociation and processing protocol to generate viable single cell suspensions in adequate quantity for scRNA-seq from needle biopsy cores. A total of 17 de-identified 18-gauge needle biopsy samples were collected in collaboration with MD Anderson Cancer Center investigators. Samples were from liver, lymph node, pelvis, abdominal, neck, and lung tissues and included matched pre- and post-treatment samples. Patient biopsies were kept in RPMI medium on ice and transferred to EMPIRI (time to capture ranged from 3 to 6.5 hours after biopsy collection). Samples were dissociated using mechanical and enzymatic methods to achieve a single cell suspension. Cells were washed and viability and cell counts were verified before capture via 10x Chromium Connect. Captured cells were processed through the 10x Genomics Chromium Next GEM Single Cell workflow before sequencing on Illumina NovaSeq 6000 sequencer. Raw sequencing data were processed through EMPIRI’s computational pipeline that includes QC steps, doublet removal, and Seurat and other downstream analyses. Recovery of viable cells from individual 18-gauge biopsy cores ranged from 2,000-300,000 cells per core, depending on the tissue type and cellularity. Cell viability was strictly monitored and was maintained above 80% for 16 of 17 samples (most samples &amp;gt;90%). Cell counts after sequencing and pipeline analysis averaged 6,466 cells per sample. The median number of UMI counts per cell was 5,580, and an average of 1,699 genes were identified per cell. Average sequencing saturation was 73%. Here, we demonstrate the feasibility of integrating scRNA-seq analysis into clinical trials or clinical studies to obtain rapid insights into on-target drug effects and anticipated cellular responses to therapies, including immunotherapies, within 3 weeks of biopsy collection. We have established a method for isolating high quality single cells for scRNA-seq from tissue biopsy core, as small as a single 18-gauge needle core. We tested our protocol on 17 human samples and showed successful isolation of highly viable cells from lung, lymph node, pelvis, abdominal, neck, and lung tissues. We were able to generate scRNA-seq data sets from all samples for analysis. Acknowledgements: We thank our MDACC collaborators, particularly Drs. Scott Kopetz and Van Morris. Citation Format: Thomas Gallup, Sang Yun, Dave Gallup, Kyuson Yun. Gain early insights from single cell RNA-sequencing of clinical trial needle biopsy cores. [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 5461.

Abstract 5601: Oncomine™ dx express CE-IVD liquid biopsy assay for non-small cell lung cancer: Performance review and analytical validation

Abstract Introduction - Clinical diagnostics assays for oncology are becoming more readily available due to the advancement and democratization of Next-Generation Sequencing (NGS). Additionally, liquid biopsy can be used in NGS to detect genetic variants in circulating tumor DNA and RNA. Liquid biopsy is a less invasive option compared to traditional biopsy methods for early detection and continuous monitoring of cancer treatment outcomes. Here, we discuss screening of over 3,500 non-small cell lung cancer (NSCLC) samples, and the analytical validation of the Oncomine™ Dx Express Test (ODxET) and Genexus™ Dx Integrated Sequencer for detection of clinically significant variants in liquid biopsy samples. Methods - Our team screened over 3,500 NSCLC liquid biopsy samples in search of high priority clinical variants. Variants selected for analytical validation studies included ERBB2 exon 20 insertion, EGFR exon 20 insertion, EGFR exon 19 deletion, EGFR T790M, KRAS G12C, BRAF V600E, and RNA fusion isoforms including ALK, NTRK1/2/3, RET, and ROS1 oncogenic drivers. Over 550 screened samples were found to be positive for these variants of interest. This screening was performed on the Genexus™ Dx Integrated Sequencer according to the user guide. Results - The Genexus™ Dx Integrated Sequencer automates library preparation, sequencing, analysis, and reporting QC metrics and variant calls. Sequencing run setup is quick and straightforward, taking less than 15 minutes to start a run and just over 24 hours to go from nucleic acid to report. Limit of detection (LoD) for DNA SNVs, insertions, and deletions at 5 ng DNA input level ranged from 0.65% to 1.82% allelic frequency (AF), depending on the variant. The higher DNA input of 30 ng resulted in a lower LoD range, from 0.31% to 0.42% AF. RNA fusion and splice variant LoD at 5 ng sample input ranged from 9.9 to 19.6 molecular counts. The higher 30 ng input resulted in a lower LoD range for RNA variants as well, ranging from 6.4 to 8.0 molecular counts. In the analytical accuracy study, the false positive rate was found to be 0.2% for SNVs, 0% for insertions/deletions, and 0% for fusion targets. In the analytical reproducibility study, the average with-in run repeatability call rate (No Calls excluded) was 99.64% for DNA variants and 98.75% for RNA variants. Conclusion - Here, we demonstrated that the ODxET workflow on Genexus™ Dx Integrated Sequencer is a fast and efficient tool for testing clinical NSCLC liquid biopsy samples with high sensitivity and specificity. For in vitro diagnostic use. Not available in all regions including the United States. Citation Format: Nicholas Siepert, Jeoffrey Schageman, Jian Gu, Thilanka Jayaweera, Madhu Jasti, Luming He, Stephen Wunch, Emilia Ostrowska, Tasha Delacour, Diarra Hassell, Thomas Bowden, Daniela Garcia, Stephanie Tong, Swasti Raut, Nader Ezzedine, Elliott Martinez, Kelli Bramlett. Oncomine™ dx express CE-IVD liquid biopsy assay for non-small cell lung cancer: Performance review and analytical validation. [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 5601.

Abstract 6700: Characterizing circulating tumor DNA release kinetics and fragment length in a chemotherapy resistant model of esophageal adenocarcinoma

Abstract Background: While the incidence of esophageal adenocarcinoma (EAC) is rapidly increasing, the 5-year survival rate of this disease remains poor at less than 25%, with a substantial number of patients exhibiting inherent or acquired resistance to chemotherapy. Circulating tumor DNA (ctDNA) isolated from liquid biopsies of EAC patients holds clinical biomarker potential in this disease, but our understanding of how chemotherapy affects ctDNA release remains limited. Methods: In order to study the impact of cancer treatment on ctDNA emission in vitro, we first sought to establish a chemo-resistant model of EAC. As such, the EAC cell line OE19 was exposed to discontinuous cisplatin treatments for three months in order to establish drug resistance. Following this, established chemo-resistant OE19 cells and parental (chemo-sensitive) OE19 cells were exposed to five daily cisplatin treatments, and cell culture supernatant was collected every day throughout treatment. ctDNA was subsequently isolated from cell culture supernatant and quantified by Qubit fluorometer dsDNA HS assay. ctDNA quantities were additionally assessed by droplet digital PCR (ddPCR) assay using primers and probes targeting the TP53 N310K mutation in the OE19 cell line. Finally, ctDNA fragment lengths were analyzed using the Agilent Bioanalyzer 2100. Results: A chemo-resistant model of the OE19 cell line was established, as exemplified by marked morphological changes in the cells and significantly increased cell viability determined by CCK8 assay after 48-hour treatments of various doses of cisplatin (p&amp;lt;0.05). ctDNA levels, as measured by both Qubit fluorometer and mutant copies detected by ddPCR assay, increased after chemotherapy treatment for both chemo-sensitive and resistant cells. Notably, ctDNA emission was higher with larger amounts cell death, with chemo-sensitive cells emitting significantly higher levels of ctDNA compared to chemo-resistant cells throughout treatment (p&amp;lt;0.05). Fragmentomics analysis revealed ctDNA peaks corresponding to apoptosis (~167 bp) and necrosis (~10,000 bp). Moreover, chemotherapy treatment caused a shift in average fragment size, with larger fragments being observed during chemotherapy treatment in both chemo-sensitive and resistant cells. Conclusion: This study reveals that in vitro cancer models can be used to successfully study ctDNA emission during pre-clinical drug analyses. ctDNA release was found to be correlated to cytotoxicity and cell death, providing us with valuable insight into how clinical liquid biopsy data should be interpreted. Finally, fragment size analysis of ctDNA reveals a potential novel biomarker for treatment response in cancer patients. Citation Format: Alexandra Bartolomucci, Sarah Tadhg Ferrier, Thupten Tsering, Xin Su, Jonathan Cools-Lartigue, Julia V. Burnier. Characterizing circulating tumor DNA release kinetics and fragment length in a chemotherapy resistant model of esophageal adenocarcinoma [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 6700.