Effusion Samples Research Articles

Autofluorescence free detection of carcinoembryonic antigen in pleural effusion by persistent luminescence nanoparticle-based aptasensors

Carcinoembryonic antigen (CEA) is an important biomarker in the diagnosis of cancer. The increase of CEA in malignant pleural effusion appears earlier and possesses higher clinical diagnostic value than that in the serum. Conventional fluorescent probes suffer from the interference of strong biotissue auto-fluorescence, which limits severely their applications in biology detection. Herein, a novel fluorescence aptasensor was designed with near-infrared persistent luminescence nanoparticles (PLNPs) for accurate detection of carcinoembryonic antigen in pleural effusion by FRET quenching and recovery mechanism. The strong background interference from the autofluorescence of pleural effusion samples can be effectively eliminated and extra increments of measured values originated from the background of different samples were ruled out, benefit from the long decay time of PLNPs and time-resolved fluorescence technology. The detection results show high accuracy of the measured values of carcinoembryonic antigen both in cancer and benign disease group with low detection limit up to 0.0851 pg mL−1. Furthermore, excellent selectivity from coexisting biomarker was achieved by the hybridization between the aptamer and the complementary DNA on PLNPs surface. Hence, the established near-infrared PLNPs-based aptasensors offer excellent performance with high selective, accuracy and signal-to-noise ratio for detection of carcinoembryonic antigen in pleural effusion.

Categorisation of serous effusions using the International System for Reporting Serous Fluid Cytopathology and assessment of risk of malignancy with diagnostic accuracy.

The International System for Reporting Serous Fluid Cytopathology (ISRSFC) standardises the reporting of serous effusion cytology under five categories: Non-Diagnostic (ND), Negative for Malignancy (NFM), Atypia of Undetermined Significance (AUS), Suspicious for Malignancy (SFM), and Malignant (M). Very few studies have been conducted so far to confirm the risk of malignancy of the different categories. The main objectives of our study were to classify serous effusions according to the ISRSFC categories and assess their risk of malignancy (ROM) and performance parameters. All serous effusion samples received from January 2019 to December 2020 were reclassified according to the ISRSFC. Using histopathological diagnosis as the gold standard, ROM and performance parameters were calculated. A total of 831 pleural effusion samples were reclassified as follows: ND, 3 (0.4%); NFM, 635 (76.4%); AUS, 65 (7.8%); SFM, 60 (7.2%); and M, 68 (8.2%). For 457 peritoneal effusion samples, the reclassifications were ND, 5 (1.1%); NFM, 368 (80.5%); AUS, 19 (4.2%); SFM, 17 (3.7%); and M, 48 (10.5%). All 12 (100%) pericardial effusions belonged to the NFM category. The ROM for the ND, NFM, AUS, SRM, and M categories was 0%, 2.1%, 33.3%, 94.1%, 100%, respectively, in pleural effusions, and 50%, 4.8%, 22.2%, 83.3%, 100%, respectively, in peritoneal effusions. The ROM was 0% for NFM in pericardial effusions. The ISRSFC is an excellent system for accurately classifying serous effusions with greater reproducibility of reports and better communication between pathologist and clinician.

A novel method for the isolation of CD45-positive and CD45-negative cells from malignant pleural effusion

Background: Patient-derived malignant pleural effusion (MPE) samples can be used to identify a patient-specific drug combination since MPE samples are readily available and cost-effective tumor cell sources. However, the isolation of target cancer cells from MPE has been inefficient because MPE samples contain a complex mixture of immune cells, non-cancerous cells, and cancer cells. Hence, new methods need to be developed to effectively isolate target cancer cells from MPE samples that can be used for 3-dimensional (3D) cell culture. Patient-derived in vitro 3D tumor models are expected to facilitate more precise drug treatment.Methods: MPE samples were obtained from Seoul St. Mary’s Hospital, The Catholic University of Korea with consent from patients previously diagnosed with lung adenocarcinoma. We isolated target cells from MPE samples using 2 different Percoll-gradient centrifugation methods.Results: The use of 40% and 75% Percoll-gradient centrifugation led to a clearer separation of CD45-positive (CD45pos) and CD45-negative (CD45neg) cells than the traditional 44% and 67% Percoll-gradient centrifugation method.Conclusion: Our findings strongly suggest that the 40% and 75% Percoll-gradient centrifugation method is more useful for the isolation of CD45pos or CD45neg cells than the previously described Percoll-gradient centrifugation method. Furthermore, our novel method was useful for the isolation of MPE-derived target cancer cells that can be used to construct in vitro patient-specific 3D tumor models.

Molecular testing on serous effusion: An update.

Cytological examination of the effusion fluid provides valuable information regarding the presence of malignancy. At times, it is challenging to diagnose malignant cells in serous effusion. The various ancillary techniques are available to solve the problem including immunocytochemistry, DNA ploidy, and multicolored flow cytometry. At present, the molecular tests on the effusion sample are of growing interest. The effusion sample is rich in cells and cell-free fluid that contains free DNA, cytokines, and extracellular vesicles. Molecular tests in effusion sample not only provide a diagnosis of malignancy but can also give valuable information that may be essential for the individualized therapy, management, and prognostic assessment. In this paper, we reviewed the application of the different molecular tests in the effusion sample.

The application value of metagenomic next-generation sequencing in children with invasive pneumococcal disease.

BackgroundA retrospective analysis was conducted to explore the sensitivity and specificity of metagenomic next-generation sequencing (mNGS) in blood, cerebrospinal fluid, and pleural effusion samples in children with invasive pneumococcal disease (IPD), and the impact of detection timing on prognosis and cost.MethodsChildren with IPD admitted to Hebei Children’s Hospital from 1 January 2017 to 1 March 2021 were allocated to 1 of 3 groups according to the clinical symptoms and lesions (Group 1: bacteremia; Group 2: meningitis; Group 3: pleurisy). Taking Alere BinaxNow® Streptococcus pneumoniae (S. pneumoniae) antigen detection and blood culture as the gold standard, receiver operating characteristic (ROC) was used to establish the diagnostic value of mNGS.ResultsA total of 96 cases were enrolled in the study, comprising Group 1 (n=65), Group 2 (n=17), and Group 3 (n=14). The positive rate of mNGS test was 62.5% (n=60), and the total coincidence rate was 75.0%. Delayed mNGS was found to have no significant effect on the 30-day survival rate; however, the species-specific read number (SSRN) of S. pneumoniae detected by mNGS in the early stage of the disease was higher, and it could significantly reduce the hospitalization days and costs (P<0.05).ConclusionsThe sensitivity and specificity of mNGS are high in the identification of S. pneumoniae in blood, cerebrospinal fluid, and pleural effusion samples, and the SSRN of S. pneumoniae is related to the interval from onset to sample collection. Early mNGS detection has no significant effect on the 30-day survival rate among children with IPD, but it can reduce hospitalization costs and duration.

Perspective on the clone library method for infectious diseases.

Recently, culture-independent molecular methods, such as DNA sequencing techniques targeting the 16S-ribosomal RNA (rRNA) gene and/or other housekeeping genes with Sanger method-based technologies, next generation sequencing (NGS), and metagenomic analysis, have been developed for detecting microorganisms in the human body; these can provide information on microbiomes of samples from individuals with or without infectious diseases. Determining the bacterial species is crucial in identifying causative bacteria of upper and lower respiratory tract infections, especially for Streptococcus species, but NGS analysis is often not precise enough to identify bacteria at the species level. This review briefly introduces previous observations of the microbiome of samples from various respiratory and other infections assessed using the clone library method with Sanger sequencing of the 16S-rRNA gene. On analysis of 16S-rRNA gene-sequence data of bronchoalveolar lavage fluid obtained from pneumonia lesions in patients with bacterial pneumonia and lung abscess, anaerobes are often detected in non-elderly patients with pneumonia, and the detection rate of Staphylococcus aureus in patients with hospital-acquired pneumonia is lower than that previously reported. Analysis of pleural effusion samples from patients with pleurisy indicated a more important role of anaerobes than previous believed. The other topics reviewed include microbiomes of nontuberculous mycobacteriosis and lower respiratory tract infections in children with permanent tracheostomy due to neuromuscular disorders, in nasal discharge, in bacterial vaginosis, in the intracystic fluid of postoperative maxillary cyst, and in bacterial conjunctivitis; urine microbiota in urethritis; fecal microbiota; and newly detected infectious organisms in the human respiratory tract.

Sodium Alginate versus Plasma Thrombin Cell Blocks in Diagnostic Cytopathology: A Comparative Analysis

Background: Cell blocks (CBs) are an essential adjunct in cytopathology practice. The aim of this study was to compare 2 techniques of CB preparation – plasma thrombin (PT) method with sodium alginate (SA) method for overall cellularity, morphological preservation, obscuring artefacts, immunocytochemistry (ICC), suitability for molecular analysis, and cost of preparation. Design: A total of 80 fine-needle aspirates from various sites and serous effusion samples were included. Of these cases, by random selection, 40 each were prepared by PT method and SA methods, respectively. The haematoxylin-eosin-stained sections from the formalin-fixed, paraffin-embedded CBs from both methods were evaluated in a blinded fashion by 2 cytopathologists and scored for cellularity, artefacts, and morphological preservation and analysed by χ² test with Yates correction. We evaluated 6 cases from each method by ICC for a range of membrane, cytoplasmic and nuclear marker expression. DNA was extracted from four cases to evaluate their utility for molecular analysis. Results: CB sections from PT and SA techniques showed comparable cellularity and excellent cytomorphological preservation. Blue gel-like artefacts were common in the SA technique but did not interfere with morphological evaluation. ICC staining results were also similar. DNA yield and utility for PCR were also comparable. The SA-CB cost half that of PT-CB (USD 0.4 vs. USD 1). Conclusion: SA technique of CB preparation is an excellent low-cost alternative to PT method for CB preparation.

Influence of deep-freezing and MGG staining on DNA and RNA quality in different types of lung adenocarcinoma cytological smears.

Preserving the optimal quality of DNA and RNA is mandatory for molecular testing in lung adenocarcinoma cytological smears (LACSs). DNA and RNA were isolated from 90 frozen unstained and 46 May Grünwald Giemsa (MGG) stained LACSs prepared from bronchial washing (BW), bronchial brushing (BB), and pleural effusion (PE) samples during 3 years. Concentrations of nucleic acids in all LACSs were assessed by spectrophotometric analysis. Fragmentation of DNA and RNA was determined by PCR amplification of selected genes. Amplicons of 100, 200, 300, 400, and 600 bp were used for DNA and 108 bp-long HPRT1 transcript fragment for RNA fragmentation analysis. Among 90 frozen LACSs, significantly lower DNA concentrations of BB and RNA concentrations of BW samples frozen for 6-10 months were observed in comparison with samples frozen for longer periods (p< .05). Among 46 paired LACSs, 44 (95.7%) frozen and 15 (32.6%) MGG-stained samples showed 600 bp-long DNA amplicons. Statistically significant difference (p< .05) in the fragmentation of DNA between frozen and MGG-stained LACSs was observed (p< .05), with DNA being less fragmented in frozen LACSs. In addition, 33 (71.7%) frozen and 36 (78.2%) MGG-stained LASCs showed HPRT1 gene amplicon of 108 bp. RNA was less fragmented in 3-year old MGG-stained samples than in LACSs frozen for 3 years. DNA and RNA extracted from frozen and MGG-stained LACSs showed different results depending on the time of storage and/or type of samples, but in general all samples had adequate quantity and quality for downstream molecular testing.

Low-cost polymer-film spiral inertial microfluidic device for label-free separation of malignant tumor cells.

We developed a low-cost polymer-film spiral inertial microfluidic device for the effective size-dependent separation of malignant tumor cells. The device was fabricated in polymer films by rapid laser cutting and chemical bonding. After fabricating the prototype device, the separation performance of our device was evaluated using particles and cells. The effects of operational flow rate, cell diameter, and cell concentration on the separation performance were explored. Our device successfully separated tumor cells from polydisperse white blood cells according to their different migration modes and lateral positions. Then, the separation of rare cells was carried out using the high-concentration lysed blood spiked with 200 tumor cells. Experimental results showed that 83.90% of the tumor cells could be recovered, while 99.87% of white blood cells could be removed. We successfully employed our device for processing clinical pleural effusion samples from patients with advanced metastatic breast cancer. Malignant tumor cells with an average purity of 2.37% could be effectively enriched, improving downstream diagnostic accuracy. Our device offers the advantages of label-free operation, low cost, and fast fabrication, thus being a potential tool for effective cell separation.

P59.13 The Prediction Performance of TP53 / RB1 Co-Mutation on Small-Cell Lung Cancer Transformation in Patients With Non-Small Cell Lung Cancer

It has been proven that patients with EGFR mutant non-small cell lung cancer (NSCLC) who harbored loss-of-function mutation in both TP53 and RB1 are more prone to small-cell lung cancer transformation (SCLC-T). This study aims to investigate the prediction performance of TP53 / RB1 mutations on SCLC-T in patients with NSCLC. Patients with pathologically confirmed SCLC-T who performed hybridization capture based next-generation sequencing of 1021 cancer-related genes using tumor tissue and / or malignant effusion samples were retrospectively enrolled in this study. Their clinicopathological characteristics, genetic profiles and treatment were reviewed. From 2016 to 2021, a total of nine patients were enrolled. The median age at diagnosis was 53 years (range 44-65 years); 55.6% (5/9) patients were male and 33.3% (3/9) patients were ever-smokers. All patients except for one with NSCLC (P5, details not known) were initially diagnosed with adenocarcinoma. One patient had pleural effusion sample sequenced at initial diagnosis and tumor tissue samples at the time of transformation (P1), and the remaining cases had only tissue and / or effusion samples at the time of transformation or after that time. Mutation in EGFR, TP53 and RB1 was detected in 100%, 88.9% and 55.6% of patients, respectively (Figure). All patients with RB1 mutations had TP53 mutations detected. All patients were previously treated with EGFR tyrosine kinase inhibitors before transformation. For P1, RB1 mutation was detected at initial diagnosis. After SCLC-T, 2 mutations disappeared and 14 mutations emerged, which may be caused by tumor heterogeneity or evolutional pressure, or related to phenotypic transformation. Copy number variants were more frequently identified in patients with RB1 mutation (100%, 5/5) than in patients without RB1 mutation (50%, 2/4). RB1 mutation was not detected in the tumor sample for P8 at the time of transformation, but was subsequently found in the cerebrospinal fluid collected after 6 months. TP53 / RB1 co-mutation was identified in about half of patients with SCLC-T and may be presented after transformation. Inferring phenotypic transformation only through the presence of TP53 / RB1 co-mutation may result in missed diagnosis of SCLC-T, which should be combined with other techniques such as serum tumor marker neuron specific enolase or pathological exanimations of re-biopsy samples.

Cytomorphological study of peritoneal fluid in suspected cases of malignancy

Cytology of body fluid is one of the oldest methods that is widely accepted. Though the pleural fluid cytology is well documented, data on peritoneal fluid cytology is sparse. It has a distinct value in confirming or disapproving malignant lesion. Most of the laboratories use cytospin smears for cytological study. While cytospin smears without doubt is a good method for cytodiagnosis, it can be complemented with cellblock for a more accurate diagnosis. Cellblock is a diagnostic modality which can help in pointing the cause for effusion and also in staging, prognosis and therapy of many malignant conditions. The aim of this study is to (1) analyse the cytomorphology of peritoneal fluid using cytospin and cellblock technique and (2) to assess the utility of cellblock method in identifying malignant cells in peritoneal effusion and wash samples. A total of 53 ascitic fluid and peritoneal wash samples that were clinically suspected of malignancy were studied. Each of the samples were processed by cytospin smear and cell block method. The results were interpreted by descriptive analysis. Sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy of the cytological test was 96.15%, 100%, 100%, 96.42% and 98.11% respectively. Cellular yield for malignancy was 3.85% more by the cellblock method. Cellblock can provide an additional information which can aid in increasing the sensitivity of cellblock. It can complement cytospin smears, especially to detect malignant cells in peritoneal fluid. A combined approach of cytospin and cellblock can help in a more accurate diagnosis.

A feasibility study investigating the utility of a point-of-care rapid immunoassay for detecting septic peritoneal effusion in dogs.

To determine the sensitivity (SN), specificity (SP), positive predictive value (PPV), negative predictive value (NPV), and accuracy of a point-of-care urine bacterial rapid immunoassay (RIA) for a diagnosis of septic peritonitis, compared to more traditional diagnostic tools, such as cytology and bacterial culture. Prospective, cross-sectional feasibility study conducted from October 2016 to June 2017. Four locations within a private practice referral hospital. Sixty-four dogs with peritoneal effusion of any etiology were enrolled. Four dogs were excluded due to absent RIA or bacterial culture results. Two additional dogs were excluded because of an inability to definitively classify them as having septic peritonitis. Abdominocentesis was performed to obtain peritoneal effusion samples for aerobic and anaerobic bacterial culture, cytology, and RIA. Cytological slides were evaluated by a clinical pathologist following enrollment. Fifty-eight dogs were included in the study, 8 of which were classified as having septic peritonitis. Compared to final diagnosis of septic peritonitis, RIA had a low PPV (36.8%) but good NPV (97.4%) and was 77.5% accurate. SN of RIA (87.5%) to diagnose septic peritonitis was similar to cytology (85.7%) and bacterial culture (87.5%); however, SP was lower (76%, 100%, and 98%, respectively). Cytology had the best overall predictive values and accuracy for diagnosing septic peritonitis (PPV 100%, NPV 97.9%, and accuracy 98%) compared to RIA and bacterial culture. RIA testing was similarly sensitive in identifying septic peritonitis compared to cytology and bacterial culture but was not very specific or accurate. As a stand-alone test, RIA commonly had false-positive test results, making it unreliable in identifying septic peritonitis. Cytology was the most accurate diagnostic test and had no false-positive results. Further investigation with a larger sample size and prevalence of septic peritonitis may prove beneficial.

Feline Coronaviruses Identified in Feline Effusions in Suspected Cases of Feline Infectious Peritonitis.

Ninety-five effusion samples were collected from cats with suspected feline infectious peritonitis in northern Taiwan; these samples showed a 47.4% (45/95) feline coronavirus (FCoV) positivity rate on immunofluorescence staining and RT-PCR. Young cats (≤24 months old) were found to have a significantly higher risk than cats >24 months old (odds ratio (OR) = 6.19, 95% confidence interval (CI) 2.54–16.00). No significant association was found between the positive rates and sex or breed. The A/G ratio in positive cases was significantly lower than the A/G ratio in negative cases. Genotyping and sequencing of the positive cases revealed 71.9% single infection with type I strains and 28.1% coinfection with types I and II. No single infections with type II strains were noted. The type I sequences had high diversity, while the type II sequences had high internal sequence identity and were more similar to CoVs from other species, such as dogs, pigs, and various small mammals. This study demonstrates the latest analysis of FCoV infection cases in northern Taiwan.

A Retrospective Study on Bacteriology, Clinicopathologic and Radiographic Features in 28 Cats Diagnosed with Pyothorax.

Simple SummaryThe cat is a popular pet in Malaysia. Pyothorax is one of the most common respiratory conditions of cats in Malaysia. The etiological agents, particularly bacteria, of local feline pyothorax cases have never been studied. Therefore, in the present study we aimed to identify the bacteria involved in feline pyothorax cases, detect the antimicrobial susceptibility of the bacteria involved and study the radiographic findings related to these cases. Clinical records of cats diagnosed with pyothorax between 2013 and 2020 were analysed. The cats were presented with respiratory signs. Blood results showed significant inflammatory patterns and radiographs revealed different findings for each cat. The common bacteria and the antimicrobial susceptibility of the bacteria were determined in this study. Reporting the most common bacteria involved will give clinicians useful information that will further help in the selection of appropriate treatment and help clinicians understand the pathogenesis of pyothorax in the Malaysian cat population.This retrospective study aimed to determine the etiological, clinicopathological, and radiographic features and outcome of feline pyothorax cases. Medical records from twenty-eight cats with pyothorax aged from 4 months to 10 years (median 10 months) diagnosed between 2013 and 2020 were reviewed. Dyspnoea (75.0%), abnormal lung sounds (75.0%) and open-mouth breathing (64.3%) were the predominant respiratory signs. Leucocytosis (61.5%), particularly monocytosis (68.0%), and hyperglobulinaemia (65.4%) were among the most prominent findings in blood analysis. Bilateral pleural effusion was found in 67.9% of the thoracic radiographs. A total of 47.4% of the cytological samples revealed the presence of bacteria, while all had positive bacterial growth. Pasteurella multocida, E. coli, Streptococcus spp., and Staphylococcus spp. were the predominant aerobic bacteria isolated from pleural effusion samples. A chest tube was placed in 64.3% of the cats and 66.7% of cats with chest tubes survived. In total, 46.4% of cats with pyothorax recovered. Amoxicillin–clavulanate was the antimicrobial of choice against aerobic bacteria found in this study and should be given in combination with antimicrobials that cover anaerobic bacteria. Chest tube placement is crucial for treatment success. Cytological results and bacterial culture may not be consistent; thus, bacterial culture should be performed for every case.

Feasibility study evaluating a veterinary point‐of‐care urine culture system for diagnosing septic peritonitis

To evaluate the use of a veterinary point-of-care urine culture system (POCUCS) for the diagnosis of septic peritonitis. Prospective feasibility study performed between August 2017 and April 2018. Private referral hospital. Twenty samples of naturally occurring canine peritoneal effusion collected via aseptic abdominocentesis. Point-of-care urine culture systems were inoculated and incubated according to manufacturer's instructions. The presence of bacterial growth, estimation of colony-forming units/mL of bacteria, and organism identification were recorded. Bacterial growth and organism identification on POCUCS were compared to an aerobic culture performed at a commercial microbiology laboratory. Serial dilution and subsequent culture on a POCUCS of a confirmed Escherichia coli infected peritoneal effusion and negative control sample were performed to determine the lowest concentration of bacteria detectable. There were 10 septic and 10 aseptic samples of peritoneal effusion confirmed by aerobic laboratory culture. Of the 10 culture-positive samples, 8 were culture-positive on the POCUCS. The sensitivity and specificity of the POCUCS for the detection of bacteria in peritoneal effusion were 80.0% and 100%, respectively. The POCUCS lowest limit of detectable bacteria in peritoneal effusion was 1000 CFUs/mL. The POCUCS evaluated in this study was less sensitive and less rapid for diagnosing septic peritonitis than blood glucose to peritoneal effusion glucose ratio and plasma lactate to peritoneal effusion glucose ratio. This POCUCS is not recommended as a tool for diagnosing septic peritonitis.

Clinical value of CD97 and CD55 levels in the differential diagnosis of tuberculous and malignant pleural effusions.

This study evaluated the clinical levels of CD97 and CD55 for the differential diagnosis of pleural effusion.Pleural effusion samples were collected from 106 patients (55 tuberculous pleural effusions [TPE] and 51 malignant pleural effusions [MPE]). CD97 and CD55 levels in pleural effusions were measured by enzyme-linked immunosorbent assay.CD97 levels were significantly higher in the TPE group than in the MPE group (P < .001), while CD55 levels in the MPE group were significantly higher than the TPE group (P < .001). The sensitivity and specificity of CD97 testing for the differential diagnosis of TPE and MPE was 80.0% and 60.8%, respectively, while the sensitivity and specificity of CD55 testing for TPE and MPE was 88.2% and 85.5%, respectively. Furthermore, the sensitivity and specificity of combinatorial CD97 and CD55 testing for TPE and MPE was 90.0% and 87.5%, respectively. Moreover, CD97 and CD55 were negatively correlated in the MPE group (r = –0.383, P = .005), while no correlations were observed in the TPE group. CD97 or CD55 showed no correlations with other inflammatory cytokines (tumor necrosis factor α, interleukin 1β, erythrocyte sedimentation rate, and C-reactive protein) in both groups (P > .05).CD97 and CD55 may be used as biological markers for the differential diagnosis of pleural effusion in clinical settings.

Abstract 558: Muti-gene ARMS PCR using both cfDNA and cfRNAin the supernatant of pleural effusion achieves rapid and accuracy driver gene mutations detection

Abstract Background Pleural effusion from patients with advanced non-small cell lung cancer has been proved valuable for molecular analysis, especially when the tissue sample not available. However, simultaneous detection of multiple driver gene alterations especially the fusions is still challenging. Methods In this study, 77 patients with advanced NSCLC and pleural effusion were enrolled, 49 of whom had matched tumor tissues. Supernatants, cell sediments, and FFPE cell blocks were prepared from pleural effusion samples for detection of driver alterations by a PCR-based 9-gene mutation detection kit. Results In addition to mutations in EGFR, KRAS and HER2 detected in genomic DNA from cell sediments, FFPE cell blocks, and in cfDNA from supernatants, and fusions in ALK detected in RNA from cell sediments and FFPE cell blocks, fusions in ALK were also successfully detected in cfRNA from supernatants. Compared with matched tumor tissue, the supernatant showed the highest overall sensitivity (81.3%), with 81.5% for SNV/Indels by cfDNA and 80% for fusions by cfRNA, followed by FFPE cell blocks (71.0%) and the cell sediments (66.7%). Within the group of treatment-naïve patients or malignant cells observed in the cell sediments, supernatant showed higher overall sensitivity (89.5%, 92.3%) with both 100% for fusions. Based on the results, an optimized driver gene mutations detection procedure of pleural effusion is proposed. With the proposed procedure, an overall sensitivity 85.3% was achieved, with 82.1% for SNV/Indels and 100% for fusions. Conclusions CfDNA and cfRNA derived from pleural effusion supernatant (PES) have been successfully tested with a PCR-based multi-gene detection kit. An optimized procedure could maximize clinical value of testing pleural effusion samples and has good potential for routine clinical application. Citation Format: Xuejing Chen, Kun Li, Zichen Liu, Fei Gai, Guanshan Zhu, Shun Lu, Nanying Che. Muti-gene ARMS PCR using both cfDNA and cfRNAin the supernatant of pleural effusion achieves rapid and accuracy driver gene mutations detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 558.

Identification and characterization of effusion tumor cells (ETCs) from remnant pleural effusion specimens.

Cancer is a leading cause of death worldwide, and patients may have advanced disease when diagnosed. Targeted therapies guided by molecular subtyping of cancer can benefit patients significantly. Pleural effusions are frequently observed in patients with metastatic cancer and are routinely removed for therapeutic purposes; however, effusion specimens have not been recognized as typical substrates for clinical molecular testing because of frequent low tumor cellularity. Excess remnant pleural effusion samples (N = 25) from 21 patients with and without suspected malignancy were collected at Stanford Health Care between December 2019 and November 2020. Samples were processed into ThinPrep slides and underwent novel effusion tumor cell (ETC) analysis. The ETC results were compared with the original clinical diagnoses for accuracy. A subset of confirmed ETCs was further isolated and processed for molecular profiling to identify cancer driver mutations. All samples were obtained with Institutional Review Board approval. The authors established novel quantitative standards to identify ETCs and detected epithelial malignancy with 89.5% sensitivity and 100% specificity in the pleural effusion samples. Molecular profiling of confirmed ETCs (pools of 5 cells evaluated) revealed key pathogenic mutations consistent with clinical molecular findings. In this study, the authors developed a novel ETC-testing assay that detected epithelial malignancies in pleural effusions with high sensitivity and specificity. Molecular profiling of 5 ETCs showed promising concordance with the clinical molecular findings. To promote cancer subtyping and guide treatment, this ETC-testing assay will need to be validated in larger patient cohorts to facilitate integration into cytologic workflow.

Multiplexed serpentine microchannels for high-throughput sorting of disseminated tumor cells from malignant pleural effusion

Accurate isolation and detection of disseminated tumor cells from the malignant pleural effusion (MPE) is of significant clinical importance. Cytology is a routine diagnostic tool for the analysis of MPE. However, its sensitivity is limited by the existence of a significant number of contaminating blood cells and relatively small and even rare number of target tumor cells in the effusion. To address this limitation, we proposed and developed multiplexed inertial microfluidic devices for fast and efficient isolation and purification of tumor cells from a large volume (> 50 mL) of pleural effusion sample in a label-free way. The rare tumor cells were separated from blood cells relying on the mechanism of size-based differential inertial focusing. The inertial focusing and separation performance of the single serpentine channel was characterized using polystyrene beads and cancer cells lines (A549 and MCF-7) spiked in blood. Subsequently, the multiplexed devices were developed by parallelizing serpentine channels in both planar and vertical directions (e.g., 8 and 24 parallel channel) and the separation was evaluated by the spiked tumor cells (recovery ratio ∼ 80 % for the 8-channel device). Finally, we validated the performance of the multiplexed device using the clinical pleural effusion samples. Malignant tumor cells (MTCs) with a concentration of 49–4806 counts/mL were detected in all 5 pleural effusion samples, with an average purity of ∼30 %. We envision that the multiplexed device is a promising disposable tool towards the clinical applications, given the superiorities of simple structure, high throughput, low cost and good efficiency.

The landscape of intergenic-breakpoint fusion in lung cancer.

e15002 Background: Kinase fusions have been proved as driver factors and drug targets for lung cancer. DNA-based next-generation sequencing (NGS) has been widely used for gene fusion detection in patients with lung cancer and intergenic-breakpoint fusions can be detected, in which breakpoints localize to intergenic regions. Theoretically, intergenic-breakpoint fusions are unlikely to be functional, but recent studies have found that intergenic-breakpoint fusions are potentially activated. However, the proportion of intergene-breakpoint fusion occurrence in lung cancer is still unclear. Hereby, next generation sequencing was performed to describe lung intergenic-breakpoint fusions for further research. Methods: Tumor tissue, pleural effusion, or blood samples of 6798 patients with lung cancer from Simceredx were collected for targeted NGS panel sequencing from June 2020 to January 2021. Gene rearrangements of ALK, ROS1, RET, NTRK(1-3) were assessed. Results: We found gene fusions in 4.43% (301/6798) patients with lung cancer totally, of which 59.1% (178/301) ALK fusions, 18.9% (57/301) RET fusions, 15.9% (48/301) ROS1 fusions and 6.64% (20/301) NTRK(1-3) fusions. The intergenic-breakpoint fusions accounted for all fusions of ALK, RET and ROS1 genes were 24.2% (43/178), 22.8% (13 /57) and 18.8% (9/48), respectively. In patients with NTRK intergenic fusions (50.0%, 10/20), NTRK1, NTRK2 and NTRK3 was involved in 2, 1, 7, respectively. Co-occurrence of different fusions was rare. Conclusions: Our data firstly revealed the specific intergenic-breakpoint fusions ( ALK, RET, ROS1, NTRK1-3) in lung cancer. The functional consequences identifying by RNA or protein assay and clinical validation need to be carried out in the future.