Biopsy-based Tests Research Articles

O-266 Multi-omics landscape of uterine fluid-derived extracellular vesicles across the menstrual cycle

Abstract Study question Do extracellular vesicles (EVs) mirror the cyclic physiological changes in endometrial tissue, reflecting the endometrial maturation along the entire menstrual cycle? Summary answer Multi-omics data highlights the importance of EVs during the mid-secretory phase and EVs cargo reflects the state of the tissue throughout the menstrual cycle. What is known already Extracellular vesicles isolated from uterine fluid (UF) during the mid-secretory phase have been characterized for their pivotal role in embryo-maternal communication, particularly in the context of microRNAs (miRNAs) and mRNA. EVs have been posited as potential biomarkers in a minimally invasive liquid biopsy approach for assessing endometrial receptivity, hopefully replacing the endometrial tissue biopsy-based testing. However, the dynamics of EVs and their cargo throughout the distinct phases of the menstrual cycle remain unknown. This is the first study to elucidate UF-EV content throughout the maturation of the endometrium. Study design, size, duration Whole-exome RNA and small-RNA sequencing was performed on 15 paired samples, comprising UF and endometrial biopsy (EB), obtained from healthy female subjects across the menstrual cycle. This initial dataset was expanded with 35 additional UF samples. Cumulatively, the analysis encompassed samples (EB and UF) across the menstrual cycle’s four distinct phases: proliferative (n = 10), early-secretory (ES) (n = 17), mid-secretory (MS) (n = 20), and late-secretory (LS) (n = 18). Additionally, 20 UF-EV samples were used for flow cytometry surface proteome analysis. Participants/materials, setting, methods Participants were excluded when infertility-associated conditions were found and included when they had at least one child born, with no hormonal medications used for three months before the sampling. EVs were extracted from UF with size-exclusion chromatography (SEC) and subsequently with glycosaminoglycan-based precipitation. The EVs were characterized with nanoparticle tracking analysis, western blot, transmission electron microscopy and flow-cytometry surface proteome analysis (MACSPlex) for 37 surface markers. Single-end RNA and small-RNA sequencing were performed on NextSeq1000. Main results and the role of chance The transcriptome, miRNome, and protein surface markers of UF-EVs were characterized throughout the menstrual cycle. Transcriptome profiling was performed in comparison to the preceding phase. During the ES phase, 5,507 differentially expressed genes (DEGs) were observed, most of them being downregulated and associated with G-coupled-protein signaling, while in the MS phase, 867 DEGs were identified associated with nucleosome assembly and cell differentiation. Interestingly in the LS phase, the histone genes associated with nucleosome assembly were upregulated compared to MS, highlighting that histone dynamics change corresponding to the menstrual cycle. The exploration of gene ontology revealed consistent enrichment of genes related to extracellular vesicles in UF-EV and EBs across the menstrual cycle. The miRNA analysis of UF-EVs and EB showed significant overlap in miRNA expression level especially in the MS phase. Furthermore, we found 16 unique miRNAs in the mid-secretory phase whose targets were associated with embryo morphogenesis and cell adhesion. MACSPlex analysis revealed that CD9 and CD63, canonical EV markers, were significantly abundant during the MS phase, indicative of an enriched secretion of EVs during the preparation of the endometrium for embryo implantation. These insights underscore that the cargo of UF-EVs reflects the dynamic changes in the endometrial tissue. Limitations, reasons for caution The research on UF-EVs faced notable challenges due to the irregular and non-standardized collection methods of UF in gynecological practice. This variability in UF collection introduces potential confounding factors. The study also operated with a limited sample size, underscoring the need for further validation. Wider implications of the findings Our study indicates that the UF-EVs reflect the menstrual cycle-related molecular changes and UF-EV analysis could be potentially used to follow the menstrual cycle-related molecular processes in the uterine environment. Trial registration number not applicable

Concordance between tissue (tumor DNA) and liquid (ctDNA) biopsy next-generation sequencing (NGS) data in detection of PIK3CA, AKT1, and PTEN alterations in breast cancer: A retrospective analysis.

e15033 Background: In the Phase 3 CAPItello-291 trial (NCT04305496), the addition of capivasertib (a potent, selective pan-AKT inhibitor) to fulvestrant significantly improved progression-free survival in patients with HR+/HER2− advanced breast cancer, especially in those with PIK3CA/AKT1/PTEN-altered tumors identified using tissue biopsy-based FoundationOneCDx. Given the growing utility of circulating tumor DNA (ctDNA) and interest in liquid biopsy-based tests to detect PIK3CA/AKT1/PTEN alterations, we performed a retrospective analytical comparison of FoundationOneCDx and blood-based FoundationOneLiquidCDx NGS data. Methods: We utilized the FoundationCORE database of breast cancer cases profiled during routine clinical care with FoundationOneCDx and/or FoundationOneLiquidCDx (covering the same genomic regions). Analytical comparison of all pathogenic PIK3CA/AKT1/PTEN alterations as well as alterations defined in the CAPItello-291 protocol (CAPItello-291 alterations) was performed in paired data from cases with both tissue and liquid biopsies, sampled within 90 days of each other. Subgroup analysis was conducted based on ctDNA tumor fraction (TF). Data from liquid biopsies were analyzed for co-occurring mutations. Results: CAPItello-291 alterations were detected in 44.9% of 35,730 tissue biopsies and 34.3% of 7056 liquid biopsies profiled from Jan 2017–Jun 2023. In paired NGS data (n=289), the overall positive percent agreement (PPA) for CAPItello-291 alterations across ctDNA TF subgroups was: TF ≥10% (n=99): 92.5%; TF 1–10% (n=66): 97.1%; TF <1% (n=124): 33.9%. The PPA for all PIK3CA/AKT1/PTEN short variants (base substitutions, indels) analyzed was: ctDNA TF ≥10%: PIK3CA, 93.9%; AKT1, 100%; PTEN, 100%, and ctDNA TF 1–10%: PIK3CA, 96.3%; AKT1, 100%; PTEN, 100%. For PTEN biallelic deletions, the PPA was 50% (2/4) in cases with ctDNA TF ≥10%. In liquid biopsies with CAPItello-291 alterations (n=2421), the most frequent co-occurring mutations were in TP53 (49.9%), ESR1 (31.2%), CDH1 (23.7%), NF1 (13.0%), RB1 (12.8%), CHEK2 (12.4%), ATM (11.9%), FGF3 (11.4%), FGF19 (11.0%), and FGF4 (10.5%). Mutations in BRCA1 (2.4%), BRCA2 (4.8%), and PALB2 (1.3%) were also observed. Conclusions: PPA between liquid and tissue biopsy NGS data for the detection of PIK3CA/AKT1/PTEN short variants was high in cases with ctDNA TF ≥1%. Liquid biopsies offer a minimally invasive approach for the detection of some CAPItello-291 alterations. However, limitations posed by ctDNA TF <1% and sample and variant types should be considered. Together, these data can help clinicians make informed decisions regarding suitable diagnostic tests to determine patient eligibility for breast cancer therapies.

Abstract PO4-06-12: Distinction of basal-like and triple-negative basal-like breast cancers utilizing a novel comprehensive single-cell liquid biopsy-based test

Abstract Introduction: Breast cancer is a highly heterogeneous disease and is a leading cause of death for women worldwide. Triple-negative breast cancer (TNBC), the most aggressive form of the disease, is clinically defined by the lack of expression of estrogen receptor (ER) and progesterone receptor (PR), as well as lack of over-expression of human epidermal growth factor receptor-2 (HER2). Approximately 70% of all TNBC cases exhibit basal-like cells, characterized by high basal expression of keratins as well as extensive genomic instability with copy number gains and losses across most chromosomes. In this study we show that novel single cell genomic evaluation coupled with immunofluorescence (IF) protein analysis of CTCs can detect TNBC with basal-like genomic features as well as basal-like tumors with expression of ER and HER2. Materials and Methods: Epic Sciences’ DefineMBC™ liquid biopsy test characterizes CTC proteomics and genomics as well as ctDNA for the treatment of metastatic breast cancer. Blood samples from 813 metastatic breast cancer patients, with either newly diagnosed or progressing disease, with historical tissue subtype [HR(+)HER2(-), HR(+/-)HER2(+), and TNBC] supplied by the referring physician were collected and tested. Up to 5 CTCs were selected and isolated from each sample based on morphology and IF signal. DNA was extracted from individual cells, and whole genome amplified and sequenced at low coverage. Copy number analysis was performed to determine copy number gains and losses across chromosomes, as well as large scale-state transitions (LSTs) and ERBB2 amplification. CTCs characterized with high chromosomal instability (e.g., exhibiting >30 LSTs; 1Q firestorm and 8Q stair step) and high levels of cytokeratin expression (mean fluorescence intensity signal >10K) were categorized as basal-like. Results: By previous metastatic tissue biopsy, 77% were HR(+), 8% were HER2(+), and 12% were TNBC. CTCs categorized in DefineMBC as basal-like subtype (9.8 % of all samples tested) showed to be highly present in patients called as TNBC (79% basal-like). Additionally, 54% of HR(+) and 23% of HER2(+) had basal-like CTCS. Patients characterized with TNBC by metastatic tissue biopsy and by DefineMBC exhibited basal-like cells in 88% of the cases. Conclusion: The results show a novel clinical assay which utilizes CTC protein detection as well as single-cell genomic analysis to identify tumors driven by basal-like cells. Generally, TNBC is classified with negative biomarker expression. We show that CTCs in TNBC display genomically basal-like cells. CTCs from HR(+) and HER2(+) tumors may express basal-like features with preserved ER and HER2 expression. The comprehensive nature of DefineMBC™ utilizing genomic analysis of detected CTCs has provided a new tool to diagnose both basal-like TNBC and basal-like hormone receptor positive and HER2 over-expressing tumors. Citation Format: Alessandra Cunsolo, David Bourdon, Ernest Lam, Giuseppe Di Caro, Nilesh Dharajiya, Timothy Pluard, Lee Schwartzberg. Distinction of basal-like and triple-negative basal-like breast cancers utilizing a novel comprehensive single-cell liquid biopsy-based test [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-06-12.

Abstract 3706: Single cell and cell-free nucleic acid analyses of liquid biopsy specimens from a blood collection tube using a new droplet digital PCR system

Abstract Introduction: Liquid biopsy-based testing has increasingly been adopted by oncologists for biomarker-driven diagnostics, therapy selection, and patient monitoring. Unlike tissue-based approaches, the target analytes in blood specimens are often rare events that must be discriminated from a background of non-tumor derived material. Droplet digital PCR (ddPCR) is an ideal platform for detection of rare nucleic acid markers in the clinic, because it is highly sensitive and specific, robust, and quantitative. Additionally, the workflow is simple to adopt and execute. The QX600 System is a new ddPCR instrument designed for advanced multiplexing which enables simultaneous detection of up to 6 unique fluorophores in a single well. This capability can decrease the sub-sampling needed for detection of multiple analytes. The objective of our study was to develop multiplexed ddPCR assays for detection of cell-based and cell-free circulating biomarkers from a single tube of whole blood. Methods: Tumor cells were enriched from the buffy coat fraction of whole blood collected in fixative-containing cfDNA BCT based on size and morphology using a microfluidics-based system. Enriched tumor cells were enumerated by immunofluorescence (DAPI+/Pan-CK+/CD45-), and gene expression was measured using the QX600 for a panel of cytokeratins (CK) and PD-L1 in CTC negative, NSCLC donor specimens (n=8). Results were compared to CTC positive, contrived specimens (whole blood spiked with breast tumor cell line MCF7 cells; n=5). For ctDNA analysis of the plasma fraction, a multiplexed ddPCR assay was designed to test for five actionable mutations prevalent in NSCLC, including EGFR del19, L858R, T790M, KRAS G12C, BRAF V600E, and wildtype EGFR. To evaluate performance of the multiplexed assay, concordance evaluation was conducted using cfDNA from 8 mutation-positive NSCLC donors on the QX600 and compared to single assays run on the QX200 system. Results: CK8 and CK19 were differentially expressed in the cells enriched from the CTC negative NSCLC cohort as compared to the contrived positives (p=0.0058, and p=0.0049, respectively). All the NSCLC cfDNA samples were positive for the reference variant, and linear regression analysis of the minor variant frequency for ctDNA biomarkers showed good concordance between QX systems (R2=0.8847). Conclusions: These studies demonstrate proof of concept for single cell and cell-free analysis from a single blood draw into a cfDNA BCT using the QX600 system. Liquid biopsy analysis of CTCs and ctDNA can enable minimally invasive, comprehensive decision making in the diagnostic workup of patients. Future studies will focus on further optimizing the cell enrichment methodology from cfDNA BCT and development of additional multiplexed ddPCR assays for NSCLC-relevant biomarkers on the QX600. Citation Format: Leisa Jackson, Colin Cochran, Claire Gould, Sarah Kreston, Jazmin Bello, Brittany D’Alessio, Janice Riley, Alisa Tubbs, Gary A. Pestano. Single cell and cell-free nucleic acid analyses of liquid biopsy specimens from a blood collection tube using a new droplet digital PCR system [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 3706.

Abstract A009: Targeting Structural Variants (DNA palindromes) in plasma cell-free DNA for Liquid Biopsy-based Cancer Detection

Abstract We aim to expand the utilities of our genome-wide detection method of chromosomal structural aberrations (structural variants, SVs) for blood-based cancer detection (liquid biopsy). SVs, such as genomic amplification, are prevalent DNA abnormalities and define tumor phenotypes. With cell-free DNA (cfDNA) in plasma, a genome-wide assay for SVs seems feasible for cancer detection. However, most current tests probe a part of the genome for cancer gene mutations and differential DNA methylation. Considering liquid biopsy's promise in clinics, approaches targeting SVs must be considered and developed. The low tumor fraction in plasma cfDNA is a major obstacle to developing liquid biopsy-based tests. Targeted assays overcome low signals by ultra-deep NGS, yet deep sequencing for the entire genome for SV detection would be costly and computationally challenging. In this regard, we have an established approach for enriching SVs (DNA palindromes or inverted repeats, also called fold-back inversions) from total genomic DNA to determine their frequency and genomic locations by sequencing (genome-wide analysis of palindrome formation, GAPF-seq). DNA palindromes arise from common events of genome instability, such as illegitimate repair of DNA double-strand breaks and telomere-telomere fusions, as an early step of genomic amplification. Therefore, a technique to identify DNA palindromes could serve as a tumor DNA detection test across tumor types, including breast cancer. The simple GAPF-seq procedure confers advantages over other methods because it simultaneously amplifies the target signal and reduces background noise. GAPF-seq exploits the inherent propensity of denatured DNA palindromes to form double-stranded DNA (dsDNA) by intra-molecular annealing. Genomic DNA will be denatured and quickly renatured to enrich DNA palindromes to favor intra-molecular annealing. Single-strand-specific nuclease S1 will eliminate the remaining single-stranded, non-palindromic DNA. The dsDNA from palindromes will be amplified by the process of preparing libraries for NGS, by which DNA palindromes appear as coverage peaks in the genome. As a proof-of-concept, we examined the read coverage of nearly 3 million 1-kb bins from low coverage NGS GAPF-seq data for 39 breast paired tumor/normal DNA. We found that the chromosomal distribution of the top 1000 high-coverage bins (GAPF profiles) could effectively differentiate breast tumor DNA, including all stage I tumor DNA, from paired normal DNA (AUROC=0.9885). Automated, three-fold cross-validation with machine learning (ML) algorithms from the STREAMLINE platform confirmed the highly accurate binary classification by GAPF profiles. The same approach was applied for 15 cfDNA from breast cancer patients and effectively differentiated cancer patients' cfDNA from healthy individuals' cfDNA (AUROC=0.900). These results support the potential of GAPF-seq as a unique, SV-targeting liquid biopsy approach for breast cancer. Citation Format: Fumie Igari, Hisashi Tanaka, Michael M Murata, Mitsue Saito, Armando E Giuliano. Targeting Structural Variants (DNA palindromes) in plasma cell-free DNA for Liquid Biopsy-based Cancer Detection [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A009.

Helicobacter pylori Eradication Rate Using Stool Antigen Test in Vietnamese Children: A Prospective Multicenter Study.

This study assessed the diagnostic value of a monoclonal immunoassay stool antigen test (HpSA) for Helicobacter pylori (H. pylori) infection and the eradication outcomes. Children undergoing digestive endoscopy at 2 Children's Hospitals in Ho Chi Minh City were recruited. Treatment was offered to H. pylori-infected children. Stool samples were collected on the same day as the endoscopy procedure and after 6 weeks post-treatment for HpSA. Diagnostic value and optimal cutoff of HpSA were assessed using biopsy-based tests as the gold standard. Eradication was defined as a negative HpSA post-treatment. Ethical approval was obtained, and informed consent was signed by the participants. In total, 394 patients participated in the study. The most common symptoms were epigastric pain (74.6%) and vomiting (37.3%). H. pylori status was positive in 78% of patients (306/394), doubtful in 10.1%, and negative in 12.2%. HpSA was positive in 73.2% (142/194). Excluding doubtful infections, the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of HpSA were 87.4%, 95.2%, 99.2%, 51.3%, and 88.4%, respectively. The optimal cutoff value of 0.148 provided similar accuracy to the recommended cutoff. The eradication rate was 56.1% in per-protocol analysis and 27.9% in intention-to-treat analysis. Treatment success was higher in boys, but lower among malnourished children and those infected with cagA+ strains. The HpSA is reliable for identifying H. pylori infection in epidemiological studies and assessing eradication outcomes. The low eradication rate highlights the need for an appropriate intervention strategy in Vietnamese children.

THE DIAGNOSTIC ACCURACY OF H. PYLORI CULTURE AND RAPID UREASE TEST IN THE DIAGNOSIS OF H.PYLORI INFECTION KEEPING HISTOPATHOLOGY AS THE GOLD STANDARD

Objective: The aim of this study was to determine the diagnostic accuracy of biopsy-based tests in the detection of Helicobacter Pylori infections keeping histopathology as a gold standard. Materials and Methods: This validation study was conducted from August 2018 to February 2019 at Khyber medical college, pathology department, Peshawar, and comprised biopsy samples of 87 patients obtained via endoscopy which was done at the gastrointestinal endoscopy department, Hayatabad medical complex (HMC). Patients with a history of prior antibiotics and PPIs were excluded from the study. Rapid urease test (RUT) and culture of gastroduodenal biopsies were validated against the histopathological examination of gastroduodenal mucosa. Results: Out of 87 patients, 57.47% (50/87) were male and 42.52% (37/87) were female patients with an age range of 18 -80 years and a mean age of 45 years. Endoscopic findings showed 64.36% (56/87) of patients with gastritis, duodenitis with 6.89% (6/87), peptic and duodenal ulcers with 22.98% (20/87), erosion with 4.59% (4/87) and adenocarcinoma of stomach with 2.29% (2/87). The sensitivity and specificity of culture were 45.45% and 90.7% respectively, while positive and negative predictive values were 62.5% and 83% respectively. The sensitivity and specificity of the rapid urease test (RUT) were 81.81% and 90% while positive and negative predictive values were 90% and 94% respectively. Conclusion: The sensitivity and specificity of the rapid urease test were 82 and 90% respectively, while that of culture was 45 and 91% respectively. Keys Words: Helicobacter Pylori, Gastritis, Rapid urease test, Adenocarcinoma

Helicobacter Pylori Infection among Patients Attending the Gastroenterology Department in Tertiary Care Hospital, Kathmandu, Nepal.

Helicobacter pylori is one of the most pathogenic organisms that cause gastritis, peptic ulcer, gastric adenocarcinoma, and mucosa-associated lymphoid tissue (MALT) lymphoma in humans. The main aim of this study was to determine the H. pylori infection among patients undergoing upper GI endoscopy and to compare the efficacy of the diagnostic method of H. pylori infection including invasive tests (biopsy-based tests like the rapid urease test (RUT), direct smear, and culture) and the noninvasive test (HpSA). A total of 100 stool samples and 200 gastric biopsy specimens were collected (2 samples from each patient) from June to November 2019. Stool samples were processed for the detection of an H. pylori stool antigen (HpSA) by a kit method. One biopsy specimen was processed for the RUT, and another was transported to the laboratory in an Eppendorf tube containing normal saline for preparation of the smear and culture. Out of 100 participants, 26% were found to be H. pylori positive by the RUT, 11% by the direct smear, 6% by the culture, and 17% by the stool antigen test. The prevalence of H. pylori infection was found to be 14%, considering at least two of the three biopsy-based tests that gave positive results. H. pylori infection was found to be higher in the age group of 46-55 years. The overall prevalence of H. pylori infection was higher in gastric ulcer cases, followed by erosive pangastritis and gastroduodenitis. Tea drinking habits and the frequency of meal consumption more than twice a day were found to be significantly associated with H. pylori infection (P < 0.05). Hence, the RUT was found to be more efficient than the direct smear and the culture method for finding H. pylori in the biopsy sample. However, none of these methods can be considered to be the gold standard alone. Thus, the RUT combined with other tests is preferable for the detection of H. pylori.

Genetics and muscle pathology in the diagnosis of muscular dystrophies: An update.

Muscular dystrophies are a clinically and genetically heterogeneous group of disorders involving the skeletal muscles. They have a progressive clinical course and are characterized by muscle fiber degeneration. Congenital muscular dystrophies (CMD) include dystroglycanopathies, merosin-deficient CMD, collagen VI-deficient CMD, SELENON-related rigid spine muscular dystrophy, and LMNA-related CMD. Childhood and adult-onset muscular dystrophies include dystrophinopathies, limb-girdle muscular dystrophies, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, and myotonic dystrophy. Traditionally, muscle biopsy and histopathology along with special pathology techniques such as immunohistochemistry or immunoblotting were used for the diagnosis of muscular dystrophies. However, recent advances in molecular genetic testing, especially the next-generation sequencing technology, have revolutionized the diagnosis of muscular dystrophies. Identification of the underlying genetic basis helps in appropriate management and prognostication of the affected individual and genetic counseling of the family. In addition, identification of the exact disease-causing mutations is necessary for accurate prenatal genetic testing and carrier testing, to prevent recurrence in the family. Mutation identification is also essential for initiating mutation-specific therapies (which have been developed recently, especially for Duchenne muscular dystrophy) and for enrolment of patients into ongoing therapeutic clinical trials. The 'genetic testing first' approach has now become the norm in most centers. Nonetheless, muscle biopsy-based testing still has an important role to play, especially for cases where genetic testing is negative or inconclusive for the etiology.

Comprehensive Approach to Distinguish Patients with Solid Tumors from Healthy Controls by Combining Androgen Receptor Mutation p.H875Y with Cell-Free DNA Methylation and Circulating miRNAs.

Simple SummaryBlood-based tests for cancer detection are minimally invasive and could be useful for screening asymptomatic patients and high-risk populations. Since a single molecular biomarker is usually insufficient for an accurate diagnosis, we developed a multi-analyte liquid biopsy-based classification model to distinguish cancer patients from healthy subjects. The combination of cell-free DNA mutations, miRNAs, and cell-free DNA methylation markers improved the model’s performance. Moreover, we demonstrated that the androgen receptor mutation p.H875Y is not only relevant in prostate cancer but had a strong predictive value for colorectal, bladder, and breast cancer. Our results, although preliminary, showed that a single liquid biopsy test could detect multiple cancer types simultaneously.Liquid biopsy-based tests emerge progressively as an important tool for cancer diagnostics and management. Currently, researchers focus on a single biomarker type and one tumor entity. This study aimed to create a multi-analyte liquid biopsy test for the simultaneous detection of several solid cancers. For this purpose, we analyzed cell-free DNA (cfDNA) mutations and methylation, as well as circulating miRNAs (miRNAs) in plasma samples from 97 patients with cancer (20 bladder, 9 brain, 30 breast, 28 colorectal, 29 lung, 19 ovarian, 12 pancreas, 27 prostate, 23 stomach) and 15 healthy controls via real-time qPCR. Androgen receptor p.H875Y mutation (AR) was detected for the first time in bladder, lung, stomach, ovarian, brain, and pancreas cancer, all together in 51.3% of all cancer samples and in none of the healthy controls. A discriminant function model, comprising cfDNA mutations (COSM10758, COSM18561), cfDNA methylation markers (MLH1, MDR1, GATA5, SFN) and miRNAs (miR-17-5p, miR-20a-5p, miR-21-5p, miR-26a-5p, miR-27a-3p, miR-29c-3p, miR-92a-3p, miR-101-3p, miR-133a-3p, miR-148b-3p, miR-155-5p, miR-195-5p) could further classify healthy and tumor samples with 95.4% accuracy, 97.9% sensitivity, 80% specificity. This multi-analyte liquid biopsy-based test may help improve the simultaneous detection of several cancer types and underlines the importance of combining genetic and epigenetic biomarkers.

Portable, handheld, and affordable blood perfusion imager for screening of subsurface cancer in resource-limited settings

Precise information on localized variations in blood circulation holds the key for noninvasive diagnostics and therapeutic assessment of various forms of cancer. While thermal imaging by itself may provide significant insights on the combined implications of the relevant physiological parameters, viz. local blood perfusion and metabolic balance due to active tumors as well as the ambient conditions, knowledge of the tissue surface temperature alone may be somewhat inadequate in distinguishing between some ambiguous manifestations of precancer and cancerous lesions, resulting in compromise of the selectivity in detection. This, along with the lack of availability of a user-friendly and inexpensive portable device for thermal-image acquisition, blood perfusion mapping, and data integration acts as a deterrent against the emergence of an inexpensive, contact-free, and accurate in situ screening and diagnostic approach for cancer detection and management. Circumventing these constraints, here we report a portable noninvasive blood perfusion imager augmented with machine learning-based quantitative analytics for screening precancerous and cancerous traits in oral lesions, by probing the localized alterations in microcirculation. With a proven overall sensitivity >96.66% and specificity of 100% as compared to gold-standard biopsy-based tests, the method successfully classified oral cancer and precancer in a resource-limited clinical setting in a double-blinded patient trial and exhibited favorable predictive capabilities considering other complementary modes of medical image analysis as well. The method holds further potential to achieve contrast-free, accurate, and low-cost diagnosis of abnormal microvascular physiology and other clinically vulnerable conditions, when interpreted along with complementary clinically evidenced decision-making perspectives.

Exploring the association with disease recurrence of miRNAs predictive of colorectal cancer.

Disease recurrence after surgery is a crucial predictor of poor prognosis in colorectal cancer, where disseminated disease at the time of intervention can also be observed in localized early-stage cases. We evaluated the ability to predict disease recurrence of miRNAs from two signatures that we have found linked to the presence of colorectal cancer (CL signature) or adenoma (HgA signature) in higher-risk subjects. miRNAs from the signatures were studied longitudinally by quantitative real-time polymerase chain reaction in plasma from 24 patients with resectable colorectal cancer collected at the time of surgery and during scheduled follow-up across 36 months. Patients either showed relapse within 36 months (alive with disease (AWD)), or remained disease-free (no evidence of disease (NED)) for the same period. Although the signatures did not predict recurrence, expression of the miRNAs from the CL signature decreased 1 year after surgery, and one miRNA of the signature, miR-378a-3p, almost reached significance in the NED subgroup (Wilcoxon signed-rank test: p-value = 0.078). Also, miR-335-5p from the HgA signature was higher in AWD patients before surgery (Kruskal-Wallis test: p-value = 0.019). These data, although from a small cohort of patients, support the possible use of miRNAs as non-invasive biomarkers in liquid biopsy-based tests to identify patients at risk of relapse and to monitor them during follow-up.

Use of Liquid Biopsy in the Care of Patients with Non-Small Cell Lung Cancer.

Recent technological advances have enabled the development of liquid biopsy-based approaches, which have revolutionized the diagnostic world. The analysis of circulating tumor DNA (ctDNA) has several clinical applications. First, ctDNA genotyping is becoming widely used for non-invasive biomarker testing. Of note, in lung cancer patients in whom biopsies are difficult to obtain, ctDNA has led to significant improvement in the diagnosis and identification of therapeutic targets. In addition, ctDNA quantification over the course of the disease can be useful for tumor response to treatment monitoring and for early detection of resistance mutations. ctDNA levels per se are also of prognostic significance and could be used to tailor treatments. Finally, improvements in assay sensitivity are facilitating the development of liquid biopsy-based tests for the detection of ctDNA at very low allele frequencies (AFs), which can be used for the measurement of minimal residual disease and ultimately for the development of strategies (by complementing imaging techniques) aimed to improve the efficiency of lung cancer screening programs.

P–162 Laser-assisted collapse of blastocysts prior to vitrification improves clinical outcomes

Abstract Study question What is the effect of artificial laser-assisted collapse before vitrification on pregnancy and implantation rates after transfer of vitrified-warmed blastocysts? Summary answer The artificial shrinkage by laser-induced collapse before vitrification significantly increased the implantation and clinical pregnancy rates after single thawed embryo transfer. What is known already Freeze all, cycle segmentation and, in general, single embryo transfer (SET) strategies (for example trophectoderm biopsy-based aneuploidy testing) have targeted blastocysts vitrification as the best option for reproductive practice worldwide. Artificial shrinkage seems to be a pre-vitrification parameter associated with an increased embryo survival after warming and implantation rate. However, the available medical evidence shows controversial results with only a limited number of prospective studies assessing the subject. Study design, size, duration This prospective cohort study evaluated 394 women who underwent a frozen blastocyst transfer at Instituto Bernabeu between July and December 2020. All patients were prepared with substitutive cycle and received single blastocyst embryo transfers. Participants/materials, setting, methods Before embryo vitrification on day 5 of development, some expanded and/or early hatching blastocysts (A/B ASEBIR categories) were artificial laser-assisted collapsed. (n = 83, study group). 311 embryos of the same quality and day of development were not collapsed (control group). We compared the embryo survival rate, clinical, implantation and miscarriage rates between groups. The statistical analysis was performed using SPSS (version 20.0). Main results and the role of chance The two groups were comparable in terms of maternal age (39.79 ± 3.83, control group; 40.21 ± 4.45, study group; p = 0.341). Embryo survival rate resulted in 100% in both groups. Regarding clinical outcomes, collapsed blastocysts significantly increased the positive pregnancy test and the clinical pregnancy and implantation rate compared to the control group, respectively (positive test: 69,9% vs 43,4%, p = 0.000018, odds ratio (OR)= 3.02 [95% CI 1.80–5.08]; clinical pregnancy and implantation: 56,6% vs 35,4%, p = 0.000041, OR = 2.39 [95% CI 1.46–3.90]). The miscarriage rate was not affected by the blastocyst collapse effect (23,6% in the control group vs 27,6% in the study group, p = 0.593, OR = 1.23 [95% CI 0.57–2.68]). Limitations, reasons for caution This is a non-randomized controlled study. Additional RCTs are warranted to corroborate our findings. Wider implications of the findings: Considering the large number of blastocyst vitrification cycles that are carried out worldwide, artificial laser-assisted collapse before vitrification has the potential to increase the clinical results in benefit of many patients. Trial registration number Not applicable

Assessment of Helicobacter pylori status by examination of gastric mucosal patterns: diagnostic accuracy of white-light endoscopy and narrow-band imaging

ObjectivesHelicobacter pylori infection is a common cause of chronic gastritis worldwide and an established risk factor for developing gastric malignancy. The endoscopic appearances predicting H. pylori status are an ongoing...

Fit-for-purpose quantitative liquid biopsy based droplet digital PCR assay development for detection of programmed cell death ligand-1 (PD-L1) RNA expression in PAXgene blood samples.

Development of a clinically applicable liquid biopsy-based test for PD-L1 mRNA expression would be beneficial in providing complementary evidence to current immunohistochemistry assays. Hence, we report the development of a fit-for-purpose assay for detection of blood PD-L1 mRNA expression using droplet digital polymerase chain reaction (ddPCR). TaqMan® assays were selected based on coverage of the PD-L1 gene and were tested for linearity and efficiency using real-time quantitative PCR. Four reference genes were analyzed in positive control cell line (A549 treated with interferon gamma, [IFN γ]) genomic DNA. The PD-L1 primer/probe sets were also evaluated in ddPCR for limit of blank, limit of detection, and precision. Finally, thirty-five healthy volunteer samples were evaluated to establish a baseline level of PD-L1 expression. In ddPCR, the limit of blank was determined to be 0 copies and the limit of detection was determined to be less than or equal to 19 copies of PD-L1. The average intra-run coefficient of variation in the ddPCR assay was 7.44% and average inter-run CV was 7.70%. Treatment of A549 cells with IFN γ resulted in a 6.7-fold increase in PD-L1 expression (21,580 copies in untreated cDNA versus 145,000 copies in treated cDNA). Analysis of healthy human samples yielded a median value of 1659 PD-L1 copies/μL with a range of 768-7510 copies/μL. The assay was transferred to an external service provider and results from our in-house experiments and those conducted externally shows a correlation of 0.994. In conclusion, a fit-for-purpose liquid biopsy-based, purely quantitative ddPCR assay for the detection of PD-L1 mRNA expression was developed and validated using PAXgene RNA blood samples. Linearity, reproducibility, limit of blank and limit of detection were measured and deemed suitable for clinical application. This ultra-sensitive liquid biopsy ddPCR assay has promising clinical potential in screening, longitudinal monitoring and disease progression detection.

Methylated markers accurately distinguish primary central nervous system lymphomas (PCNSL) from other CNS tumors

BackgroundDefinitive diagnosis of primary central nervous system lymphoma (PCNSL) requires invasive surgical brain biopsy, causing treatment delays. In this paper, we identified and validated tumor-specific markers that can distinguish PCNSL from other CNS tumors in tissues. In a pilot study, we tested these newly identified markers in plasma.ResultsThe Methylation Outlier Detector program was used to identify markers in TCGA dataset of 48 diffuse large B-cell lymphoma (DLBCL) and 656 glioblastomas and lower-grade gliomas. Eight methylated markers clearly distinguished DLBCL from gliomas. Marker performance was verified (ROC-AUC of ≥ 0.989) in samples from several GEO datasets (95 PCNSL; 2112 other primary CNS tumors of 11 types). Next, we developed a novel, efficient assay called Tailed Amplicon Multiplexed-Methylation-Specific PCR (TAM-MSP), which uses two of the methylation markers, cg0504 and SCG3 triplexed with ACTB. FFPE tissue sections (25 cases each) of PCNSL and eight types of other primary CNS tumors were analyzed using TAM-MSP. TAM-MSP distinguished PCNSL from the other primary CNS tumors with 100% accuracy (AUC = 1.00, 95% CI 0.95–1.00, P < 0.001). The TAM-MSP assay also detected as few as 5 copies of fully methylated plasma DNA spiked into 0.5 ml of healthy plasma. In a pilot study of plasma from 15 PCNSL, 5 other CNS tumors and 6 healthy individuals, methylation in cg0504 and SCG3 was detectable in 3/15 PCNSL samples (20%).ConclusionThe Methylation Outlier Detector program identified methylated markers that distinguish PCNSL from other CNS tumors with accuracy. The high level of accuracy achieved by these markers was validated in tissues by a novel method, TAM-MSP. These studies lay a strong foundation for a liquid biopsy-based test to detect PCNSL-specific circulating tumor DNA.

Efficacy of sequential therapy versus standard triple therapy versus quinolone-based triple therapy for eradication of Helicobacter pylori infection

Background: Helicobacter pylori colonization is a risk factor for Adenocarcinomas of the distal (noncardia) stomach. The presence of Helicobacter pylori is strongly associated with primary gastric lymphoma. The urea breath test, the stool antigen test, and biopsy-based tests can all be used to assess the success of treatment. Helicobacter pylori is susceptible to a wide range of antibiotics in vitro, monotherapy is not usually successful, probably because of inadequate antibiotic delivery to the colonization niche. Current regimens consist of a PPI or H2 blocker, bismuth citrate and two or three antimicrobial agents given for 7-14 days. Research on optimizing drug combinations to increase efficacy continues. Efficacy of Sequential Therapy versus Standard Triple Therapy versus Quinolone-based Triple Therapy for eradication of Helicobacter pylori infection is done in this study.Methods: This study had been conducted on 150 patients divided into three groups randomly 50 Patients each and were treated with Sequential, Standard and Quinolone based triple therapy respectively. Patients were followed up no sooner than four weeks of completing therapy by rapid urease test to confirm eradication.Results: There was no significant difference with regards to presence of GERD, Gastric Ulcers, Duodenal Ulcers (p value&gt;0.05) except for presence of erosive gastritis which was significantly higher in patients in quinolone group (p value&lt;0.05). The eradication rate was 90%, 86%, 82% in Sequential therapy group, Triple therapy group and Fluroquinolone group respectively. However, there was no statistically significant difference in eradication rates in these groups (p value&gt;0.05).Conclusions: Sequential therapy group had better eradication rates (90%) as compared to standard triple therapy group (86%) and fluroquinolone therapy group (82%) but results were not statistically significant when all three groups were compared together.

Abstract 2275: Detection of IDH1 mutations in plasma cell-free circulating tumor DNA (ctDNA) from patients with cholangiocarcinoma

Abstract Somatic mutations in the isocitrate dehydrogenase 1 (IDH1) gene are detected in 13-15% of cholangiocarcinoma (CC) cases overall and up to ~25% of intrahepatic CC cases. Ivosidenib (AG-120) is a first-in-class, oral, potent, reversible, targeted inhibitor of the mutant IDH1 (mIDH1) protein, and AG-881 is an oral, potent inhibitor of both mIDH1 and mIDH2. Phase 1 studies for both molecules in patients with mIDH1/2 solid tumors, including CC, have completed enrollment, and analyses are ongoing (AG120-C-002 [NCT02073994]; AG881-C-002 [NCT02481154). Patients with mIDH1 CC were enrolled into the clinical studies based on local tissue testing for the IDH1 mutation. In this work, we examined the feasibility of mIDH1 detection in ctDNA from patients with mIDH1 CC, and explored the correlation with detection by tissue-based next-generation sequencing (NGS) assays and with plasma levels of the oncometabolite D-2-hydroxyglutarate (2-HG). Baseline plasma samples were collected from a total of 41 patients, including 28 from AG120-C-002 and 13 from AG881-C-002. A BEAMing digital PCR platform (Sysmex) was used for the detection and quantification of five mIDH1 alleles, including R132C, R132H, R132L, R132S, and R132G, with 0.02% analytical sensitivity (0.04% for R132H). Tumor tissues were available from 35 of 41 patients and analyzed using the FoundationOne and/or Personalis ACE cancer research targeted panel. Additionally, baseline plasma levels of 2-HG were measured and correlated with plasma mIDH1 ctDNA. Detection of mIDH1 in plasma ctDNA was concordant with IDH1 mutation status by central testing of tissue in 32 of 35 patients (91.4%), with 31 mIDH1-positive results from both plasma and tissue (88.6%). One patient was mIDH1-negative in both plasma and tumor. In all 31 mIDH1-positive cases, the mIDH1 allele detected was concordant. In general, mIDH1 variant allele frequency (VAF) was lower in plasma than in tumor tissue (mean VAF 2.9% in plasma, 26.2% in tumor). Additionally, the level of plasma ctDNA mIDH1 VAF was significantly correlated with baseline plasma 2-HG concentration (Spearman R=0.535, p=0.0009). Correlation with clinical outcome is currently being explored and will be presented. Taken together, our results demonstrate the feasibility of IDH1-R132 mutation detection in plasma from CC patients, with a 91.4% concordance rate with detection in tumor tissue. These results provide a rationale for exploring liquid biopsy-based testing methods when the feasibility of repeated biopsies or sample exhaustion limits the ability to detect actionable mutations through tissue-based NGS panels, which can be a major challenge for trial participation in this indication. Confirmatory liquid biopsy studies will be conducted with baseline and longitudinal samples collected from the ongoing phase 3 study of ivosidenib in mIDH1 CC (NCT02989857). Citation Format: Elia Aguado-Fraile, Sung Choe, Camelia Gliser, Lori Steelman, Liewen Jiang, Bin Fan, Kha Le, Maeve A. Lowery, Susan Pandya, Bin Wu. Detection of IDH1 mutations in plasma cell-free circulating tumor DNA (ctDNA) from patients with cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2275.

The Diagnostic Tests for Detection of Helicobacter pylori Infection.

Helicobacter pylori causes one of the most common infections in human populations. The role of this bacterium in chronic gastritis, gastric ulcer, gastric cancer, as well as extra-digestive diseases such as ischemic heart disease and chronic obstructive pulmonary diseases, is well known. Prevention and control of these diseases can occur by early diagnosis and eradication of H. pylori infection. At present, different methods have been established to detect H. pylori infection. The biopsy-based tests, which are known as invasive methods, such as rapid urease test and histology, have the highest specificity among the others. Similarly, culture of biopsy samples is used for diagnosis of H. pylori infection. It has a high specificity value, and also allows us to perform antibiotic sensitivity testing. On the contrary, polymerase chain reaction and other molecular methods have good sensitivity and specificity, and can be used for detection of H. pylori infection, its virulence factors, and eradication success after treatment. While serological tests are more appropriate for epidemiological studies, their main weakness for clinical use is low specificity. Overall, specificity and sensitivity, cost, usefulness, and limitation of tests should be considered for selection of detection methods of H. pylori in each country.