Tumor Biomarkers Research Articles

Prognostic significance of hemoglobin, albumin, lymphocyte and platelet score in solid tumors: a pooled study

ObjectiveThe high hemoglobin, albumin, lymphocyte, and platelet (HALP) score has been reported to be a good prognostic indicator for several malignancies. However, more evidence is needed before it can be introduced into clinical practice. Here, we systematically evaluated the predictive value of HALP for survival outcomes in patients with solid tumors.MethodsThis study was performed according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) and Assessing the Methodological Quality of Systematic Reviews (AMSTAR) Guidelines. In March 2024, an electronic literature search was performed for articles regarding the prognostic role of HALP in solid tumors. Data from studies with reported risk ratios (HRs) and 95% confidence intervals (CIs) were pooled in a meta-analysis. Study bias was assessed using the QUIPS tool.ResultsOf the 729 articles reviewed, 45 cohorts including data from 17,049 patients with cancer were included in the pooled analysis. The pooled results demonstrated that elevated HALP score was significantly associated with favorable overall survival (HR = 0.60, 95% CI 0.54-0.67, p < 0.01), cancer-specific survival (HR = 0.53, 95% CI 0.44- 0.64, p < 0.01), progression-free survival (HR = 0.62, 95% CI 0.54-0.72, p < 0.01), recurrence-free survival (HR = 0.48, 95% CI 0.30-0.77, p < 0.01), and disease-free survival (HR = 0.72, 95% CI 0.57-0.82, p < 0.01). Subgroup analyses based on various confounding factors further revealed the consistent prognostic impact of HALP on overall survival in patients with solid tumors.ConclusionsOur findings suggest that high HALP is associated with better survival outcomes in patients. The HALP score is a potential prognostic biomarker in solid tumors, but it needs to be further studied whether it can improve the established prognostic model.

Plasma mutation profile of precursor lesions and colorectal cancer using the Oncomine Colon cfDNA Assay

BackgroundColorectal cancer (CRC) is the second leading cause of cancer death worldwide. Early detection of precursor lesions or early-stage cancer could hamper cancer development or improve survival rates. Liquid biopsy, which detects tumor biomarkers, such as mutations, in blood, is a promising avenue for cancer screening.AimTo assess the presence of genetic variants in plasma cell-free tumor DNA from patients with precursor lesions and colorectal cancer using the commercial Oncomine Colon cfDNA Assay.Material and methodsCell-free DNA (cfDNA) samples from the plasma of 52 Brazilian patients were analyzed. Eight patients did not have any significant lesions (five normal colonoscopies and three hyperplastic polyps), 24 exhibited precursor lesions (13 nonadvanced adenomas, 10 advanced adenomas, and one sessile serrated lesion), and 20 patients with cancer (CRC). The mutation profile of 14 CRC-associated genes were determined by next-generation sequencing (NGS) using the Oncomine Colon cfDNA Assay in the Ion Torrent PGM/S5 sequencer.ResultsThirty-three variants were detected in eight genes (TP53, PIK3CA, FBXW7, APC, BRAF, GNAS, KRAS, and SMAD4). No variants were detected in the AKT1, CTNNB1, EGFR, ERBB2, MAP2K1 and NRAS genes. All variants were considered pathogenic and classified as missense or truncating. The TP53 gene harbored the most variants (48.48%), followed by the KRAS gene (15.15%) and the APC gene (9.09%). It was possible to detect the presence of at least one pathogenic variant in cfDNA in 60% of CRC patients (12/20) and 25% of precursor lesions (6/24), which included variants in three patients with nonadvanced adenoma (3/13 – 23.08%) and three with advanced adenomas (3/10 – 30%). No variants were detected in the eight patients with normal findings during colonoscopy. The detection of mutations showed a sensitivity of 60% and a specificity of 100% for detecting CRC and a sensitivity of 50% and a specificity of 100% for detecting advanced lesions.ConclusionThe detection of plasma NGS-identified mutations could assist in early screening and diagnostic of CRC in a noninvasive manner.

Cell-Free Carbonic Anhydrase IX mRNA in Urine as Biomarker for Urogenital Cancers: The Relationship Between Urinary Extracellular RNA and Tumor-Cell-Associated RNA

Circulating tumor cells and cell-free nucleic acids are novel diagnostic, prognostic and predictive tools for non-invasive and cost-effective cancer detection in liquid biopsy. Carbonic anhydrase IX (CAIX) has been proposed as a biomarker in urogenital tumors and urine sediment. Our aim was to evaluate CAIX full-length percentage (CAIX FL%) in urine-cell-free RNA (cfRNA) and its relationship with tumor-cell-associated RNA (TC-RNA). CAIX FL% was quantified by reverse transcription quantitative polymerase chain reaction (RT-qPCR) in patients with prostate, kidney or bladder carcinoma. When cfRNA and TC-RNA were analyzed, CAIX FL% was significantly higher in urine samples from cancer patients than from controls. Using a 10% cutoff for CAIX FL%, specificity, sensitivity, positive and negative predictive values, as well as accuracy for TC-RNA were higher than for cfRNA in all urogenital cancers, but varied according to tumor type. CAIX FL% distribution in TC-RNA differed significantly (p < 0.001) between control and tumor samples (37.5% and 96.2%, respectively); similar results were obtained for each tumor type. Additionally, the 10% cutoff showed a 77.9% concordance between TC-RNA and cfRNA. In conclusion, urine is proposed as an alternative biofluid for investigating CAIX FL% in urogenital cancers, and this parameter can be reliably measured as cfRNA and TC-RNA with different predictive capabilities depending on tumor type.

Mesothelin expression prediction in pancreatic cancer based on multimodal stochastic configuration networks.

Predicting tumor biomarkers with high precision is essential for improving the diagnostic accuracy and developing more effective treatment strategies. This paper proposes a machine learning model that utilizes CT images and biopsy whole slide images (WSI) to classify mesothelin expression levels in pancreatic cancer. By combining multimodal learning and stochastic configuration networks, a radiopathomics mesothelin-prediction system named RPMSNet is developed. The system extracts radiomic and pathomic features from CT images and WSI, respectively, and sends them into stochastic configuration networks for the final prediction. Compared to traditional radiomics or pathomics, this system has the capability to capture more comprehensive image features, providing a multidimensional insight into tissue characteristics. The experiments and analyses demonstrate the accuracy and effectiveness of the system, with an area under the curve of 81.03%, an accuracy of 73.67%, a sensitivity of 71.54%, a precision of 76.78%, and a F1-score of 72.61%, surpassing both single-modality and dual-modality models. RPMSNet highlights its potential for early diagnosis and personalized treatment in precision medicine.

Complete remission after pembrolizumab monotherapy in a non-small cell lung cancer patient with PD-L1 negative, high tumor mutational burden, and positive tumor-infiltrating lymphocytes: A case report.

Immune checkpoint inhibitors have been used to treat cancer patients. Non-small cell lung cancer (NSCLC) patients with a high expression level of programmed cell death ligand-1 (PD-L1) could benefit from immune checkpoint inhibitor monotherapy. However, treating NSCLC patients with PD-L1 negative is still a clinical challenge. The utilization of new-type tumor markers as predictive indicators of therapeutic efficacy, with the aim of guiding clinical medication strategies, has emerged as a paramount focus of clinical investigation and interest. We reported a 72-year-old male with cough diagnosed as poorly differentiated metastatic lung adenocarcinoma (cT3N2M1, stage IV). He tested negative for driver gene mutations, and PD-L1 negative (＜1%), but a high tumor mutational burden (30.9 and 39.1 mutations/Mb in the lung tissue and blood, respectively), and positive tumor-infiltrating lymphocytes. The patient received pembrolizumab monotherapy. After 8 treatment cycles over 5 months, repeat examinations showed significantly reduced lung mass and circulating tumor DNA abundance. The patient reached clinical complete remission and had long-term survival with no significant adverse events. A comprehensive evaluation of multiple tumor biomarkers should be considered in NSCLC patients. Pembrolizumab monotherapy could benefit NSCLC patients with negative driver genes, PD-L1 negative, a high tumor mutational burden, and positive tumor-infiltrating lymphocytes.

Circulating Extracellular Vesicles as Promising Biomarkers for Precession Diagnostics: A Perspective on Lung Cancer.

Extracellular vesicles (EVs) have emerged as promising biomarkers in liquid biopsy, owing to their ubiquitous presence in bodily fluids and their ability to carry disease-related cargo. Recognizing their significance in disease diagnosis and treatment, substantial efforts have been dedicated to developing efficient methods for EV isolation, detection, and analysis. EVs, heterogeneous membrane-encapsulated vesicles secreted by all cells, contain bioactive substances capable of modulating recipient cell biology upon internalization, including proteins, lipids, DNA, and various RNAs. Their prevalence across bodily fluids has positioned them as pivotal mediators in physiological and pathological processes, notably in cancer, where they hold potential as straightforward tumor biomarkers. This review offers a comprehensive examination of advanced nanotechnology-based techniques for detecting lung cancer through EV analysis. It begins by providing a brief overview of exosomes and their role in lung cancer progression. Furthermore, this review explores the evolving landscape of EV isolation and cargo analysis, highlighting the importance of characterizing specific biomolecular signatures within EVs for improved diagnostic accuracy in lung cancer patients. Innovative strategies for enhancing the sensitivity and specificity of EV isolation and detection, including the integration of microfluidic platforms and multiplexed biosensing technologies are summarized. The discussion then extends to key challenges associated with EV-based liquid biopsies, such as the standardization of isolation and detection protocols and the establishment of robust analytical platforms for clinical translation. This review highlights the transformative impact of EV-based liquid biopsy in lung cancer diagnosis, heralding a new era of personalized medicine and improved patient care.

Advances and prospects of precision nanomedicine in personalized tumor theranostics

Tumor, as the second leading cause of death globally, following closely behind cardiovascular diseases, remains a significant health challenge worldwide. Despite the existence of various cancer treatment methods, their efficacy is still suboptimal, necessitating the development of safer and more efficient treatment strategies. Additionally, the advancement of personalized therapy offers further possibilities in cancer treatment. Nanomedicine, as a promising interdisciplinary field, has shown tremendous potential and prospects in the diagnosis and treatment of cancer. As an emerging approach in oncology, the application of nanomedicine in personalized cancer therapy primarily focuses on targeted drug delivery systems such as passive targeting drug delivery, active targeting drug delivery, and environmentally responsive targeting drug delivery, as well as imaging diagnostics such as tumor biomarker detection, tumor cell detection, and in vivo imaging. However, it still faces challenges regarding safety, biocompatibility, and other issues. This review aims to explore the advances in the use of nanomaterials in the field of personalized cancer diagnosis and treatment and to investigate the prospects and challenges of developing personalized therapies in cancer care, providing direction for the clinical translation and application.

Genome-wide DNA methylation analysis reveals a unique methylation pattern for pleural mesothelioma compared to healthy pleura and other lung diseases

BackgroundPleural mesothelioma (PM) is a rare and aggressive cancer type, typically diagnosed at advanced stages. Distinguishing PM from other lung diseases is often challenging. There is an urgent need for biomarkers that can enable early detection. Interest in the field of epigenetics has increased, particularly in the context of tumour development and biomarker discovery. This study aims to identify specific changes in DNA methylation from healthy pleural tissue to PM and to compare these methylation patterns with those found in other lung diseases.ResultsEPIC methylation array data (850 K) were generated for 11 PM and 29 healthy pleura in-house collected samples. This is the first time such a large dataset of healthy pleura samples has been generated. Additional EPIC methylation array data (850 K) for pleural mesothelioma and other lung-related diseases were downloaded from public databases. We conducted pairwise differential methylation analyses across all tissue types, which facilitated the identification of significantly differentially methylated CpG sites. Extensive differential methylation between PM and healthy pleura was observed, identifying 81,968 differentially methylated CpG sites across all genomic regions. Among these, five CpG sites located within four genes (MIR21, RNF39, SPEN and C1orf101) exhibited the most significant and pronounced methylation differences between PM and healthy pleura. Moreover, our analysis delineated distinct methylation patterns specific to PM subtypes. Finally, the methylation profiles of PM were distinctly different from those of other lung cancers, enabling accurate differentiation.ConclusionsDNA methylation analyses provide a robust method for distinguishing PM from healthy pleural tissues, and specific methylation patterns exist within PM subtypes. These methylation differences underscore their importance in understanding disease progression and may serve as viable biomarkers or therapeutic targets. Moreover, differential methylation patterns between PM and other lung cancers highlights its diagnostic potential. These findings necessitate further translational studies to explore their clinical applications.

CT-based habitat radiomics for predicting treatment response to neoadjuvant chemoimmunotherapy in esophageal cancer patients.

We used habitat radiomics as an innovative tumor biomarker to predict the outcome of neoadjuvant therapy for esophageal cancer. This was a two-center retrospective clinical study in which pretreatment CT scans of 112 patients with esophageal cancer treated with neoadjuvant chemoimmunotherapy and surgery between November 2020 and July 2023 were retrospectively collected from two institutions. For training (n = 85) and external testing (n = 27), patients from both institutions were allocated. We employed unsupervised methods to delineate distinct heterogeneous regions within the tumor area. To represent the prediction effect of different models, we plotted the AUC curves. The AUCs of the habitat models were 0.909 (0.8418-0.9758, 95% CI) and 0.829 (0.6423-1.0000, 95% CI) in the training and external test cohorts, respectively. The AUCs of the nomogram models were 0.914 (0.8483-0.9801, 95% CI) and 0.849 (0.6752-1.0000, 95% CI) in the training and external test cohorts, respectively. The results revealed that the model based on habitat data outperforms traditional radiomic analysis models. In addition, when the model is combined with clinical features, it improves the predictive accuracy of pathological complete response in patients undergoing neoadjuvant chemoimmunotherapy.

Personalised PDAC chip with functional endothelial barrier for tumour biomarker detection: a platform for precision medicine applications

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer characterised by poor survival rates and an increasing global incidence. Advances in the staging and categorization of pancreatic tumours, along with the discovery of functional mutations, have made precision treatments possible, which may lead to better clinical results. To further improve customized treatment approaches, in vitro models that can be used for functional drug sensitivity testing and precisely mimic the disease at the organ level are required. In this study, we present a workflow for creating a personalised PDAC chip utilising primary tumour-derived human pancreatic organoids (hPOs) and Human Umbilical Vein Endothelial Cells (HUVECs) to simulate the vascular barrier and tumour interactions within a PDMS-free organ-on-a-chip system. The patient PDAC tissue, expanded as tumour hPOs, could be cultured as adherent cells on the chip for more than 50 days, allowing continuous monitoring of cell viability through outflows from tumour and endothelial channels. Our findings demonstrate a gradual increase in cell density and cell turnover in the pancreatic tumor channel. Tumour-specific biomarkers, including CA-19.9, TIMP-1, Osteopontin, MIC-1, ICAM-1 and sAXL were consistently detected in the PDAC chip outflows. Comparative analyses between tissue culture plates and microfluidic conditions revealed significant differences in biomarker secretion patterns, highlighting the advantages of the microfluidics approach. This PDAC chip provides a stable, reproducible tumour model system with a functional endothelial cell barrier, suitable for drug sensitivity and secretory biomarker studies, thus serving as a platform for functional precision medicine application and multi-organ chip development.

The CRISPR/Cas system-mediated function of Hg2+ on urease activity for colorimetric detection of the tumor biomarker in clinical samples

Detecting tumor biomarkers in blood samples is essential in cancer diagnosis and prognosis. This study proposes a CRISPR/Cas system-mediated colorimetric assay to detect the tumor biomarker in human serum samples. It involves using the tumor biomarker as a switch of the CRISPR/Cas system to modulate the function of Hg2+, which is a typical urease inhibitor. The detection of carcinoembryonic antigen (CEA) is demonstrated as an example. The CEA aptamer is used to activate the CRISPR/Cas system, allowing for the cleavage of a single-stranded DNA (ssDNA) rich in thymine (T) nucleotides, thereby causing the inhibition of the urease activity by Hg2+. However, in the presence of CEA, the CRISPR/Cas system cannot be activated due to the specific binding of CEA to its aptamer, which leads to the capture of Hg2+ owing to the formation of a DNA hairpin structure with T-Hg2+-T coordination bonds. Thus, the urease activity is remained. Since the enzymatic hydrolysis of urea by urease increases the pH of the urea solution containing phenol red and changes the solution from yellow to pink, CEA can be detected with the assistance of the colorimetric assay. This method shows high sensitivity and selectivity for CEA with a detection limit of 0.028 ng/mL. In addition, serum samples from patients with normal and abnormal levels are tested to verify the validity of the proposed method for practical applications. Overall, this study provides a simple, fast, and stable colorimetric assay to detect the tumor biomarker in human serum and has great potential for early cancer screening and prognostic determination.

Bladder sparing management for muscle-invasive bladder cancer after a complete clinical response: ready for prime time?-a narrative review.

A standard of care for muscle-invasive bladder cancer (MIBC) is cisplatin-based neoadjuvant chemotherapy (NAC) followed by radical cystectomy (RC). Given recent improvements in NAC and the morbidity associated with RC, bladder-sparing therapy has been investigated as a promising treatment for patients with MIBC who experience a complete clinical response (CCR) to systemic therapy. However, clinical staging is unreliable, making it challenging to determine ideal candidates for bladder-sparing therapy. Our primary objective is to review the efficacy of NAC, strategies for determining a CCR as a surrogate for a complete pathologic response, and the emerging role of imaging, tumor genomics, and biomarkers in selecting candidates for bladder-sparing therapy. We surveyed the literature for studies investigating the outcomes of current treatment modalities for MIBC and methods for determining a CCR following systemic therapy as well as the impact this has on pathologic staging. Studies employing imaging, tumor biomarkers, and genomics were included. Clinical staging with cystoscopy or transurethral resection shows significant discordance with final pathology, with high rates of understaging. Multiparametric magnetic resonance imaging (mpMRI) has shown strong utility in determining the presence of MIBC, but it has yet to reliably identify CCR. Meanwhile, somatic DNA damage repair mutations and biomarkers such as circulating and urinary tumor DNA are strong predictors of recurrence, showing promise in predicting and monitoring a CCR to systemic therapy. Multiple ongoing trials are currently assessing the use of biomarkers and genomic analyses in determining eligibility for bladder-sparing therapy. While no one method has reliably demonstrated the ability to detect a true CCR, a multimodal approach involving imaging, biomarkers, and genomic analyses holds promise. We eagerly await the results of clinical trials investigating these tools, which may allow for the safe recommendation of bladder-sparing therapy.

Intratumoral Transforming Boron Nanosensitizers for Amplified Boron Neutron Capture Therapy.

Boron neutron capture therapy (BNCT) is an advanced binary tumor-cell-selected heavy-particle radiotherapy used for treating invasive malignant tumors. However, its clinical applications have been impeded by the rapid metabolism and insufficient tumor-specific accumulation of boron agents. To tackle this issue, we develop a smart boron nanosensitizer (BATBN) capable of transforming its size in response to cancer biomarker for optimal balance between penetration and retention of boron-10 for BNCT. BATBN comprises an ultrasmall boron quantum dots (BQD) core (4 nm) conjugated with cell-penetrating peptides, which facilitates its cellular uptake and deep tumor penetration. In the tumor microenvironment, the tumor biomarker can specifically initiate a self-condensation reaction of BATBN, leading to the formation of larger-sized nanoaggregates. Due to such a specific intratumoral transformation, BATBN demonstrates a 2.4-fold increase in intratumoral boron concentration and a 5.0-fold increase in tumor retention time compared to the BQDs. Thus, the tumor volume of the BATBNs-treatment group is 2.7-fold smaller than that of BQDs in preclinical tumor models after 21 days of neutron irradiation treatment. This study presents a supramolecular strategy to endow BNCT agents with the biomarker-activated size interconversion, permitting precise and efficient BNCT for cancer treatment.

Dual-site responsive module-triggered CRISPR/Cas12a switch with enhanced reaction kinetics for specific detection of PSA

Despite advances in DNA-based nanodevices for tumor biomarker detection, the pursuit of enhanced sensitivity and specificity remains a profound challenge. In this study, we designed an integrated dual-site responsive module as the AND logic gate that responded to both prostate-specific antigen (PSA) and apurinic/apyrimidinic endonuclease 1 (APE1), triggering a tetrahedral DNA nanostructure (TDN)-accelerated CRISPR/Cas12a signal switch for PSA detection. In detail, the dual-site responsive module was a T-shaped probe comprised of three DNA strands, which contained PSA aptamer and apurinic/apyrimidinic (AP) sites, significantly improving the specificity of prostate cancer screening by orthogonal recognition of PSA and APE1. When both PSA and APE1 were present, PSA could specifically bind with the aptamer, while APE1 catalyzed and cleaved the AP sites, releasing a part of one auxiliary strand as the mimic target (MT). To enhance the trans-cleavage efficiency of the CRISPR/Cas12a signal switch, we parallelly constructed the TDN with extended non-target strands (NTS) at each vertex, which could capture the MT to form double-stranded DNA (dsDNA) activators at the TDN vertices (TDN-activators) for triggering the CRISPR/Cas12a signal switch. Benefiting from the rigid framework of TDN, the TDN-activators not only increased the local concentration of Cas12a but also enabled a dominant conformation that was more favorable for recognizing and activating the Cas12a-crRNA complex, which demonstrated a 4.06-fold increase in trans-cleavage efficiency compared to that of dsDNA-activated Cas12a. This work applies the T-shaped probes and TDN-accelerated CRISPR/Cas12a signal switch to achieve the specific and sensitive electrochemiluminescence (ECL) detection of PSA from 1.0 × 10−5 to 10 μg/mL with a low limit of detection (LOD) of 4.5 × 10−6 μg/mL, offering a promising tool for early clinical screening of prostate cancer.

The application of hepatopancreatoduodenectomy in advanced gallbladder carcinoma: Patients’ selection, surgical outcome and influence on survival compared to radical cholecystectomy

Aimto investigate hepatopancreatoduodenectomy (HPD) application in gallbladder carcinoma (GBC) and compare it to radical cholecystectomy (RC) regarding patients’ characteristics, surgical outcome and survival. MethodsPatients treated in our center were included. Three groups (HPD, RC, no-surgery) were compared. Comparisons included basic characteristics, liver function, tumor biomarkers, surgical complications, outcome, and survival. Differences in severity, surgical risks and survival were investigated. ResultsIn total, 19 patients in HPD, 89 in RC and 20 in no-surgery groups were included. Median follow-up was 42.0 months. Compared to RC, HPD group revealed increased severity represented by significantly higher rates of elevated ALT, AST, TB, DB, preoperative biliary decompression, T3/4, N1/2, stage III/IV, and MVI. Similar severity was observed between HPD and no-surgery groups. Additionally, HPD presented higher risks of perioperative bleeding, transfusion and complications (grade III/IV). Pancreatic fistula and delayed gastric emptying were most encountered in HPD and only one mortality was recorded. Although HPD had shorter survival in general population (HPD: 17 ± 5.804, RC: 49 ± 11.433,P < 0.001); however, in matched (PSM) advanced stages, its benefits matched RC (HPD: 17 ± 4.793, RC: 11 ± 1.169, P = 0.966). Significant survival advantages were observed when comparing HPD to no-surgery group (HPD: 17 ± 5.804, no-surgery: 7 ± 0.836, P = 0.013). ConclusionCases benefiting from HPD are usually severe and comparable to unresectable cases. Although HPD complexity and complication risks are higher than RC, the survival benefits of HPD in advanced GBC stages are not inferior. Thus, HPD is a valuable option in such patients and can be the only approach to achieve R0 resection.

Nanochannel confined graphene quantum dots/platinum nanoparticles boosts electrochemiluminescence of luminal-O2 system for sensitive immunoassay

Sensitive detection of tumor biomarkers is of great significance for early cancer diagnosis, treatment evaluation, and recurrence monitoring. Development of convenient electrochemiluminescence (ECL) immunosensor using dissolved oxygen (O2) as an endogenous co-reactant of luminol combined with efficient nanocatalysts to boost ECL signal in neutral media is highly desirable. Herein, sensitive detection of tumor biomarker using ECL of luminal-O2 enhanced by confinement of nitrogen-doped graphene quantum dots (N-GQDs) and platinum nanoparticles (PtNPs) on nanochannel array was demonstrated. A high-density nanochannel array-modified electrode was achieved by rapidly growing an amino-functionalizedvertically-ordered mesoporous silica film (NH2-VMSF) on the inexpensive and readily available indium tin oxide (ITO) electrode. Through simple electrodeposition, N-GQDs were confined and PtNPs were in-situ synthesized in nanochannels of NH2-VMSF. These confined nanocomposites catalyzed the electroreduction of O2 at negative potentials to generate a large amount of reactive oxygen species (ROS) and facilitated luminol oxidation, enhancing the ECL signal of luminol by 25 times. Two immunosensors were fabricated after covalent immobilization of the recognition antibody of carbohydrate antigen 199 (CA199) or carbohydrate antigen 125 (CA125) on the outer surface of the NH2-VMSF and blocking of non-specific sites. When tumor biomarkers bind to the corresponding antibodies on the recognition interface, the formed immunocomplex hindered the diffusion of luminol to the underlying electrode, resulting in a decrease in the ECL signal and sensitive detection of tumor biomarker. The constructed CA199 immunosensor exhibited a linear detection range for CA199 from 0.5 mU mL−1 to 50 U mL−1, with a detection limit (DL) of 0.03 mU mL−1. For CA125 detection, linear detection ranged from 0.5 mU mL−1 to 500 U mL−1, with a DL of 0.005 mU mL−1. The fabricated immunosensors demonstrated good selectivity and high reproducibility. This study provides great potential for the development of efficient luminol ECL systems in neutral media and expands the biological application in sensitive detection of tumor biomarker.

Abstract B017: The double-edged sword of cystatin B in luminal-like and triple-negative breast cancers

Abstract The complex protease network of cathepsins and their inhibitors, cystatins, intricately regulates numerous cellular processes, and its dysregulation is frequently implicated in cancer pathogenesis. Abnormal cathepsin activity, subcellular localization, and a disrupted balance between cathepsins and cystatins have been identified in cancer. While diminished cystatin levels typically correlate with more aggressive cancer phenotypes, few findings challenge this paradigm. In this context, our latest study revealed elevated expression of cystatin B (CSTB) in luminal-like breast cancer (BC) patients experiencing relapse within five years post-surgery. Furthermore, computational analysis of publicly available datasets supports our idea of CSTB as a potential negative prognostic biomarker in luminal-like tumors. Intriguingly, a converse relationship is observed in basal-like/triple negative BC patients, where increased CSTB expression seems to be associated with improved prognosis. In addition, the cysteine/cathepsin network intricately intersects with immune functions, adding another layer of complexity. Cathepsins are indeed involved in the induction of an efficient anti-tumor immune response, being key in antigen processing and presentation by antigen-presenting cells and in the cytotoxic activities of NK and T cells. On the other hand, myeloid cells rely on cathepsins for the degradation of ECM proteins that may favor migration and invasion of tumor cells. All these pieces of evidence led to the hypothesis that the functional impact of CSTB may vary depending on the tumor intrinsic characteristics and on its microenvironment and therefore that CSTB might fulfill distinct functional roles in luminal-like and basal-like BC. To start addressing our hypothesis, we silenced CSTB expression in the 4T1 mouse cell line, a TNBC model spontaneously metastasizing to the lungs upon intra-mammary fat pad injection. Preliminary in vivo experiments to investigate the effect on primary tumor growth and metastatic dissemination showed that 4T1 shCSTB lines were indeed characterized by increased tumor growth and metastatic dissemination than the two sh-control lines. Interestingly, expression of stemness- related genes (Nanog, Sox2 and Oct4) were up-modulated upon CTSB silencing, in agreement with a more aggressive phenotype. On the other hand, preliminary in vitro assays with a luminal- like BC cell line support the hypothesis of an opposite effect of CSTB down-modulation in luminal BC subtype. Moreover, CSTB silencing in RAW264.7 macrophagic cell line, impaired M1 in vitro polarization, while it did not appear to affect M2 skewing. Overall, although preliminary, these data suggest that CSTB may affect BC aggressiveness by both interfering with tumor cell-intrinsic features and microenviroment-related traits. Citation Format: Anna Zaccaria, Chiara Ratti, Laura Botti, Sabina Sangaletti, Mario P. Colombo, Claudia Chiodoni. The double-edged sword of cystatin B in luminal-like and triple-negative breast cancers [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B017.

Abstract PR013: Highly accurate detection of early-stage colorectal cancer using tumor and immune extracellular vesicles biomarkers

Abstract Introduction: Colorectal cancer (CRC) is one of the most common cancers worldwide, and early detection is critical for successful treatment and improved survival rates. However, precancerous and early-stage CRC present significant diagnostic challenges due to the small size of lesions and the very low expression of tumor-specific biomarkers in the bloodstream. Current genomics-based diagnostic methods struggle to detect these lesions, making it imperative to develop more sensitive and specific approaches. Circulating extracellular vesicles (EVs) are emerging as a promising solution for early-stage CRC detection, since EVs are produced by tumor cells as well as the tumor microenviroment and host immune cells. Thus, EVs may offer the means to detect and monitor small early-stage tumors through both direct detection of tumor- associated biomarkers as well through tumor specific host response and tumor microenvironment biomarkers. Methods: To identify novel early-stage CRC specific biomarkers, we performed proteomics analysis on EVs purified from patient plasma using size exclusion chromatography and a proprietary buffer system that enhances EV and corona protein recovery. TrueDiscovery™ Data-independent acquisition (DIA) mass spectrometry (MS) analysis was conducted on 24 pre- cancer/stage 0 and 25 stage 1 CRC patients and 75 normal patient plasma samples. An in-house developed machine learning pipeline was used to identify differentially expressed proteins and model candidate multiplexes to identify those with extremely high diagnostic accuracy (&amp;gt; 0.99). Results: An average of ∼2,500 proteins were identified per sample and included in bioinformatics analysis. Comparative analysis between control patient EVs and either pre- cancerous/Stage 0, or Stage 1 colon cancer EVs using an in-house Machine Learning pipeline identified 336 and 493 differentially expressed proteins for each group (FDR adjusted p-value &amp;lt; 0.001). Of these, the best 17 pre/stage 0 proteins and 53 stage 1 proteins were trained and tested using Support Vector Machine to assess all potential 3-plexes in order to identify those with mean diagnostic accuracy &amp;gt; 99%. This yielded 5 3-plexes in precancer/stage 0 and 34 3-plexes in stage 1 with near perfect diagnostic accuracy. These plexes are currently being assessed in single analyte and multiplex immunoassays with the goal of translating candidate 3-plexes to the clinical. Conclusions: Key biomarkers from CRC patient EVs indicate the potential to detect and diagnose early-stage and low tumor burden cancers using our methods. Interestingly, the most accurate 3-plexes consist of proteins from immune, inflammatory and metabolic processes, suggesting that for the detection of early stage cancer it may be critical to include both tumor and host specific biomarkers. Citation Format: Poorva Mudgal, Cheryl Bandoski, Zachary Opheim, Martin Mehnert, Valesca Anschau, Issa Isaac, Alan Ezrin, Todd Hembrough. Highly accurate detection of early-stage colorectal cancer using tumor and immune extracellular vesicles biomarkers [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr PR013.

Abstract A063: Highly accurate detection of early-stage colorectal cancer using tumor and immune extracellular vesicles biomarkers

Abstract This abstract is being presented as a short talk in the scientific program. A full abstract is printed in the Proffered Abstracts section (PR013) of the Conference Program/Proceedings. Citation Format: Poorva Mudgal, Cheryl Bandoski, Zachary Opheim, Martin Mehnert, Valesca Anschau, Issa Isaac, Alan Ezrin, Todd Hembrough. Highly accurate detection of early-stage colorectal cancer using tumor and immune extracellular vesicles biomarkers [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A063.

Abstract A055: Tumor muscle invasion promotes tumor heterogeneity and normal muscle reprogramming

Abstract Aggressive human prostate tumors traverse a contractile smooth muscle pseudo-capsule, in a process termed extracapsular extension (ECE), defining the pT3 pathologic stage associating with increased biochemical recurrence, bone metastases, and cancer-specific mortality. While T3 lesions can be detected by non-invasive mpMRI imaging, how the tumor invades into and through the contractile muscle is unknown. Using a mouse xenograft model system of ECE, we investigated the transcriptomic consequences of human tumor invasion into and through the contractile smooth muscle of the mouse diaphragm. Both human and mouse gene transcripts were documented and analyzed. Tumor cells were intraperitoneally injected into male NSG mice and 6 weeks later, three tumor compartments (pre-invasive, muscle-invasive, and super-invasive cells that had reached the superior diaphragm surface) were analyzed for differential bulk human and mouse gene expression. Whole genome transcriptomic sequencing and GO pathway analysis revealed that approximately 414 human genes were differentially expressed between the non- invasive, muscle-resident, and super-invasive tumor populations and at least 5 enriched pathways were involved. The unique expression gene patterns of muscle-resident tumor cells were reversible when compared to the pre-invasive and super-invasive expression patterns. Significant increases in g-H2AX and nuclear deformation were observed in the muscle-resident tumor clusters as compared to the non-invasive tumor mass. No differences in tumor cell proliferation, as detected by Ki67 staining, were found between the tumor clusters in the three compartments. Immunohistochemistry staining for a damage response cytokeratin (KRT6A) and integrin (CD49f) revealed heterogeneity within the muscle resident tumors as compared to the non- invasive cells. Taken together, these data suggest a hostile contractile muscle environment elicits specific responses by the invading tumor. Single cell analysis within each of the tumor compartments is currently underway to define the spatial heterogeneity of gene expression in invasive tumor cell clusters within the contractile muscle. A dynamic reciprocity of the tumor/muscle microenvironment is suggested by the analysis of the bulk transcriptomic sequencing demonstrating a significant increase in mouse muscle bio-synthetic gene transcription as a consequence of the human tumor penetrating the tissue. Taken together, these data indicate that human tumors, during the act of traversing the contractile muscle layer, respond to this unique microenvironment by transiently altering transcription, while sustaining nuclear damage which results in the reprogramming of the muscle. This new information suggests that novel tumor or muscle biomarkers might indicate early muscle invasion events and assist new high-resolution image analysis for precision diagnostic and/or therapeutic decisions. (Supported in part by P30 CA23074; F30 CA143924, UACC Team Science Award). Citation Format: Kendra D Marr, Beatrice S Knudsen, Rafael Sainz, Kelvin W Pond, Noel E Warfel, Belinda E Sun, Anne E Cress. Tumor muscle invasion promotes tumor heterogeneity and normal muscle reprogramming [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A055.