High Specificity Research Articles

Xpert-Ultra assay on stool and urine to improve tuberculosis diagnosis in children: the Médecins Sans Frontières experience in Guinea-Bissau and South Sudan

Abstract Background Over half of childhood tuberculosis (TB) remains undiagnosed yearly. WHO recommends Xpert-Ultra as a first paediatric diagnosis test, but microbiological confirmation remains low. We aimed to determine the diagnostic performance of Xpert-Ultra on stool and urine in presumptive paediatric TB cases in two high-TB burden settings. Methods This Médecins sans Frontières cross-sectional multicentric study took place at Simão Mendes hospital, Guinea-Bissau (July 2019 to April 2020) and in Malakal hospital, South Sudan (April 2021 to June 2023). Children 6 months to 15 years with presumptive TB underwent clinical and laboratory assessment, with one respiratory and/or extrapulmonary sample (gold standard (GS)), one stool and one urine specimen analysed with Xpert-Ultra. Results A total of 563 children were enrolled in the study, 133 from Bissau, 400 from Malakal; 30 were excluded. Confirmation of TB was achieved in 75 (14.1%) while 248 (46.5%) had unconfirmed TB. Of 553 with GS specimen, the overall diagnostic yield was 12.4% (66/533). A total of 493 and 524 samples were used to evaluate Xpert-Ultra on stool and on urine, respectively. Compared to GS, sensitivity and specificity of Xpert-Ultra on stool were 62.5%(95%CI:49.4-74) and 98.3%(95%CI:96.7-99.2), whereas on urine were 13.9%(95%CI:7.5-24.3) and 99.4%(95%CI:98.1-99.8), respectively. Nine patients were positive on stool and/or urine but negative on GS. Conclusions Xpert-Ultra on stool showed moderate to high sensitivity and high specificity when compared to GS and an added diagnostic yield when GS was negative. Xpert-Ultra on stool was useful in extrapulmonary cases. Xpert-Ultra in urine showed low test performance. Trial registration NCT06239337

Abstract PO2-07-12: Analysis Of Malignity Rates Of Percutaneous Biopsy In Lymph Nodes Of Breast Cancer Patients

Abstract BACKGROUND: The evaluation of regional lymph nodes in patients with breast cancer is one of the main predictive and prognostic factors for treatment. The methods of percutaneous biopsies of suspicious lymph nodes frequently used are fine-needle aspiration cytology (FNA) and core needle biopsy (CORE). According to the international literature, CORE and FNA are considered diagnostic methods with high specificity (98% vs. 99%), however, the FNA may present up to 21% of inconclusive results by insufficient material. Although CORE is well established as a percutaneous method for diagnostic evaluation of suspected breast lesions, the literature is scarce on the use of this technique for the evaluation of suspicious lymph nodes in breast cancer patients. OBJECTIVE: Analyze the positivity of FNA and CORE performed in suspicious lymph nodes for breast cancer metastasis according to the anatomical location of biopsies and the type of needle used, verifying which technique was preferred. METHODS: A retrospective study was conducted by evaluating the database of patients treated in a public hospital in São Paulo, Brazil. Women submitted to ultrasound-guided percutaneous biopsy of lymph nodes from May 2015 to November 2019 were included in the study. The data were analyzed using IBM-SPSS version 27 and Microsoft EXCEL version 2010. RESULTS: A total of 499 biopsies were performed and the mean age of the women was 54.2 years (SD± 11.9) in the CORE group and 53.4 years (SD± 11.8) in the FNA group (p=0.619). According to the anatomical location, 385 were axillary (77.2%), 62 supraclavicular (12.4%), 48 cervical (9.6%) and 4 infraclavicular (0.8%). Regarding the type of needle, 393 were CORE (78.8%) and 106 were FNA (21.2%). When analyzing the results of the FNA, 38 (35.8%) did not present enough material, 31 (29.2%) were positive, 32 (30.2%) were negative and 5 (4.8%) showed atypical cells. Among the 393 CORE performed, 255 (64.9%) were positive, 132 (33.6%) were negative, 1 (0.3%) showed atypical cells and 5 (1.3%) had no representative material. No complications were reported after the procedures. CONCLUSION: CORE was the preferred diagnostic technique in our center, being considered a feasible procedure to evaluate lymph nodes in different sites and with low rates of inconclusive results by insufficient material. In the future, studies evaluating indirect costs may confirm the feasibility of CORE in patients with suspicious lymph nodes in terms of obtaining greater agility and resolutive conducts in the public healthcare system. Citation Format: Marina Diogenes, Andressa Amorim, ANDRE MATTAR, MARCELO ANTONINI, Luiz Henrique Gebrim, REGINALDO COELHO LOPES, Luciana Damous. Analysis Of Malignity Rates Of Percutaneous Biopsy In Lymph Nodes Of Breast Cancer Patients [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 PO2-07-12.

Abstract PO1-18-06: Phase 1/2a Open-label Clinical Trial of BI-1607, an Fc Engineered Monoclonal Antibody to CD32b (FcγRIIB), in Combination with Trastuzumab in Subjects with HER2-positive Advanced Solid Tumors – CONTRAST

Abstract Background The introduction of trastuzumab has dramatically changed outcomes in patients with human epidermal growth factor receptor 2 positive (HER2+) cancer. However, primary or acquired resistance to trastuzumab has been increasingly recognized as a major obstacle in the clinical management of this disease. Combination of anti-HER2 antibodies with other immunotherapies is likely to improve the quantity and quality of responses. BI-1607 is a human monoclonal antibody (mAb) targeting FcRIIB (CD32b) with antagonistic function capable of blocking the inhibitory function of FcRIIB on immune effector cells. BI-1607 has been engineered to lack a glycan in position N297Q in the constant domain (Fc), and thus cannot interact with FcγRs through its Fc. Given its high specificity and affinity for FcγRIIB, BI-1607 blocks other antibodies’ binding to FcγRIIB. As a result, BI-1607 is expected to shift tumor cells coated antibodies (here anti-HER2) to selectively engage activating FcγRs, thus augmenting FcγR-dependent therapeutic activity (ADDC, ADCP). Tumor-associated macrophages express high levels of FcγRIIB and are a major target of BI-1607 in the tumor microenvironment. This concept was demonstrated in preclinical in vivo models showing increased efficacy of the combination therapy with the murine surrogate of BI-1607 and an anti-HER2, an anti–cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and an anti-CD20 (rituximab) as compared to monotherapy. It important to note that BI-1607 will have no single-agent activity. Instead, its clinical use will be in combination with other immunotherapies and tumor-targeting antibodies such as trastuzumab. The choice of trastuzumab as the combination agent in this trial was based on promising preclinical studies, a recognized need for additional options for those patients who fail to respond or stop responding to trastuzumab, and promising results from the newly approved Fc-engineered anti-HER2 mAb margetuximab. Ultimately, if shown to be safe and effective in combination with trastuzumab, BI-1607 can also be used in combination with other cytotoxic or immunomodulatory antibodies for cancer treatment. Methods This is a Phase 1/2a, first-in-human, open-label, multicenter, dose-escalation, consecutive-cohort study of BI-1607 in combination with trastuzumab in subjects with HER2+ advanced solid tumors. Phase 1 aims to assess safety and tolerability and to determine the RP2D of BI-1607 in combination with trastuzumab. Phase 2a will explore efficacy at RP2D of BI-1607 in combination with trastuzumab in two separate expansion cohorts, a) in subjects with locally advanced or metastatic HER2+ breast cancer and b) in subjects with HER2+ metastatic gastric or gastroesophageal junction adenocarcinoma. Eligible patents must have a HER2+ locally advanced unresectable or metastatic solid tumors and have received standard of care or be intolerant to standard of care antineoplastic therapy, with progressive disease after the last line of treatment. Accrual/Planned Accrual As of June 222d, 2023 10 patients have been treated into trial and dosed with BI-1607 and trastuzumab at doses from 75-900 mg every 3 weeks (Q3W) in Phase 1. Phase 2a will enroll 15 subjects each in two cohorts of HER2+ advanced/metastatic breast cancer or HER2+ gastric/GEJ adenocarcinoma respectively. For information regarding the study, please contact: anna.ropenga@bioinvent.com Citation Format: Javier Cortés, Araceli Priego, Elena Garralda Cabanas, Katerin Rojas Lamito, Simon Lord, Thorsten Goetze, Sherko Küemmel, Simon Crabb, Marie Borggren, Ingrid Karlsson, Linda Mårtensson, Anna Ropenga, Ingrid Teige, Johan Wallin, Björn Frendeus, Andres McAllister. Phase 1/2a Open-label Clinical Trial of BI-1607, an Fc Engineered Monoclonal Antibody to CD32b (FcγRIIB), in Combination with Trastuzumab in Subjects with HER2-positive Advanced Solid Tumors – CONTRAST [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 PO1-18-06.

Kinase Inhibition via Small Molecule-Induced Intramolecular Protein Cross-Linking.

Remarkable progress has been made in the development of cysteine-targeted covalent inhibitors. In kinase drug discovery, covalent inhibitors capable of targeting other nucleophilic residues (i.e. lysine, or K) has emerged in recent years. Besides a highly conserved catalytic lysine, almost all human protein kinases possess an equally conserved glutamate/aspartate (e.g. E/D) that forms a K-E/D salt bridge within the enzyme active-site. Electrophilic ynamides were previously used as effective peptide coupling reagents and to develop E/D-targeting covalent protein inhibitors/probes. In the present study, we report the first ynamide-based small-molecule inhibitors capable of inducing intramolecular cross-linking of various protein kinases, leading to subsequent irreversible inhibition of kinase activity. Our strategy took advantage of the close distance between the highly conserved catalytic K and E/D residues in a targeted kinase, thus providing a conceptually general approach to achieve irreversible kinase inhibition with high specificity and desirable cellular potency. Finally, this ynamide-facilitated, ligand-induced mechanism leading to intramolecular kinase cross-linking and inhibition was unequivocally established by using recombinant ABL kinase as a representative.

Abstract PO3-08-01: Decision curve analysis to compare breast cancer risk predictions for a polygenic integrated clinical risk model with those of a gold standard

Abstract Risk-based guidelines have been developed to provide advice on options that are available to women who are at increased risk of breast cancer. We and others are focused on improving risk prediction models for general population use. For breast cancer, polygenic risk has been identified as important for risk prediction, especially when combined with clinical risk factors such as family history, anthropometric measures, breast imaging measures, and hormonal and reproductive risk factors. When comparing the performance of risk prediction models, the AUC is most often used but it focuses on performance in terms of distinguishing between affected and unaffected individuals and does not take into account differences in the specificity (true negative rate) and sensitivity (true positive rate) of the models being compared. In contrast, decision curve analysis takes sensitivity and specificity into account and enables the comparison of the net benefit of risk prediction tools at the clinical risk thresholds that are used to guide clinical management decisions. Herein, we use decision curve analysis to compare breast cancer risk prediction guidelines for BRISK and the Breast Cancer Risk Assessment Tool (BCRAT) in the UK Biobank, and for BRISK and IBIS version 7 in the Nurses’ Health Study. We evaluated the net benefit at the 5-year risk thresholds (1.67% and 3%) that are used to guide clinical management decisions around risk-reducing medication and at the remaining lifetime risk of 20% that is used to guide supplemental MRI surveillance. In the UK Biobank, BRISK showed a net benefit over BCRAT at the 5-year risk thresholds of 1.67% and 3.0%. The AUCs were 0.649 (95% CI = 0.640, 0.695) for BRISK and 0.567 (95% CI = 0.556, 0.577) for BCRAT and showed that, overall, BRISK discriminated between affected and unaffected women better than BCRAT (P < 0.001). In the Nurses’ Health Study, BRISK showed a net benefit over IBIS at the remaining lifetime risk threshold of 20%. The AUCs were 0.647; 95% CI = 0.627, 0.668 for BRISK and 0.571; 95% CI = 0.546, 0.595 for IBIS, a statistically significant improvement for BRISK over IBIS (P < 0.0001). We also looked at sensitivity, specificity, positive predictive value and negative predictive value even though these risk models are one-step removed from breast cancer diagnosis by screening/risk-reduction options. The positive predictive values are higher for the BRISK (27.1%) model compared to BCRAT (5.5%) in the UK Biobank dataset and higher for BRISK (15.4%) compared to IBISv7 (14.0%) in the Nurses’ dataset. We have shown the application of a statistical tool that can be used to directly compare risk models at clinically relevant thresholds. We have shown that BRISK, which comprises polygenic risk and clinical risk factors, shows higher net benefit at both 1.67% and 3% thresholds compared to BCRAT and at 20% remaining lifetime risk for IBIS version 7. In breast cancer risk prediction, a tool with high specificity (and therefore a necessary trade-off for low sensitivity) is preferred because we do not want to incorrectly classify women as not being at high risk and deny them the opportunity for intervention in the form of more frequent screening, alternative modes of screening or risk-reducing medication. This improvement in general population risk stratification that BRISK can provide has the potential to have a clinically significant effect on women’s health. Citation Format: Erika Spaeth, Bernard A Rosner, Gill Dite. Decision curve analysis to compare breast cancer risk predictions for a polygenic integrated clinical risk model with those of a gold standard [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 PO3-08-01.

Kinase Inhibition via Small Molecule‐Induced Intramolecular Protein Cross‐Linking

Remarkable progress has been made in the development of cysteine‐targeted covalent inhibitors. In kinase drug discovery, covalent inhibitors capable of targeting other nucleophilic residues (i.e. lysine, or K) has emerged in recent years. Besides a highly conserved catalytic lysine, almost all human protein kinases possess an equally conserved glutamate/aspartate (e.g. E/D) that forms a K‐E/D salt bridge within the enzyme active‐site. Electrophilic ynamides were previously used as effective peptide coupling reagents and to develop E/D‐targeting covalent protein inhibitors/probes. In the present study, we report the first ynamide‐based small‐molecule inhibitors capable of inducing intramolecular cross‐linking of various protein kinases, leading to subsequent irreversible inhibition of kinase activity. Our strategy took advantage of the close distance between the highly conserved catalytic K and E/D residues in a targeted kinase, thus providing a conceptually general approach to achieve irreversible kinase inhibition with high specificity and desirable cellular potency. Finally, this ynamide‐facilitated, ligand‐induced mechanism leading to intramolecular kinase cross‐linking and inhibition was unequivocally established by using recombinant ABL kinase as a representative.

CXCL13 as a biomarker in the diagnostics of European lyme Neuroborreliosis - A prospective multicentre study in Austria.

'Definite Neuroborreliosis (NB)' is diagnosed with the presence of NB-specific symptoms, cerebrospinal fluid (CSF) pleocytosis and an elevated Borrelia Burgdorferi antibody index. However, some diagnostic uncertainties exist. The B-cell chemokine CXCL13 represents an emerging biomarker for the diagnosis and treatment of NB because its intrathecal concentration rises prior to the Borrelia antibody index and drops rapidly after antibiotic therapy. Nevertheless, due to lacking prospective data, a definite CXCL13 cut-off for the diagnosis of NB is still pending. Definition of a CSF CXCL13 cut-off for the diagnosis of acute and untreated NB in a prospective study setting. This multicentre prospective study involved 6 neurological departments treating patients in the Lower Austria district (1.7 million inhabitants). The controls were patients scheduled for a spinal tap but not clinically diagnosed with NB. Demographic data, clinical characteristics and blood counts, as well as inflammatory CSF values and CSF CXCL13-concentration were analysed. We recruited 440 adult patients, of whom 42 have been diagnosed as having an acute and untreated 'definite NB'. Three hundred ninety-eight patients were assigned to the control group. The median intrathecal CXCL13 concentration was 2384 pg/ml for patients with NB and 0 pg/ml for controls. The difference was highly statistically significant (P ≤ .001). A CSF CXCL13 cut-off of 271 pg/ml resulted in a sensitivity of 95.2% and a specificity of 97.2% for the confirmation or exclusion of NB. Based on our results, we propose a CSF CXCL13 cut-off of 271 pg/ml with Euroimmun-Elisa for the diagnosis of acute and untreated NB. Due to its high sensitivity and specificity, CXCL13 is a strong candidate biomarker for routine NB assessment, especially in clinically unclear cases.

Abstract PO1-25-06: Integrated analysis of single-cell RNA and VDJ-sequencing data to identify T cell marker expressed on antigen-specific T cells

Abstract Background In recent years, the advancement of adoptive cell transfer-based immunotherapies has significantly contributed to the evolution of cancer treatment. The crucial step in achieving successful treatment lies in the identification of T cells that exhibit reactivity towards tumor-specific antigens. However, the heterogeneous composition of tumor antigen-specific T cells poses a challenge as there are no standardized biomarkers available for their identification. To overcome this limitation, we investigated alternative approaches, employing single cell RNA and VDJ sequencing to identify antigen-specific T cells. Methods First, peripheral blood mononuclear cells (PBMCs) obtained from healthy human donors were stained with carboxyfluorescein succinimidyl ester (CFSE) to facilitate the identification of proliferating antigen-specific T cells. Then the PBMCs were exposed to 1 ug/ml of CMV pp65 peptide. After a 2-day incubation period with the CMV peptide, interleukin-2 (IL-2) was administered to stimulate T cell expansion. At various time points (0, 1, 2, 3, 5, and 7 days) following the CMV peptide treatment, CD8+ T cells were isolated through fluorescence-activated cell sorting (FACS) analysis. On day 7, the CD8+ T cells were further segregated into CFSE- and CFSE+ cell populations. The single-cell transcriptomes and T cell receptor (TCR) repertoire of the CD8+ T cells were subsequently examined using the Chromium Single Cell Reagent Kit. Results The TCR repertoire analysis of CD8+ CFSE- T cells revealed the dominance of 10 major clones, accounting for 93.9% of the total cells. These clones were confirmed to be CMV-specific through various in vitro assays, including NFAT-luciferase, IFN-γ ELISA, and cytotoxicity assay. In contrast, CD8+ CFSE+ T cells did not exhibit significant overlap with the major TCR clones found in CFSE- CD8+ T cells, comprising only 0.02% of the total cells. Gene set enrichment analysis demonstrated that the upregulated genes in CFSE- CD8+ T cells, when compared to CFSE+ T cells, were related to cell cycle proliferation and T cell cytotoxicity. Furthermore, these genes exhibited a matching upregulation pattern with CFSE- T cells from another batch of the same donor, as well as from two other healthy donors. In addition, there was a moderate increase in the expression of the upregulated genes in CFSE- CD8+ T cells at day 3 compared to day 0-2. In this end, we selected a set of nine genes that were consistently upregulated in CFSE- CD8+ T to serve as markers for antigen-specific T cells, employing a logistic regression model. This gene set showed high sensitivity (97.7%) and specificity (95.6%) in identifying T cell clones that respond to CMV antigen at day 3. Conclusions We have discovered potential markers for antigen-specific CD8+ T cells. These results can offer the promising opportunity for earlier isolation of T cells that exhibit reactivity towards tumor-specific antigens. This breakthrough has significant implications, as it can potentially contribute to cost savings in adoptive cell transfer-based immunotherapies. Citation Format: Byung-Kwan Jeong, Young-Ae Kim, Jungwook Park, Baknoon Ham, Jiheyong Kim, Chae Lyul Lim, Hee Jin Lee. Integrated analysis of single-cell RNA and VDJ-sequencing data to identify T cell marker expressed on antigen-specific T cells [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 PO1-25-06.

Detection of left ventricular systolic dysfunction in patients with ischemic heart disease using spatial and frontal QRS-T angles of the electrocardiogram

Aim. To evaluate the possibilities of the spatial QRS-T angle (sQRS-Ta) and the frontal QRS-T angle (fQRS-Ta) to detect low left ventricular ejection fraction (LVEF) in patients with chronic coronary heart disease. Materials and methods. We analyzed the data of 287 patients with chronic coronary artery disease, in 80 cases complicated by chronic heart failure with low LVEF. fQRS-Ta was calculated as absolute value of the difference between the frontal plane QRS and T axes. sQRS-Ta was calculated using the synthesized vectorcardiogram as a spatial angle between the integral QRS and T vectors. Results. The fQRS-Ta values in the group were 70 [25; 141]°. sQRS-Ta values in the group were 96 [55; 148]°. There were correlations between fQRS-Ta and LVEF (r=-0.58; p0.0001) and sQRS-Ta with LVEF (r=-0.63; p0.0001). According to the ROC analysis, fQRS-Ta with a threshold value of 90° made it possible to identify patients with LVEF≤40% with a sensitivity of 85% and a specificity of 75% (area under the ROC curve 0.85±0.03) and patients with LVEF≤35% with 87% sensitivity and 69% specificity (area under the ROC curve 0.84±0.04). sQRS-Ta with a threshold value of 125° made it possible to identify patients with LVEF≤40% with a sensitivity of 86% and a specificity of 82% (area under the ROC curve 0.89±0.03) and patients with LVEF≤35% with sensitivity 93% and specificity 76% (area under the ROC curve 0.89±0.02). Conclusion. In patients with chronic coronary artery disease, as the LVEF decreases, the frontal and spatial angles between the QRS and T vectors increase. These indicators with sufficiently high sensitivity and specificity make it possible to identify patients with reduced LVEF among patients with chronic coronary artery disease.

Abstract PO4-26-09: The Advantage of Artificial-Intelligence in HER2 IHC 0 and 1+ Scoring in Breast Cancer

Abstract Background: Trastuzumab deruxtecan has substantially changed the treatment of HER2-low breast cancer, emphasizing the need for accurate differentiation between HER2 immunohistochemistry (IHC) scores of 0 and 1+. However, the current accuracy of HER2 IHC 0 and 1+ scoring in real-world is inadequate. Artificial intelligence (AI) has emerged as a potential solution to improve interpretation accuracy. We developed an AI algorithm based on whole slide images (WSI) to quantitatively assess HER2 expression and its role in interpreting HER2 IHC scores of 0 and 1+. Methods: Our three-phase AI analysis framework involved segmenting tumor areas (excluding ductal carcinoma in situ), detecting and classifying tumor cells based on membrane staining patterns, and grading HER2 IHC scores according to the 2018 ASCO/CAP HER2 guideline. The AI tool was trained using 6012 patches annotated by experienced pathologists. Performance evaluation was conducted using a test dataset comprising 265 slides. A total of 141 HER2 IHC slides (27 IHC 0 and 114 IHC 1+) from patients diagnosed with invasive breast cancer at Fudan University Shanghai Cancer Center in 2021 were included. Two pretrained expert pathologists independently rescored the HER2 slides, followed by analysis using the AI tool. All inconsistent cases were also reviewed by a third senior pathologist. Interobserver agreement between the pathologists and concordance between the pathologists and the AI interpretation results were assessed. We also explored potential ranges where HER2 interpretation by pathologists exhibited inconsistency or inaccuracy. Results: The HER2 AI algorithm showed high performance in interpreting all levels of HER2 expression, with an overall kappa value of 0.85. For HER2 IHC 0 and 1+ cases, the AI model demonstrated high sensitivities (0.839 and 0.914) and specificities (0.983 and 0.890) (Table 1). After the rescoring process, 51 cases were reclassified as IHC 0 and 90 cases as IHC 1+. The overall agreement rate between the historical and re-scoring results was 73.05% (103/141). The agreement rate between the AI and re-scoring results was 91.49% (129/141) (Table 2), indicating comparable interpretation capabilities between the AI model and well-trained pathologists. The interobserver agreement between the two pathologists was 83.69% (118/141), with agreement rates of 88.23% (45/51) for IHC 0 and 81.11% (73/90) for IHC 1+. Analyzing the inconsistent cases (n=12) between IHC 0 and IHC 1+, we examined the percentage of weak and incomplete expression provided by the AI. In 9 out of 12 cases, the AI provided the same results as the third senior pathologist, suggesting that AI could assist in interpretation within this range. We identified a range of 2.83% to 24.98% as a "grey-zone," where even well-trained pathologists provided inconsistent results. Conclusion: Our study demonstrates the excellent performance of an AI-based tool for scoring HER2 IHC 0 and 1+. The AI model exhibits comparable capabilities to well-trained pathologists in interpreting HER2 expression. Additionally, we identified a "grey-zone" among pathologists, highlighting the limitations of manual subjective interpretation. AI assistance within this zone may help mitigate subjective variations. Table 1. Correlation between pathologist read and AI read of HER2 immunohistochemistry. Table 2. Correlation between pathologist rescoring and AI read of HER2 immunohistochemistry. Citation Format: Ming Li, Hong Lv, Yizhi Zhao, Chenglu Zhu, Hansheng Li, Mingzhen Lin, Wen-Tao Yang. The Advantage of Artificial-Intelligence in HER2 IHC 0 and 1+ Scoring in Breast Cancer [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 PO4-26-09.

Abstract PO4-04-08: Design and Development of the APIS ESR1 Mutations Kit to Detect Eleven Mutations Relevant to Acquired Endocrine Therapy Resistance

Abstract Introduction: Estrogen receptor 1 (ER/ESR1) mutations have arisen as key biomarkers for endocrine therapy resistance in ER positive (ER+) breast cancer (BC) patients: detecting these mutations is key to guiding researchers to better understand acquired resistance during treatment. Ongoing clinical trials, which explore the prevalence of ESR1 mutations under various treatments, commonly use next-generation sequencing (NGS)-based assays. However, the NGS panels available are costly, can have long turnaround times and require specialist equipment and software. Research use only digital PCR assays are also available but have similar drawbacks to NGS. A targeted qPCR-based assay is more time and cost effective, and does not require specialist equipment. Here, we demonstrate the APIS ESR1 Mutations Kit, a targeted, sensitive, and robust qualitative qPCR assay able to detect eleven ESR1 mutations across ESR1 exons 5 (E380Q), 7 (S463P) and 8 (P535H, L536R, L536Q, L536H, L536P, Y537C, Y537S, Y537N and D538G). The assay is designed to assess circulating-free DNA (cfDNA) samples, reducing the need for invasive procedures in clinical settings and can make use of standard cfDNA extraction kits. Therefore, the APIS ESR1 Mutations Kit provides the flexibility for researchers in any conventional molecular biology laboratory to explore ESR1 mutations with high sensitivity and specificity. Methods: The APIS ESR1 Mutations Kit provides all components and controls required to assess samples. For kit development, mutation-specific DNA fragments were spiked into wildtype background DNA (either cfDNA extracted from pooled healthy donor plasma, human genomic DNA, or DNA fragments); the Limit of Blank (LoB) and Limit of Detection (LoD) were determined using these samples. To determine linearity, a dilution series of DNA fragments ranging from 5 to 10,000 copies per reaction was assessed. All PCR runs were performed using a QuantStudio™5 Dx (ThermoFisher) instrument. Results: The performance studies showed the APIS ESR1 Mutations Kit can detect D538G at 0.4 % mutant allele frequency (MAF) and Y537S at 0.06 % MAF. All other mutations can be detected at ≤ 1.0 % MAF. By using clamp and blocking technologies, each design is mutation specific, enabling identification of specific amino acid mutations. In addition, the assays can perform in high wild-type backgrounds, enabling lower limit of blanks with ≥95% confidence and threshold cut-offs to enhance the sensitivity of the assay. The linearity of each target is within 90-110% from 50 to 10,000 copies per reaction. Conclusions: The ESR1 Mutations Kit demonstrated high sensitivity and performance as a qualitative qPCR assay to detect eleven ESR1 mutations. Table. Mutations detected with the APIS ESR1 Mutations Kit Citation Format: Anna Gasior, Colette Whitfield, Anita Campbell, Christine Hoy, Sally Holdsworth, Jesse Mukose, Ryan Nana, Sahar Shammakhi, Benjamin Scales, Joanna Gorniak. Design and Development of the APIS ESR1 Mutations Kit to Detect Eleven Mutations Relevant to Acquired Endocrine Therapy Resistance [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 PO4-04-08.

Abstract PO5-27-11: A COMPARATIVE STUDY OF DIAGNOSTICS MADE BY FINE NEEDLE ASPIRATION (FNA) BIOPSY VERSUS TRU-CUT FOR BREAST PATHOLOGY IN A ONE STOP CLINIC PROGRAM IN MEDELLIN, COLOMBIA

Abstract Breast cancer is the leading cause of morbidity and mortality among women in Colombia due to non-communicable chronic diseases. This has prompted health insurance companies to develop innovative initiatives to mitigate these negative outcomes. Since September 2018, Ayudas Diagnósticas SURA, in alliance with the Gustave Roussy Hospital and General Electric Healthcare has promoted a “One-stop clinic” program called: “TIEMPO PARA TI”. One of the primary challenges was to demonstrate that breast cytology, based on the Yokohama guidelines approved by The International Academy of Cytology has a high sensitivity, specificity, and diagnostic accuracy. The Yokohama system for classifying breast cytology is comprised of five categories: C1 not satisfactory, C2 benign, C3 atypical, C4 malignancy suspected, and C5 malignant. From September 2018 to December 2022, consecutive patients referred for breast biopsy and suspected of having breast cancer underwent triple assessment of breast tumor and were included in this retrospective study. Descriptive analysis was made using STATA v17 software to compare fine needle aspiration (FNA) biopsy versus Tru-cut. A total of 14,501 patients were included, 14.398 FNA and 14.401 trucut biopsies were performed. The comparative analysis showed a 99% concordance between categories 2, which correspond to benign lesions and 96.7% between categories 5, which correspond to malignant lesions. Our findings demonstrate that a well-executed FNA biopsy is not inferior to a trucut biopsy, as revealed by the Yokohama system, and offers significant advantages, such as cost-effectiveness and prompt diagnosis. Both techniques should be used with caution, considering their advantages and disadvantages. Citation Format: Sara Gil, Carlos Agudelo, Jorge Castaño, Melissa Naranjo, Clara Piedrahita, Daniel Wilches, Kelly Corredor. A COMPARATIVE STUDY OF DIAGNOSTICS MADE BY FINE NEEDLE ASPIRATION (FNA) BIOPSY VERSUS TRU-CUT FOR BREAST PATHOLOGY IN A ONE STOP CLINIC PROGRAM IN MEDELLIN, COLOMBIA [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 PO5-27-11.

Abstract PO2-13-08: Cell-free orphan noncoding RNAs and AI enable early detection of invasive breast cancer and ductal carcinoma in-situ

Abstract Background Earlier detection of breast cancer through mammography screening has reduced disease-specific mortality; however, confounding issues such as technical challenges, breast density, and tumor size can result in false negatives and ultimately later stage diagnosis. Next generation liquid biopsy has the potential to complement mammography and enable earlier detection for more women. We have previously demonstrated high sensitivity and specificity for early detection of invasive breast cancer (IBC) by utilizing a novel category of cancer-associated small RNAs, termed orphan noncoding RNAs (oncRNAs), through a liquid biopsy platform. Here, we further improve the ability to detect breast cancer in a larger, multi-source cohort through an AI-driven approach and demonstrate potential for detection of ductal carcinoma in-situ (DCIS). Methods We utilized The Cancer Genome Atlas (TCGA) small RNA-seq database to discover a library of 20,538 oncRNAs, through a female-specific analysis, that were significantly enriched among 1,103 breast tumors compared to 349 normal tissue samples spanning multiple tissue sites. The diagnostic performance of these oncRNAs were assessed in an independent cohort of archived serum samples from 663 female individuals, sourced from Indivumed (Hamburg, Germany), Proteogenex (Inglewood, CA), and MT Group (Los Angeles, CA), including 279 breast cancer patients of various stages (221 IBC and 58 DCIS; mean age: 57.0 ± 13.8 years; ever-smoker: 25.8%) and 304 age-matched controls (mean age: 58.5 ± 13.9 years; ever-smoker: 23.4%) without breast cancer. All samples were collected between 2010–2022 at time of diagnosis for breast cancer patients. We sequenced the small RNA content of these samples at an average depth of 25.28 ± 9.37 million 50-bp single-end reads. We detected 18,025 (87.8%) unique breast cancer-specific oncRNA species within at least one sample from the study cohort. We then trained a generative AI model using 5-fold cross-validation to predict cancer status for all samples. Results Our oncRNA-based model achieved an overall AUC of 0.95 (95% CI, 0.93–0.97) for prediction of IBC versus cancer-free controls with a sensitivity of 0.87 (0.82–0.91) at 90% specificity. We observed high sensitivities, also at 90% specificity, across all tumor stages and tumor sizes (Table 1). Sensitivities for the earliest stage and smallest tumor size were 0.87 (0.78–0.93) and 0.81 (0.61–0.93) for Stage I (n=83) and T1a–b ( >1mm to ≤10mm; n=26), respectively. Additionally, in a small single-source cohort, we also saw high model accuracy and sensitivity for DCIS, which we aim to confirm in additional cohorts. While our overall cancer cohort primarily consisted of individuals with luminal breast cancer, our model had high sensitivities across all breast cancer subtypes at 0.90 (0.84–0.94), 0.73 (0.59–0.85), and 0.86 (0.42–1.0) for luminal (n=181), HER2 positive (n=49), and triple negative (n=7), respectively. Conclusions We further demonstrate the potential utility of oncRNAs as a blood-based biomarker using an AI algorithm for sensitive and accurate early detection of breast cancer in a large cohort. Additionally, we have shown that this oncRNA-based assay performs well in detecting small, early-stage invasive breast tumors, with potential to detect precursors of breast cancer. Table 1: Model sensitivity in breast cancer by tumor stage and size For each tumor stage and size, as defined by the AJCC 7th Edition breast cancer staging system, sensitivity and 95% Pearson-Clopper confidence intervals (CI) are reported at 90% specificity for the number of samples (N). Citation Format: Noura Tbeileh, Taylor Cavazos, Mehran Karimzadeh, Jeffrey Wang, Alice Huang, Dung Ngoc Lam, Seda Kilinc, Jieyang Wang, Xuan Zhao, Andy Pohl, Helen Li, Lisa Fish, Kimberly Chau, Marra Francis, Lee Schwartzberg, Patrick Arensdorf, Hani Goodarzi, Fereydoun Hormozdiari, Babak Alipanahi. Cell-free orphan noncoding RNAs and AI enable early detection of invasive breast cancer and ductal carcinoma in-situ [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 PO2-13-08.

Abstract PO1-20-05: SURVIVE study – a multicenter, randomized, controlled phase 3 superiority trial, evaluating liquid biopsy guided intensified follow-up surveillance in women with intermediate-to high-risk early breast cancer

Abstract Background: Current guidelines limit routine breast cancer follow-up to clinical examinations and breast imaging. This is based on the results of two large cohort studies conducted in the 1980s that showed no improvement in overall survival (OS) by an intensified screening for distant metastases. Thus, imaging for distant metastases should currently only be performed in patients with specific symptoms. To detect distant relapse in a pre-symptomatic stage, we suggest the evaluation of a liquid biopsy-guided intensified surveillance, analyzing tumor markers (CA 27.29, CA 125 and CEA), circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA). Trial design: Funded by the German Federal Ministry of Education and Research, the SURVIVE study is the first large, randomized breast cancer surveillance trial to investigate the potential survival benefit of a liquid biopsy guided follow-up care in intermediate- to high-risk early breast cancer patients. The trial is a German multicenter, controlled phase 3 superiority study, in which 3500 patients are randomized in a 1:1 ratio to standard vs. liquid biopsy guided intensified follow-up care. At the time of writing 150 patients have been enrolled since December 2022. The total study duration is 144 months. Stratification factors are study site as well as HR-, HER2- and nodal status at surgery. Both study arms receive standard follow-up according to national guidelines. Additional blood samples (year 1-3 every 3 months, year 4-5 every 6 months) and standardized Quality of Life (QoL) questionnaires (EORTC-QLQ-C30/PA-F12, every 6 months) will be collected. While the standard surveillance arm samples will only be stored in a biobank, samples collected in the intensive surveillance arm will be tested for standard tumor markers, CTCs and ctDNA. Pre-specified abnormal findings of any of the liquid biopsy markers indicative of minimal residual disease trigger complete staging. In the event of confirmed disease recurrence, guideline-based treatment follows, otherwise, liquid biopsy testing continues. If applicable, study participants may enter interventional trials. Eligibility criteria: Eligible patients are adult females or males with histologically confirmed primary invasive intermediate to high-risk early breast cancer, defined as an indication for (neo-)adjuvant chemotherapy, and/or large tumor size ( > 50 mm), and/or positive lymph nodes (≥ pN1mi), and/or high grade (≥ G3). Primary anti-tumor therapy (surgery, adjuvant chemo- or radiotherapy) must have been completed ≤ 24 months (≤ 60 months for luminal carcinoma) previously. Patient enrollment during adjuvant endocrine- or antibody-therapy, CDK4/6-/PARP- or PI3K-inhibitors and antibody-drug conjugates is allowed. Statistical methods: The two primary objectives are to determine whether intensified, liquid biopsy-guided surveillance leads to better OS or an overall lead-time effect, compared to standard surveillance. The primary endpoint OS will be analyzed in the ITT population using Kaplan Meier methods and cox regression models, the overall lead time effect is a purely descriptive composite measure. Secondary objectives include invasive disease-free survival (IDFS), distant disease-free survival (DDFS), distant recurrence free survival (DRFS), breast cancer-specific survival (BCSS) and quality of life (QoL). Specific aims: To improve OS and the QoL during breast cancer follow-up, we propose a liquid biopsy guided follow-up method, with high sensitivity and specificity for the earlier detection of distant (oligo-)metastases, to enable earlier initiation of therapy. Conclusion: The SURVIVE-study is a long-awaited trial in early breast cancer follow-up, based on promising liquid biopsy markers. If successful, the results will lead to a paradigm shift in current follow-up care of intermediate to high-risk early breast cancer survivors. Clinical trial information: NCT05658172 Citation Format: Franziska Mergel, Sophia Huesmann, Thomas Friedl, Kerstin Pfister, Tatjana Braun, Angelina Fink, Forca Mehmeti, Tanja Fehm, Volkmar Müller, Klaus Pantel, Andreas Hartkopf, Elisabeth Wiesmüller, Brigitte Rack, Wolfgang Janni. SURVIVE study – a multicenter, randomized, controlled phase 3 superiority trial, evaluating liquid biopsy guided intensified follow-up surveillance in women with intermediate-to high-risk early breast cancer [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 PO1-20-05.

The road to evolution of ProTx2: how to be a subtype-specific inhibition of human Nav1.7.

The human voltage-gated sodium channel Nav1.7 is a widely proven target for analgesic drug studies. ProTx2, a 30-residue polypeptide from Peruvian green tarantula venom, shows high specificity to activity against human Nav1.7, suggesting its potential to become a non-addictive analgesic. However, its high sensitivity to human Nav1.4 raises concerns about muscle side effects. Here, we engineered three mutants (R13A, R13D, and K27Y) of ProTx2 to evaluate their pharmacological activities toward Nav1.7 and Nav1.4. It is demonstrated that the mutant R13D maintained the analgesic effect in mice while dramatically reducing its muscle toxicity compared with ProTx2. The main reason is the formation of a strong electrostatic interaction between R13D and the negatively charged amino acid residues in DII/S3-S4 of Nav1.7, which is absent in Nav1.4. This study advances our understanding and insights on peptide toxins, paving the way for safer, effective non-addictive analgesic development.

Abstract PO4-06-03: A comparative study between RECIST 1.1 using MRI and PERCIST 1.0 using PET/CT to evaluate the response in patients of carcinoma breast receiving neoadjuvant chemotherapy

Abstract Introduction: Breast cancer is the most common cancer in women leading to the major public health problem with an estimated new cases of 2,261,419 (11.7%). Depending upon the stage at presentation, the primary modalities vary. Presently, the primary option for HER2-positive, Triple negative cancers of size more than 2cm or nodal involvement and locally advanced breast cancer is neoadjuvant chemotherapy (NACT). Accurate assessment of the tumor response and residual cancer after undergoing NACT is considered crucial for reducing the number of local recurrence cases and to predict the prognosis as patients who achieve pathological complete response (pCR) after NACT have a longer disease free period and better overall survival as compared to non-responders. In our study we have compared the two commonly used criteria i.e. RECIST 1.0 and PERCIST 1.0. Methodology: 70 patients diagnosed with breast cancer requiring neoadjuvant chemotherapy were recruited in the study conducted in the department of surgical disciplines, AIIMS Delhi from January 2021 to December 2022. Each patient underwent a contrast enhanced MRI breast and whole body FDG PET/CT at the beginning of chemotherapy and after completion of chemotherapy within a stipuated time of two to three weeks. For RECIST 1.1 criteria, response assessment was done using MRI and PERCIST used PET/CT. The response was compared with the gold standard histopathological size. Results: Concordance between the RECIST 1.1 and PERCIST 1.0 response classifications was seen in 27 (39.1%) cases, while discordance was seen in 42 (60.8%). A significant difference was observed between RECIST 1.1 and PERCIST 1.0 (k=0.1309, p< 0.0001) for response classification. Tumor response was upgraded in 32 and downgraded in 2 patients using PERCIST 1.0 as compared to RECIST 1.1. Sensitivity, specificity, positive predictive value, negative predictive value, and accuracy to predict pCR along with comparison among different molecular subtypes is shown in the table below. Conclusion: RECIST 1.1 showed high specificity, PPV and accuracy in predicting pathological complete response but tends to underestimate it whereas PERCIST 1.0 showed higher sensitivity with a tendency to overestimate. RECIST 1.1 had a high level of specificity and NPV as compared to the PERCIST 1.0, which showed a high level of sensitivity and PPV. Thus, they act as complementary modalities for predicting pathological complete response. The accuracy to predict pCR was higher for the triple-negative phenotype and Her2 neu enriched with PERCIST 1.0 as compared to RECIST 1.1, while that of PERCIST 1.0 was lower for luminal type. Table. Comparison of RECIST 1.1 and PERCIST 1.0. Citation Format: Anita Dhar, Kanika Sharma. A comparative study between RECIST 1.1 using MRI and PERCIST 1.0 using PET/CT to evaluate the response in patients of carcinoma breast receiving neoadjuvant chemotherapy [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 PO4-06-03.

Multimodal analysis of cfDNA methylomes for early detecting esophageal squamous cell carcinoma and precancerous lesions

Detecting early-stage esophageal squamous cell carcinoma (ESCC) and precancerous lesions is critical for improving survival. Here, we conduct whole-genome bisulfite sequencing (WGBS) on 460 cfDNA samples from patients with non-metastatic ESCC or precancerous lesions and matched healthy controls. We develop an expanded multimodal analysis (EMMA) framework to simultaneously identify cfDNA methylation, copy number variants (CNVs), and fragmentation markers in cfDNA WGBS data. cfDNA methylation markers are the earliest and most sensitive, detectable in 70% of ESCCs and 50% of precancerous lesions, and associated with molecular subtypes and tumor microenvironments. CNVs and fragmentation features show high specificity but are linked to late-stage disease. EMMA significantly improves detection rates, increasing AUCs from 0.90 to 0.99, and detects 87% of ESCCs and 62% of precancerous lesions with >95% specificity in validation cohorts. Our findings demonstrate the potential of multimodal analysis of cfDNA methylome for early detection and monitoring of molecular characteristics in ESCC.

Abstract PS06-06: Analysis of ctDNA for the detection of minimal residual disease (MRD) using a tissue-free, multiomic assay in patients with early-stage breast cancer

Abstract Background: The detection of ctDNA in patients with early-stage breast cancer after completion of adjuvant therapy is associated with a high risk of recurrence. Current clinical guidelines do not recommend routine screening for metastatic disease unless there are clinical signs and symptoms in patients who completed adjuvant therapy. However, ctDNA may serve as an early biomarker of recurrence which may allow effective identification of patients with asymptomatic distant metastases or molecular relapse only, who could benefit from early treatment intervention. Herein, we utilized a tissue-free, multiomic assay for the sensitive and specific detection of MRD in patients with early-stage breast cancer. Methods: Plasma samples were collected from patients with stage I-III breast cancer who were enrolled in the SUCCESS-A phase 3 clinical trial (NCT02181101) between 2006 and 2007. All patients received adjuvant chemotherapy +/- endocrine therapy and/or anti-HER2 therapy. Samples were collected approximately two years after completion of adjuvant chemotherapy, and plasma samples selected for analysis were from patients without evidence of disease recurrence prior to sample collection. Presence of MRD was assessed using Guardant Reveal powered by Infinity, which evaluates the epigenomic signals associated with cancer versus normal DNA for the detection of ctDNA. Samples with the presence of ctDNA are characterized for somatic alterations with common sources of interference such as clonal hematopoiesis excluded. An analytically validated bioinformatics pipeline was used for the detection of breast cancer ctDNA. Samples were analyzed blinded to the clinical data. Median survival times were estimated using the Kaplan-Meier method and hazard ratios were calculated based on univariable cox regression models. Results: A total of 311 plasma samples from 311 patients were evaluable. ctDNA was detected in 34% (13/38) of patients who subsequently developed distant recurrence and in none (0/7) of the patients who had local or contralateral breast cancer recurrence. ctDNA was detected in 60% (9/15) of samples that were collected within one year before recurrence. In the ctDNA detected samples from patients who had disease recurrence, the median time from sample collection to recurrence was 7.9 months (range, 1.4-28.6 months). ctDNA was detected in 6 patients who did not have a documented disease recurrence, resulting in a specificity of 97.7% (260/266). In the overall cohort, ctDNA detection was prognostic for recurrence-free survival (RFS; HR 11.0, 95% CI 2.28-53.6; p< 0.0001), distant recurrence-free survival (D-RFS; HR 13.7, 95% CI 2.52-74.9; p< 0.0001), and overall survival (OS; HR 17.4, 95% CI 1.33-227; p< 0.0001). Conclusion: Detection of ctDNA after adjuvant chemotherapy was highly prognostic and demonstrated high specificity in an early-stage breast cancer cohort. Larger, prospective studies are needed to confirm the prognostic value of ctDNA in the post-treatment setting and assess the clinical utility of MRD detection in this population. Citation Format: Wolfgang Janni, Thomas Friedl, Brigitte Rack, Peter A. Fasching, Andreas Hartkopf, Hans Tesch, Ralf Lorenz, Georg Heinrich, Jens-Uwe Blohmer, Tanja Fehm, Volkmar Müller, Andreas Schneeweiss, Matthias Beckmann, Matthias Ruebner, Nadia Harbeck, Klaus Pantel, Derek Dustin, Mingyang Cai. Analysis of ctDNA for the detection of minimal residual disease (MRD) using a tissue-free, multiomic assay in patients with early-stage breast cancer [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 PS06-06.

Membraneless organelles assembled by AuNPs-enzyme integration in non-photosynthetic bacteria: Achieving high specificity and selectivity for solar hydrogen production

The emergence of semiartificial photosynthesis holds great promise for improving light energy capture and conversion efficiency. However, achieving high product specificity and selectivity in semiartificial photosynthetic systems remains a challenge. In this study, we employed genetic engineering techniques to construct a membraneless organelle in E. coli, serving as the photoreaction carrier. The self-assembly of photosensitizers and hydrogenase within the carrier has successfully established a semiartificial photosynthetic model characterized by high product specificity and selectivity. In this model system, electrons were directly transferred to hydrogenase upon AuNPs light excitation in membraneless organelle, eliminating the need for additional electron mediators and bypassing the use of intracellular NADH/NAD+ as a reducing agent to avoid byproduct formation. The semiartificial photosynthetic system demonstrated an 11782-fold and 40-fold increase in hydrogen production compared to bare AuNPs and the empty vector E. coli hybrid system, respectively. This membraneless organelle holds the potential to robustly support the specific synthesis of high-energy-density, value-added compounds through solar energy conversion.

Development of a quantum dots based immunochromatographic strip for rapid and on-site detection of African swine fever virus

African swine fever (ASF) is a lethal disease caused by ASF virus (ASFV), severely impacting the global swine industry. Though nuclear acid-based detection methods are reliable, they are laboratory-dependent. In this study, we developed a device-independent, user friendly and cost-effective quantum dots based immunochromatographic strip (QDs-ICS) with high specificity and sensitivity for the rapid and on-site detection of ASFV antigen. For the preparation of the QDs-ICS, we generated a monoclonal antibody (mAb) mAb-8G8 and polyclonal antibody (pAb) against ASFV-p72 protein. The pAb was labelled with QDs to be used as the detection probe and the mAb-8G8 was coated on the nitrocellulose membrane as the test line. Our results proved that the strip displayed no cross-reactivity with other swine viruses and detection limit of the QDs-ICS was down to 1 ng/mL for the ASFV-p72 protein with great reproducibility. The strip also exhibited high stability with a storage period up to 12 months under room temperature. Twenty blind samples and one hundred clinical samples were examined by the QDs-ICS, conventional PCR and real-time PCR method, respectively. Results showed that the agreement rate between the QDs-ICS and PCR method was 100%, and the agreement rate between the strip and real-time PCR was 94%. The novel QDs-ICS developed here would be an effective tool for on-site detection of ASFV.