Multiplex Platform Research Articles

Single-Droplet Microsensor for Ultra-Short Circulating EFGR Mutation Detection in Lung Cancer Based on Multiplex EFIRM Liquid Biopsy.

Liquid biopsy is a rapidly emerging field that involves the minimal/non-invasive assessment of signature somatic mutations through the analysis of circulating tumor DNA (ctDNA) shed by tumor cells in bodily fluids. Broadly speaking, the unmet need in liquid biopsy lung cancer detection is the lack of a multiplex platform that can detect a mutation panel of lung cancer genes using a minimum amount of sample, especially for ultra-short ctDNA (usctDNA). Here, we developed a non-PCR and non-NGS-based single-droplet-based multiplexing microsensor technology, "Electric-Field-Induced Released and Measurement (EFIRM) Liquid Biopsy" (m-eLB), for lung cancer-associated usctDNA. The m-eLB provides a multiplexable assessment of usctDNA within a single droplet of biofluid in only one well of micro-electrodes, as each electrode is coated with different probes for the ctDNA. This m-eLB prototype demonstrates accuracy for three tyrosine-kinase-inhibitor-related EGFR target sequences in synthetic nucleotides. The accuracy of the multiplexing assay has an area under the curve (AUC) of 0.98 for L858R, 0.94 for Ex19 deletion, and 0.93 for T790M. In combination, the 3 EGFR assay has an AUC of 0.97 for the multiplexing assay.

FEN1-aided recombinase polymerase amplification (FARPA) for one-pot and multiplex detection of nucleic acids with an ultra-high specificity and sensitivity

Recombinase polymerase amplification (RPA) running at 37–42 °C is fast, efficient and less-implemented; however, the existing technologies of nucleic acid testing based on RPA have some limitations in specificity of single-base recognition and multiplexing capability. Herein, we report a highly specific and multiplex RPA-based nucleic acid detection platform by combining flap endonuclease 1 (FEN1)-catalysed invasive reactions with RPA, termed as FEN1-aided RPA (FARPA). The optimal conditions enable RPA and FEN1-based fluorescence detection to occur automatically and sequentially within a 25-min turnaround time and FARPA exhibits sensitivity to 5 target molecules. Due to the ability of invasive reactions in discriminating single-base variation, this one-pot FARPA is much more specific than the Exo probe-based or CRISPR-based RPA methods. Using a universal primer pair derived from tags in reverse transcription primers, multiplex FARPA was successfully demonstrated by the 3-plex assay for the detection of SARS-CoV-2 pathogen (the ORF1ab, the N gene, and the human RNase P gene as the internal control), the 2-plex assay for the discrimination of SARS-CoV-2 wild-type from variants (Alpha, Beta, Epsilon, Delta, or Omicrons), and the 4-plex assay for the screening of arboviruses (zika virus, tick-borne encephalitis virus, yellow fever virus, and chikungunya virus). We have validated multiplex FARPA with 103 nasopharyngeal swabs for SARS-CoV-2 detection. The results showed a 100% agreement with RT-qPCR assays. Moreover, a hand-held FARPA analyser was constructed for the visualized FARPA due to the switch-like endpoint read-out. This FARPA is very suitable for pathogen screening and discrimination of viral variants, greatly facilitating point-of-care diagnostics.

Expanded PCR Panel Testing for Identification of Respiratory Pathogens and Coinfections in Influenza-like Illness.

While COVID-19 has dominated Influenza-like illness (ILI) over the past few years, there are many other pathogens responsible for ILI. It is not uncommon to have coinfections with multiple pathogens in patients with ILI. The goal of this study was to identify the different organisms in symptomatic patients presenting with ILI using two different high throughput multiplex real time PCR platforms. Specimens were collected from 381 subjects presenting with ILI symptoms. All samples (nasal and nasopharyngeal swabs) were simultaneously tested on two expanded panel PCR platforms: Applied Biosystems™ TrueMark™ Respiratory Panel 2.0, OpenArray™ plate (OA) (32 viral and bacterial targets); and Applied Biosystems™ TrueMark™ Respiratory Panel 2.0, TaqMan™ Array card (TAC) (41 viral, fungal, and bacterial targets). Results were analyzed for concordance between the platforms and for identification of organisms responsible for the clinical presentation including possible coinfections. Very good agreement was observed between the two PCR platforms with 100% agreement for 12 viral and 3 bacterial pathogens. Of 381 specimens, approximately 58% of the samples showed the presence of at least one organism with an important incidence of co-infections (~36-40% of positive samples tested positive for two and more organisms). S. aureus was the most prevalent detected pathogen (~30%) followed by SARS-CoV-2 (~25%), Rhinovirus (~15%) and HHV6 (~10%). Co-infections between viruses and bacteria were the most common (~69%), followed by viral-viral (~23%) and bacterial-bacterial (~7%) co-infections. These results showed that coinfections are common in RTIs suggesting that syndromic panel based multiplex PCR tests could enable the identification of pathogens contributing to coinfections, help guide patient management thereby improving clinical outcomes and supporting antimicrobial stewardship.

A membrane biosensor based on colour differences encoded upconversion silica nanoparticles for multiplex nucleic acids detection

Upconversion luminescent nanomaterials provide a powerful tool for multiplex nucleic acids detection simultaneously. However, the conventional encoding methods based on emission spectrum or intensity have limitations due to spectral overlap and cross-relaxation quenching among various activators. Herein, we present a facile method for the preparation of high-capacity upconversion luminescent coding nanoparticles by multiple regulating the synthesis kinetics of lanthanide ions doped CaF2 nanocrystals in mesoporous silica nanotemplets (named C@S). The secondary growth of nanocrystals with different lanthanide ions inside mesopores has a similar performance to that of core/multi-shell-structured upconversion nanoparticles prepared through complicated processes. The textural properties and chemical conformations are maintained after repeated recombination, which is beneficial for reproducible multiplex bioassays. A membrane biosensor is constructed using various probe-modified C@S and a flexible graphene oxide-coated film to detect and identify multiple molecular species in one sample. The colour differences of encoded C@S derived from CIE-L*a*b* colour coordinate system are applied to quantify the divergence of upconversion luminescence for the first time, which allows the upconversion nanoparticles with similar spectra and luminescence colours to be used in the same multiplex detection platform and avoids subjective judgement. This work opens new opportunities for upconversion luminescent materials in the field of automatic high-throughput detection.

New genotyping rescues old phenotypes: beneficial traits in Heterorhabditis bacteriophora wild type material and association to single nucleotide polymorphisms

Summary The entomopathogenic nematode (EPN), Heterorhabditis bacteriophora, is a biocontrol agent with almost world-wide distribution, thus providing a huge source of biodiversity available for exploitation in breeding programmes to improve beneficial traits. For management of economically important insect pests, the nematode is mass-produced in liquid culture, formulated with inert carriers, and transported under controlled temperature conditions to maintain its quality. The biocontrol potential of nematode materials is defined by their genetic background. Previous reports have assessed the phenotypic plasticity of H. bacteriophora. Major beneficial traits are field efficacy influenced by their virulence, longevity and field persistence and tolerance to abiotic stresses like oxidative stress, desiccation and extreme low and high temperatures. For several of these traits high heritability values (h2) have been reported, providing a platform for trait improvement by selection. Modern breeding uses molecular markers to track beneficial traits. Recent studies reported hundreds of single nucleotide polymorphisms (SNPs) in H. bacteriophora. This investigation revived a large set of cryopreserved wild types, hybrids and phenotyped materials of H. bacteriophora. Additional phenotypic data on the virulence (LC50) of H. bacteriophora strains against mealworm (Tenebrio molitor) were produced ranging from 1.4 ± 0.3 to 8.0 ± 3.6 nematodes per insect. Markers linked to beneficial traits were used for a comprehensive genotype analysis transferring reported SNP sequence information into the multiplex platform SeqSNP. Specific alleles associated to beneficial traits were identified and 65 materials were classified for the presence of relevant SNPs associated to beneficial traits. This information provides a valuable platform for exploitation of the nematode’s biodiversity for future breeding and selection approaches.

Cellular and Molecular Mechanisms of Liver Fibrosis in Patients with NAFLD.

The expression of immune- and cancer-related genes was measured in liver biopsies from 107 NAFLD patients. The strongest difference in overall gene expression was between liver fibrosis stages F3 and F4, with 162 cirrhosis-associated genes identified. Strong correlations with fibrosis progression from F1 to F4 were observed for 91 genes, including CCL21, CCL2, CXCL6, and CCL19. In addition, the expression of 21 genes was associated with fast progression to F3/F4 in an independent group of eight NAFLD patients. These included the four chemokines, SPP1, HAMP, CXCL2, and IL-8. A six-gene signature including SOX9, THY-1, and CD3D had the highest performance detecting the progressors among F1/F2 NAFLD patients. We also characterized immune cell changes using multiplex immunofluorescence platforms. Fibrotic areas were strongly enriched in CD3+ T cells compared to CD68+ macrophages. While the number of CD68+ macrophages increased with fibrosis severity, the increase in CD3+ T-cell density was more substantial and progressive from F1 to F4. The strongest correlation with fibrosis progression was observed for CD3+CD45R0+ memory T cells, while the most significant increase in density between F1/F2 and F3/F4 was for CD3+CD45RO+FOXP3+CD8- and CD3+CD45RO-FOXP3+CD8- regulatory T cells. A specific increase in the density of CD68+CD11b+ Kupffer cells with liver fibrosis progression was also observed.

Inflammatory panel cytokines are elevated in the neocortex of late-stage Alzheimer’s disease but not Lewy body dementias

BackgroundChronically dysregulated neuroinflammation has been implicated in neurodegenerative dementias, with separate studies reporting increased brain levels of inflammatory mediators and gliosis in Alzheimer’s disease (AD) as well as in Lewy body dementias (LBD). However, it is unclear whether the nature and extent of neuroinflammatory responses in LBD are comparable to those in AD. In this study, we performed head-to-head measurements of a panel of cytokines in the post-mortem neocortex of AD versus the two major clinical subtypes of LBD, namely, dementia with Lewy bodies (DLB) and Parkinson’s disease dementia (PDD).MethodsPost-mortem tissues from the mid-temporal cortex (Brodmann area 21) of a cohort of neuropathologically well-defined AD, PDD and DLB patients were processed and measured for a comprehensive range of cytokines (IL-1α, IL-1Ra, IL-8, IL-10, IL-12p70, IL-13, IFN-γ, GM-CSF and FGF-2) using a multiplex immunoassay platform. Associations between inflammation markers and neuropathological measures of neuritic plaques, neurofibrillary tangles as well as Lewy bodies were also performed.ResultsWe found IL-1α, IFN-γ, GM-CSF and IL-13 to be elevated in the mid-temporal cortex of AD patients. In contrast, none of the measured cytokines were significantly altered in either DLB or PDD. Similar cytokine changes were observed in two other neocortical areas of AD patients. Furthermore, increases of IL-1α, IFN-γ, GM-CSF, IL-10 and IL-13 associated with moderate-to-severe neurofibrillary tangle burden, but not with neuritic plaques or Lewy bodies. Our findings of elevated neocortical pro- and anti-inflammatory cytokines in AD, but not in DLB or PDD, suggest that neuroinflammatory responses are strongly linked to neurofibrillary tangle burden, which is higher in AD compared to LBD. In conclusion, neuroinflammation may not play a prominent role in the pathophysiology of late-stage LBD.

52 Influence of Saccharomyces cerevisiae CNCM I-1077 on the intestinal environment, gut permeability, and markers of systemic inflammation in horses fed a high starch diet

Probiotics may balance the hindgut environment and relieve systemic inflammation in horses fed high starch concentrates to meet energy requirements. Therefore, 30 mature Quarter Horse geldings (545 ± 58 kg BW) were used in a randomized complete design for 32-d to test the hypothesis that supplemental live Saccharomyces cerevisiae CNCM I-1077 (SC; Levucell® SC Advantage) stabilizes the intestinal environment, reduces gut permeability, and decreases inflammation when horses are fed a high starch diet. Horses were stratified by BW, age, and body condition score (BCS) and randomly assigned treatments consisting of either a concentrate formulated with 2g starch • kg BW−1 • meal−1 (CON, n = 15) or the same concentrate top-dressed with 25 g/d SC(TRT, n = 15) containing 20 billion cfu. Horses were fed individually in stalls (3 × 3 m) every 12 h. Between meals, horses were housed in adjacent dry lots with ad libitum access to Coastal bermudagrass hay and water. On d 0 and 32, BW and BCS were recorded and blood samples were collected immediately before feeding (h 0) and at h 2, 8, 16, and 24 post meal. Samples were analyzed for serum D-lactate using a colorimetric assay, chemokine (CCL2) and cytokine (TNFα) concentrations were measured using a multiplex platform, and whole blood 16s rRNA sequencing was performedusing Illumina MiSeq. Fecal samples were obtained on d 0, 16, and 32 via rectal palpation before the morning meal and at h 8, 16, and 24 post meal. Fecal pH was determined using a portable pH meter and fecal starch was measured via concentration analysis. Data were analyzed using PROC MIXED of SAS v9.4. Non-normal data (CCL2) were log-transformed. From d 0 to 32 BW increased (P ≤ 0.01), independent of treatment, with no change in BCS (P = 0.97). Fecal starch was undetectable indicating nearly complete digestion of dietary starch regardless of treatment. On d 0, fecal pH declined to h 16 (P ≤ 0.01) in both groups and returned to baseline by h 24 post meal. At h 0, CON had lower fecal pH on d 32 than d 0 (P ≤ 0.01). D-lactate peaked at h 8 on d 0, and CON was greater (P ≤ 0.01) than TRT. On d 32, D-lactate tended to be higher in TRT (P = 0.10) at 16 h post meal compared with CON. LogCCL2 and TNFα declined (P ≤ 0.02) across treatments to d 32. Fold change of percent reads from d 0 in bacterial 16s rRNA was not different between treatment groups. A high starch diet initially reduced fecal pH and increased BW but after 32 d, there was no difference in intestinal inflammation or gut permeability, regardless of SC supplementation.

82 Responses to a combined exercise and transport stressor following dietary supplementation of anti-endotoxin IgY in mature horses

Immunoglobulin Y elicits positive effects in domestic livestock with gastrointestinal or inflammatory diseases; however, there are limited studies in horses. To test the hypothesis that dietary anti-endotoxin IgY (IgY) has a positive effect on gastrointestinal health and mitigates systemic inflammation in horses undergoing exercise, 30 stock-type horses (578 ± 59 kg BW; 14 ± 3 yr) were used in a completely randomized design. Horses were stratified by BW, age, and sex and then randomly assigned to treatments: 0 g/d IgY (CON; n = 10), 1 g/d IgY (TRT1; n = 10), or 2 g/d IgY (TRT2; n = 10). Horses were fed every 12 h, with dietary treatments (Camas Inc., Le Center, MN) top-dressed on the morning concentrate for 33 d. Horses were maintained in dry lots with ad libitum access to Coastal bermudagrass hay. Horses were exercised 5 d/wk for 1 h. On d 0 and 30, BW and BCS were recorded, and blood was collected for complete blood cell count (CBC) and serum blood chemistry. On d 32, horses were transported 270 km and underwent a 3.2 km exercise stressor on concrete at a walk with maximum 15 min of trot. Whole blood, serum, and fecal samples collected via rectal palpation were obtained pre-, post-, and 24 h post-trailering. Fecal pH was determined by a portable meter, and serum cytokine (TNFα, IL-4, IL-10, IL-1β, IL-17, IFN-α, IFN-γ) and chemokine (CCL2, 3, 5 and 11) concentrations weremeasured using a multiplex platform. Whole blood DNA was extracted using a commercial DNA extraction kit. For gut permeability analysis, 16s rDNA was analyzed via qPCR and calculated as fold change from d 0. Data were analyzed using PROC MIXED of SAS v9.4. Dietary IgY did not affect BW, BCS, blood chemistry, or CBC (P ≥ 0.17). Fecal pH decreased over time (P ≤ 0.01). There was no treatment × time interaction for 16S rDNA (P = 0.45). Dietary treatment affected circulating 16S rDNA (P < 0.01) where TRT2 (1.89-fold) was greater than CON (0.70-fold), and TRT1 (1.24-fold) was intermediate. A significant time effect (P = 0.001) was observed for 16S rDNA where fold change decreased after the stressor from 1.90-fold for pre-, to 1.02 for post-, and 0.92 for 24 h post- trailering. There tended to be a treatment × time interaction (P = 0.08) for CCL3, as CON was greater at 24 h and TRT2 declined post-trailering, returning to baseline by 24 h. Levels of CCL5 decreased (P < 0.01) and TNFα tended to decline (P = 0.06) through 24 h post trailering in all treatment groups. Other cytokines and chemokines were non-detectible. Despite greater circulating 16s rDNA with higher treatment dosages, there was no effect on systemic inflammatory markers.

Rapid and Accurate Detection of SARS-CoV-2 Using an iPad-Controlled, High-Throughput, Portable, and Multiplex Hive-Chip Platform (HiCube).

Rapid and accurate detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is one of the most effective measures to control the coronavirus disease 2019 (COVID-19) pandemic. However, there is still lack of an ideal detection platform capable of high sample throughput, portability, and multiplicity. Herein, by combining Hive-Chip (capillary microarray) and reverse transcriptional loop-mediated isothermal amplification (RT-LAMP), we developed an iPad-controlled, high-throughput (48 samples at one run), portable (smaller than a backpack), multiplex (monitoring 8 gene fragments in one reaction), and real-time detection platform for SARS-CoV-2 detection. This platform is composed of a portable Hive-Chip device (HiCube; 32.7 × 29.7 × 20 cm, 5 kg), custom-designed software, and optimized Hive-Chips. RT-LAMP primers targeting seven SARS-CoV-2 genes (S, E, M, N, ORF1ab, ORF3a, and ORF7a) and one positive control (human RNase P) were designed and prefixed in the Hive-Chip. On-chip RT-LAMP showed that the limit of detection (LOD) of SARS-CoV-2 synthetic RNAs is 1 copy/μL, and there is no cross-reaction among different target genes. The platform was validated by 100 clinical samples of SARS-CoV-2, and the results were highly consistent with those of the traditional real-time PCR assay. In addition, on-chip detection of 6 other respiratory pathogens showed no cross-reactivity. Overall, our platform has great potential for fast, accurate, and on-site detection of SARS-CoV-2.

Abstract CT082: Next-generation sequencing (NGS) and cytokine assessment from a phase III study of copanlisib in combination with rituximab in patients with indolent non-Hodgkin lymphoma (iNHL) - associations with survival endpoints

Abstract Introduction: The PI3K inhibitor copanlisib (C) plus the anti-CD20 antibody rituximab (R) was superior to R plus placebo (P) in patients (pts) with relapsed iNHL (Matasar et al. Lancet Oncol 2021). We previously reported that C+R improved median progression-free survival (PFS) in the iNHL, follicular lymphoma (FL), and non-FL groups, with a statistically significant improvement in the PTEN-positive population (Shalini et al., AACR 2022). Here we conducted NGS analysis from pts treated with either C+R or P+R for possible impact on PFS outcomes. We also determined if there was an association between plasma cytokine levels at baseline with survival. Methods: Adult pts with relapsed B-cell iNHL were randomly assigned 2:1 to either C+R or P+R; standard dosing on a 28-day cycle applied. Either fresh or archival tumor tissues were collected for central pathology review and biomarker analysis. NGS was conducted using the TSO500 platform for 476 genes. Analyses included EZH2 and BCL2 mutation status in FL subset, and other genes for FL subsets and overall iNHL, as prognostic and predictive markers. The effect of mutations on PFS and overall survival (OS) outcomes was assessed. Cytokines from baseline plasma were assessed using the multiplex MSD platform (71-plex). Association between cytokine levels and PFS and OS were explored. Results: Baseline tumor samples were evaluable by NGS from 200 pts; 127 C+R and 73 P+R. Variant alleles with frequencies &amp;gt;10% were analyzed. One quarter (25.7%) of FL pts were determined to have EZH2 mutations. C+R treatment significantly improved PFS for FL pts with in mutant EZH2 (p=0.0066) compared to P+R. FL pts with mutant BCL2 had a statistically improved PFS compared to WT BCL2 when receiving C+R treatment. Plasma samples were available for 71-plex assessment in 373 pts; 246 C+R and 127 P+R. Pts were assigned as high or low baseline cytokine groups based on the median values. For majority of cytokines, the low cytokine group correlated with better PFS for all pts, FL and Other iNHL pts in the C+R arm. For OS, there were too few events at the time of primary data cutoff to make any associations. However, with longer follow up (2 years from primary completion) there was a significant improvement in OS in iNHL pts with low IL2 values at baseline receiving C+R compared to pts receiving P+R; HR 0.495 (95%CI: 0.270,0.908), p=0.0230. This association was only seen for pts with low (or undetected) levels of IL2 at baseline. Conclusions: Mutation status was associated with improvements in PFS in FL pts with mutant BCL2 or mutant EZH2 treated with C+R. iNHL pts with low plasma levels of IL2 levels treated with C+R showed a significant improvement in OS. Citation Format: Shalini Chaturvedi, Anke Weispfenning, David Bauer, Rong Du, Tine Descamps, Sara Bellinvia, Teresa Lunt, Lidia Mongay Soler, Barrett H. Childs, Pier Luigi Zinzani. Next-generation sequencing (NGS) and cytokine assessment from a phase III study of copanlisib in combination with rituximab in patients with indolent non-Hodgkin lymphoma (iNHL) - associations with survival endpoints [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT082.

Abstract LB162: A multiplex organoid avatar drug testing platform for precision medicine

Abstract The promise of precision medicine is to improve patient outcomes by making better therapeutic choices based on cancer causing somatic mutations. This strategy has shown limited success in colorectal cancer, in part because the genetic rules governing resistance and sensitivity are not fully elucidated. Direct drug sensitivity testing of individual patient-derived organoids is an attractive addition to this decision process because it does not require understanding of all drug-somatic mutation interactions. Individual patients avatars can be challenged with a variety of drugs and response used to inform patient care. We have developed a panel of colon cancer organoid avatars and uniquely tagged each one with a lentiviral vector possessing identifying DNA sequence bar codes flanked by common PCR primers. Each bar-code is uniquely identifiable and quantifiable from a mixture of organoids by nanopore sequencing of a common PCR product. Treatment of the mixture of organoid avatars over time under multiple drug conditions and sampling at different time points allowed the real-time monitoring of relative Darwinian fitness of each organoid in the mixture. We demonstrated the utility of this approach at identifying both expected and novel drug responses for individual organoids. Resistance to both nutlin and irinotecan were correctly predicted by TP53 somatic mutations while sensitivity to both lapatinib and ibrutinib were best predicted by mutations in EGFR signaling pathway. Patterns of drug response revealed unexpected common mechanisms. Future work will focus on informing patient care decisions with these results and determining if improvements in patient outcomes are realized. Citation Format: Shrey Patel, Sana Khalili, Victoria Moy, Emma Gray, Sawyer Lyons, Riley Brents, Carolyn Banister, Phillip J. Buckhaults. A multiplex organoid avatar drug testing platform for precision medicine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB162.

Multiplex PCR-ASE functionalized microfluidic diagnostic platform for the detection of clarithromycin resistance mutations in Helicobacter pylori

With the application of clarithromycin for Helicobacter pylori treatment, the number of resistant strains has increased greatly and has become a problem for treatment. Here, a novel multiplex PCR- allele-specific extension (PCR-ASE) functionalized microfluidic diagnostic platform with advantages of low-cost, easy-to-use and high sensitivity was firstly fabricated and proposed. A biphasic amplification multiplex PCR–ASE assay was integrated and used for the rapid detection of different mutations at positions 2142/2143 in the 23S rRNA gene in a single tube, and the results can be read with the naked eye using a nucleic acid detection strip (NADS). Our data revealed good performance for the multiplex diagnostic platform, which had an analytical sensitivity of approximately 50 copies/reaction and the ability to detect hetero-resistance at proportions as low as 0.5 %. Furthermore, it had no cross-reactivity with 10,000 copies of the wild-type template. The performance of platform for detection of clarithromycin resistance in 25 tissue biopsies was compared to that of Sanger sequencing and found to be in complete concordance. Due to its efficiency and simplicity, the multiplex PCR-ASE functionalized detection platform has great advantages for clinical application using a conventional thermal cycler for the detection of clarithromycin resistance within 2 h.

Abstract 79: Study of tumor microenvironment of ovarian clear cell carcinoma

Abstract Background: Clear cell ovarian carcinoma (CCOC) is characterized by a distinct histologic and molecular profile, and associated with very poor responses to standard treatment consisting of surgery and carboplatin:taxol chemotherapy. CCOC is chemo-resistant at the time of diagnosis and response to chemotherapy in the recurrent setting is less than 10%. Literature suggests a potential role for immune checkpoint inhibitors (ICI). However, only a subgroup of patients (20%) responds to ICI and little is known about the mechanisms of response and resistance to therapy. We postulate that a better understanding of CCOC tumor microenvironment (TME) could help predict patients’ response to ICI. Objective: The aim of this study is to investigate the relationship between TME immune infiltrate and clinical/outcome in 22 CCOC cases and identify subsets of CCOC who may benefit from immunotherapy. Material &amp; Methods: We characterized the immune landscape of 11 early and 11 advanced CCOC through a multiplex IHC Discovery Platform. Spatial single-cell proteomics analyses (cyclic-IF) and spatially-resolved RNAseq in 10 CCOC cases using an OC tissue microarray (TMA) were performed. Results were corelated with clinical and treatment outcome. Results: The percent of CD8, CD4, CD20 B cells Tregs, PD1, PDL1, monocytes and M2 monocytes and myeloid cells was significantly higher in advanced than early-stage cancers. Recurrent cancers were more immunosuppressive than cases with no recurrence. Tumor infiltrate was dense in 4 cases. Four patients with early-stage disease had a high number of CD8 naïve cells and experienced no recurrence. TME analysis of single case of advanced CCOC (before and after chemotherapy), revealed that hot tumor changed to cold tumor, suggesting the resistance to treatment. Cyclic IF analyses identified 3 tumor phenotype groups in a subset of 10 CCOC present on the OC TMA. The majority of CCOC had high expression of CCNE and high PI3K-AKT-mTor activity, low expression of hormone receptors (AR, ERa, PRg) and low cell cycle activity. Some tumors were also high for HER2. The stromal compartment was enriched in collagen VI, aSMA and PDGFR. The immune monitoring of several of those samples also revealed an immunosuppressive microenvironment, including the presence of M2 macrophages and expression of immune checkpoint proteins PD-L1 and B7-H4. Conclusion: Our study shows that various phenotypes of CCOC are defined by the cancer cell profiles and TME content. Importantly, a strong immunosuppressive microenvironment was detected in many samples, suggesting a potential response to ICI. Furthermore, we detected the expression of several therapeutic targets (MAPK, CCNE, PI3K-AKT-mTOR, HSP90, HER2), including oncogenic signaling pathways (RTK, MAPK). Different tumor phenotypes identified across our CCOC samples suggest that clear cell carcinoma could be subclassified into subtypes that should be treated differently. Citation Format: Tanja Pejovic, Sonali Joshi, Shawn Campbell, Dhanir Tailor, Joanna Pucilowska, Benjamin Tate, Pierre-Valérien Abate, Korina Mouzakitis, Marilyne Labrie, Elizabeth Munro, Jenna Emerson, Sanjay V. Malhotra. Study of tumor microenvironment of ovarian clear cell carcinoma [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 79.

Abstract 4624: Deciphering the tumor microenvironment at single-cell resolution using a workflow combining RNA transcript and protein detection with Brightplex®, a sequential chromogenic multiplex assay

Abstract Tumor-infiltrating immune cells play an important role against cancer and are critical to control tumor growth and spread. Immunotherapy and immune checkpoint blockade, which aim to reinvigorate exhausted T cells have revolutionized cancer therapy. Despite inducing long-term response in many cancer types, these therapies remain ineffective for a majority of the patients. A better understanding of the tumor microenvironment, and in particular the characterization of immune cells and other stromal cells, along with their abundance and distribution, may help to better stratify patients and understand mechanisms of resistance to immunotherapy. Brightplex® is a chromogenic multiplex technology allowing the detection of several biomarkers on a single FFPE slide to identify and quantify complex cell populations such as T-cells expressing immune checkpoints, M1 and M2 macrophages, Treg cells and myeloid-derived suppressor cells. However, the exquisite characterization of some cell types within the tumor microenvironment or their activation status may require the identification of soluble proteins that cannot be detected by IHC. For example, the detection of secreted proteins such as cytokines or activation factors would allow one to determine the activation status of immune cells or cancer-associated fibroblasts (CAF). In that case, the detection of RNA transcripts corresponding to soluble biomarkers by In-Situ hybridization can be used as a substitute to protein detection.Here, we propose a new multiplex platform combining In Situ Hybridization (ISH) and immunohistochemistry (IHC) using Brightplex® technology. This platform allows the detection of several biomarkers that can be either RNA transcripts or proteins on a single FFPE tissue section. Thanks to that technique it is possible to: 1) detect and quantify TGFb in cancer associated fibroblasts, or IL-10 in immunosuppressive cells, 2) Quantify the level of RNA expression by individual cells, 3) assess the spatial distribution of activated cells based on complex phenotypes within the tumor or other regions of interest. This new workflow combining RNA transcript detection and proteins detection using Brightplex® is automated on Bond RX platform. Following staining and slide digitization, images are fused to create a virtual multi-channel image where cells of interest are detected by digital pathology (DP). Following cell detection, their densities and spatial distribution are obtained using R-studio software.Integrated into an Immunogram, an analytics platform which integrates multi-omics datasets from Veracyte Biopharma Atlas, this new tool could be a powerful solution to understand the tumor landscape and predict response to immunotherapy and patient outcome. Citation Format: Alex Trinh, Marion Olive, Aurélie Collignon, Maité Chamourin, Georgia Culley, Clemence Jaume, Vanina Leca, Assil Benchaaben, Giovanni Bussotti, Alboukadel Kassambara, Jerome Galon, Jacques Fieschi. Deciphering the tumor microenvironment at single-cell resolution using a workflow combining RNA transcript and protein detection with Brightplex®, a sequential chromogenic multiplex assay. [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 4624.

Rapid and Visual Detection of Oxytetracycline Using Mesoporous Silica Nanochannels Modified Interdigital Electrode

Oxytetracycline (OTC) is one of the widely used antibiotics in veterinary practices because of its broad-spectrum antibacterial activity and low cost. However, drug abuse has triggered adverse effects on human health, which brings a growing demand for on-site diagnosis of OTC residues in animal-derived foods. In this study, we demonstrated the combined use of interdigital electrode microarray and electrochemiluminescence (ECL) imaging for parallel multiplex measurement of OTC. Well-ordered and vertically aligned mesoporous silica nanochannels modified on microarray substrate could exert a strong electrostatic attraction to the ECL luminophores and accelerate their mass transport to generate enhanced ECL signal. The performance of the integrated ECL microarray sensor was fully validated with respect to linearity (0.5 μM to 50 μM), sensitivity (limit of detection 0.26 μM), accuracy (recovery rate between 96.78% and 106.1%), low operating sample volume (480 nL), short assay time (1.5 min), and antifouling ability toward complex media. The multiplex microarray platform can serve as a simple, fast, and low-cost tool for the detection of a wide spectrum of antibiotics in the field of food safety.

Beneficial Effect of Toxoplasma gondii Infection on the Prognosis of Breast Cancer Was Modified by Cytokines.

Animal experiments have shown the anticancer activity of Toxoplasma gondii (T. gondii), but its effect on the prognosis of cancer patients is unclear. Thus, the present study aimed to investigate the prognostic role of anti-T. gondii IgG in breast cancer patients and the modification effect of cytokines. A total of 1121 breast cancer patients were recruited between 2008 and 2018 and followed up until December 31, 2021. Anti-T. gondii IgG and cytokines were measured using an enzyme-linked immunosorbent assay (ELISA) kit and a multiplex assay platform. Endpoints were overall survival (OS) and progression-free survival (PFS). Survival and multiplicative interaction analyses were performed using multivariate Cox regression models. According to the cutoff value of optical density (OD=0.111), 900 (80.29%) and 221 (19.71%) patients were divided into two groups: low or high anti-T. gondii IgG. Compared to patients with a low anti-T. gondii IgG level, the adjusted hazard ratios (HRs) of OS and PFS for patients with high anti-T. gondii IgG levels were 0.60 (95% confidence interval (CI): 0.37-0.99) and 0.67 (0.46-0.98), respectively. These associations were profound among patients with a high cytokine score (HR=0.29, 95% CI: 0.10-0.82 for OS; HR=0.30, 95% CI: 0.13-0.69 for PFS), accompanied by a significant interaction between the level of anti-T. gondii IgG and the cytokine score (P interaction=0.019 for PFS); interleukin-17 (IL-17) and interleukin-9 (IL-9) were the main contributors to the interaction. Anti-T. gondii IgG was found to be beneficial to breast cancer survival, especially in women with systematic inflammation and high IL-17 or IL-9 levels, suggesting the potential of T. gondii as a prognostic marker and a novel immunotherapy approach for cancer patients.

Point-of-Care Diagnosis of Malaria Using a Simple, Purification-Free DNA Extraction Method Coupled with Loop-Mediated Isothermal Amplification-Lateral Flow.

We propose a protocol suitable for point-of-care diagnosis of malaria utilizing a simple and purification-free DNA extraction method with the combination of loop-mediated isothermal amplification assay and lateral flow (LAMP-LF). The multiplex LAMP-LF platform developed here can simultaneously detect Plasmodium knowlesi, P. vivax, P. falciparum, and Plasmodium genus (for P. malariae and P. ovale). Through the capillary effect, the results can be observed by the red band signal on the test and control lines within 5 min. The developed multiplex LAMP-LF was tested with 86 clinical blood samples on-site at Hospital Kapit, Sarawak, Malaysia. By using microscopy as the reference method, the multiplex LAMP-LF showed 100% sensitivity (95% confidence interval (CI): 91.4 to 100.00%) and 97.8% specificity (95% CI: 88.2% to 99.9%). The high sensitivity and specificity of multiplex LAMP-LF make it ideal for use as a point-of-care diagnostic tool. The simple and purification-free DNA extraction protocol can be employed as an alternative DNA extraction method for malaria diagnosis in resource-limited settings. By combining the simple DNA extraction protocol and multiplex LAMP-LF approach, we aim to develop a simple-to-handle and easy-to-read molecular diagnostic tool for malaria in both laboratory and on-site settings.

Signal amplification strategies for optical biodetection at the liquid crystal–solid interface

ABSTRACTLiquid crystals (LCs) are reliable signal transducers for biosensors with chemical and biological sensing potentials demonstrated on the basis of their anisotropy. Thanks to the high sensitivity and fast response to analytes, the LC–solid, LC–aqueous and LC droplet interfaces facilitate sensing platforms to be established without the requirement of sophisticated and costly analytical instruments. In this mini-review, we summarise strategies of optical signal amplification to improve the detection limit of biological analytes at the LC–solid interface. Because approaches towards signal amplification for a single target analyte are seldom compared by discussing various LC-based biosensing platforms, this mini-review may provide implications for integrating current technologies to promote the development of multiplex platforms based on the LC–solid interface.

Syndromic testing for sexually transmitted infection: current and future demand

Sexually transmitted infections (STIs) are a major global public health problem, with significant social burden worldwide. Accurate and appropriate diagnosis and treatment of STIs are important for preventing the transmission of STIs as well as major health consequences of untreated STIs, such as infertility and certain cancer. For diagnosis of STIs, the application of conventional culture and immunoassays is limited by their low sensitivity and long turnaround time. Nucleic acid amplification tests for STIs allow for syndromic tests for multiple pathogens simultaneously and show high sensitivity with a short turnaround time. In this review, we discuss the characteristics of commercially available multiplex molecular platforms and the features needed in next-generation syndromic tests for STIs.