Liquid Biopsy Markers Research Articles

Extracellular Vesicle microRNAs as Possible Liquid Biopsy Markers in HNSCC—A Longitudinal, Monocentric Study

Background: Biomarkers for HNSCC are still lacking. Biomolecules obtained via liquid biopsy are being investigated for diagnosis, prognosis, and therapy monitoring, including extracellular vesicles (EVs) and EV-cargo, e.g., proteins, RNA, and microRNA. This study aims to understand localization-dependent EV-microRNA expression in blood sera, their dynamics over time (12 months FU), and insights into their potential in diagnostics and therapy monitoring. Methods: Via liquid biopsy, blood serum was taken from 50 patients with HNSCC and 16 controls. Extracellular vesicles were isolated from serum by precipitation, and the contained microRNA-21, -1246, -200c, -let-7a, -181a, and -26a were amplified by reverse transcription and determined with real-time PCR. Expression ratios (HNSCC to healthy controls) were collated with the patients’ clinical parameters. A second liquid biopsy was carried out avg. 12 months later in the tumor aftercare. A sub-analysis with the Oropharynx subsite was implemented. Results: EV-mir-21, -let-7a, and -181a were 2.5–3-fold higher expressed in HPV/p16+ than in HPV/p16- HNSCC. Different expressions of EV-mir-181a and -26a could be demonstrated depending on the therapy modality. Conclusions: EV-microRNA could be a promising biomarker in the diagnosis and therapy monitoring of HNSCC. A systematic comparison of EV- and tissue microRNA expression in different HNSCC-subsites is needed.

Extracellular Hsp70 and Circulating Endometriotic Cells as Novel Biomarkers for Endometriosis.

Stress-inducible heat shock protein 70 (Hsp70), which functions as a molecular chaperone and is frequently overexpressed in different cancer cell types, is present on the cell surface of tumor cells and is actively released into the circulation in free and extracellular lipid vesicle-associated forms. Since the exact pathomechanism of endometriosis has not yet been elucidated (although it has been associated with the development of endometrial and ovarian cancer), we asked whether extracellular Hsp70 and circulating endometriotic cells (CECs) reflect the presence and development of endometriosis. Therefore, circulating levels of free and lipid microvesicle-associated Hsp70 were measured using the Hsp70-exo ELISA, and the presence of circulating CECs in the peripheral blood of patients with endometriosis was determined using membrane Hsp70 (mHsp70) and EpCAM monoclonal antibody (mAb)-based bead isolation approaches. Isolated CECs were further characterized by immunofluorescence using reagents directed against cytokeratin (epithelial marker), CD45 (leukocyte marker), CD105/CD44 (mesenchymal stemness markers) and by comparative RNA analysis. Similar to the situation in patients with cancer, the levels of circulating Hsp70 were elevated in the blood of patients with histologically proven endometriosis compared to a healthy control cohort, with significantly elevated Hsp70 levels in endometriosis patients with lesions outside the uterine cavity. Moreover, CECs could be isolated using the cmHsp70.1 mAb-based, and to a lesser extent EpCAM mAb-based, bead approach in all patients with endometriosis, with the highest counts obtained using the mHsp70-targeting procedure in patients with extra-uterine involvement. The longevity in cell culture and the expression of the cytokeratins CD105 and CD44, together with differentially expressed genes related to epithelial-to-mesenchymal transition (EMT), revealed similarities between mHsp70-expressing CECs and circulating tumor cells (CTCs) and suggest a mesenchymal stem cell origin. These findings support the involvement of mHsp70-positive stem cell-like cells in the development of endometriotic lesions. In summary, elevated levels of Hsp70 and CECs in the circulation could serve as liquid biopsy markers for endometriosis with extra-uterine involvement and help to elucidate the underlying pathomechanism of the disease.

High Expression of the Tumor Suppressor Protein ITIH5 in Cholangiocarcinomas Correlates with a Favorable Prognosis.

Background: Cholangiocarcinoma (CCA) are aggressive bile duct cancers with a poor prognosis for which there are only few established prognostic biomarkers and molecular targets available. The gene ITIH5, a known class II tumor suppressor gene (C2TSG), encodes a secreted protein of the extracellular matrix mediating tumor suppressive properties. Recently, it was surprisingly found that the ITIH5 protein is specifically upregulated in CCAs and that ITIH5 detection in blood could be an excellent liquid biopsy marker for indicating the presence of a CCA tumor in a patient. We therefore investigated whether patients with CCAs with abundant versus low ITIH5 protein expression also differ in their prognosis. Methods: To clarify this question, a large CCA cohort (n = 175) was examined using immunohistochemistry on a tissue microarray (TMA). Results: Abundant ITIH5 expression in CCA was associated with favorable survival, a low UICC stage and the absence of perineural invasion (PNI). Conclusions: ITIH5 has biomarker potential not only for the early detection of CCA from blood-based liquid biopsies but also as a prognostic tissue biomarker for risk stratification. Our results suggest that the upregulation of ITIH5 is particularly abundant in intrahepatic CCAs (iCCA). The mechanisms mediating the strong initial upregulation of ITIH5 during the oncogenic transformation of bile duct cells are still unclear.

Potential liquid biopsy markers of exosomal microRNAs in renal interstitial fibrosis blood and urine

ABSTRACT Objective: To explore more and better liquid biopsy markers of exosomal microRNAs (exo-miRNAs) in renal interstitial fibrosis (RIF) and to preliminary investigate the biological functions and signaling pathways involved in these markers. MATERIALS AND METHODS: High-throughput miRNA sequencing was performed on blood and urine exo-miRNAs from three RIF patients and three healthy volunteers, and differential expression analysis and bioinformatic processing were performed. Results: There were 13 differentially expressed exo-miRNA (DEexo-miRNA) between RIF and healthy blood, and 20 DEexo-miRNAs in urine. These were various DEexo-miRNAs in different specimens, and the former included PC-3p-213532_58, hsa-miR-338-5p_R-1, PC-5p-34127_410, pal-miR-9993a-3p_L+2R-1, and hsa-miR-26a-1-3p with intermediate expression levels; the latter involved hsa-miR-126-3p, hsa-miR-217-5p, hsa-miR-199b-3p_R-1, mmu-miR-5106_R-4_1ss1AG, and PC-5p-39041_356, and others, while hsa-miR-378a-3p, hsa-miR-143-3p_R+1, hsa-miR-183-5p, hsa-miR-126-3p, hsa-miR-155-5p_R-1, mmu-miR-5106_R-4_1ss1AG, hsa-miR-126-5p, and hsa-miR-199b-3p_R-1 had high expression levels. Bioinformatics analysis of the up-regulated DEmiRNA with high expression in urine showed that there are 291 target corresponding mRNAs for six of eight DEexo-miRNAs, with mmu-miR-5106_R-4_1ss1AG and hsa-miR-199b-3p_R-1 having no target gene found in TargetScan and miRanda. GO annotation revealed that GO:0005515 (protein binding) had the lowest P value, involving the most genes. KEGG analysis revealed that the signaling included the mTOR signaling pathway, autophagy - animal, etc., and hsa05200 (pathways in cancer) had a lower P value, involving the most genes. Conclusions: Urine is a better sample for RIF exo-miRNA detection than blood. Urinary exosomal hsa-miR-143-3p_R+1, hsa-miR-183-5p, hsa-miR-126-3p, mmu-miR-5106_R-4_1ss1AG, hsa-miR-126-5p, and hsa-miR-199b-3p_R-1 are novel liquid biopsy markers for RIF. These DEexo-miRNA may be associated with the occurrence and development of RIF and may participate in specific biological processes by regulating the expression of their target mRNA. Further research may require exploring the specific functions and mechanisms of these miRNAs in RIF, as well as whether they can serve as diagnostic or therapeutic targets for RIF.

Plasma Exosome miR-203a-3p is a Potential Liquid Biopsy Marker for Assessing Tumor Progression in Breast Cancer Patients.

Timely detection of tumor progression in breast cancer (BC) patients is critical for therapeutic management and prognosis. Plasma exosomal miRNAs are potential liquid biopsy markers for monitoring tumor progression, but their roles in BC remain unclear. In the TCGA database, we first screened for miRNAs significantly associated with BC progression by comparing miRNA expression in para-carcinoma tissues, stage I BC tissues, and stage II-III BC tissues (n = 1026). Cox regression analyses and survival analyses were performed on candidate miRNAs to explore their prognostic value (n = 848). KEGG, GO, and PPI analyses were used to identify enriched pathways associated with cancer. Finally, the potential of candidate miRNAs as liquid biopsy markers was evaluated by sequencing and analyzing plasma exosomal miRNAs from our collection of 45 BC patients (14 in stage I, 31 in stage II-III) and 5 healthy controls, combined with qRT-PCR analysis to assess the correlation of candidate gene expression in plasma exosomes and BC tissues. We found that only miR-203a-3p was progressively elevated with BC progression and was associated with poor prognosis in the TCGA dataset. Its potential target genes were enriched in pathways related to tumor progression, and the downregulation of 48 of these genes was associated with poor prognosis. More importantly, plasma exosomal miR-203a-3p was also found to gradually increase with BC progression, and its expression was positively correlated with miR-203a-3p in BC tissues. This result suggests that plasma exosomal miR-203a-3p may reflect the expression of miR-203a-3p in tumor tissues and serve as a potential liquid biopsy marker for monitoring BC progressions. We found for the first time that elevated miR-203a-3p was associated with BC progression and poor prognosis. Our findings suggested that plasma exosomal miR-203a-3p could hold potential as a liquid biopsy marker for evaluating BC progression in patients.

Identification of an autophagy- and macropinocytosis-related prognostic signature for the prediction of prognosis and therapeutic response in gastric cancer.

Traditional liquid biopsy markers show a low rate of positivity and accurate in gastric cancer. With the rapid advancement of sequencing technology, scientists have identified promising research avenues in this field. Autophagy and macropinocytosis utilize diverse pathways and mechanisms to supply resources and fuel for tumor growth. Nonetheless, their potential interplay introduces an untapped avenue for the discovery of novel tumor biomarkers. To develop an innovative prognostic signature based on autophagy- and micropinocytosis-related genes, with the aim to predict the outcome and therapeutic response of gastric cancer patients. Additionally, to validate the prognostic impact of this signature, and elucidate the role of representative molecules in gastric cancer. To construct and validate a prognostic signature for gastric cancer, bioinformatics methods such as COX regression, LASSO regression, survival analysis, ROC curve, and nomogram were utilized based on the sequencing and clinical data of gastric cancer patients retrieved from the TCGA and GEO databases. GSEA functional enrichment analyses were employed to predict the biological functions. Meanwhile, qRT-PCR and Western blot experiments were utilized to quantify the mRNA and protein expression levels. Furthermore, the EdU assay and colony formation assay were utilized to examine the cell proliferation ability while the Transwell assays were conducted to assess the migration and invasion abilities of gastric cancer cells. Through consistency clustering and univariate COX analyses, potential prognostic genes involved in both autophagy and macropinocytosis were identified. Based on these genes, a 9-gene signature was constructed, which demonstrated high accuracy in predicting gastric cancer patients' survival period, immunotherapeutic response, and chemotherapy drug tolerance. Furthermore, qRT-PCR analyses of gastric cancer tissue samples showed that the representative genes of this signature were aberrantly overexpressed in gastric cancer, with MATN3, as the most notable molecule, exhibiting significant carcinogenic effects on cancer cells by actively regulating their proliferation, migration, and invasion abilities. Our newly created prognostic signature possesses significant potential as a biomarker for gastric cancer, while MATN3 is identified as an oncogenic factor in gastric cancer. This brings to light new perspectives, which can contribute to enhancing the diagnosis and treatment of gastric cancer.

Extracellular vesicles carry transcriptional 'dark matter' revealing tissue-specific information.

From eukaryotes to prokaryotes, all cells secrete extracellular vesicles (EVs) as part of their regular homeostasis, intercellular communication, and cargo disposal. Accumulating evidence suggests that small EVs carry functional small RNAs, potentially serving as extracellular messengers and liquid-biopsy markers. Yet, the complete transcriptomic landscape of EV-associated small RNAs during disease progression is poorly delineated due to critical limitations including the protocols used for sequencing, suboptimal alignment of short reads (20-50nt), and uncharacterized genome annotations-often denoted as the 'dark matter' of the genome. In this study, we investigate the EV-associated small unannotated RNAs that arise from endogenous genes and are part of the genomic 'dark matter', which may play a key emerging role in regulating gene expression and translational mechanisms. To address this, we created a distinct small RNAseq dataset from human prostate cancer & benign tissues, and EVs derived from blood (pre- & post-prostatectomy), urine, and human prostate carcinoma epithelial cell line. We then developed an unsupervised data-based bioinformatic pipeline that recognizes biologically relevant transcriptional signals irrespective of their genomic annotation. Using this approach, we discovered distinct EV-RNA expression patterns emerging from the un-annotated genomic regions (UGRs) of the transcriptomes associated with tissue-specific phenotypes. We have named these novel EV-associated small RNAs as 'EV-UGRs' or "EV-dark matter". Here, we demonstrate that EV-UGR gene expressions are downregulated by ∼100 fold (FDR<0.05) in the circulating serum EVs from aggressive prostate cancer subjects. Remarkably, these EV-UGRs expression signatures were regained (upregulated) after radical prostatectomy in the same follow-up patients. Finally, we developed a stem-loop RT-qPCR assay that validated prostate cancer-specific EV-UGRs for selective fluid-based diagnostics. Overall, using an unsupervised data driven approach, we investigate the 'dark matter' of EV-transcriptome and demonstrate that EV-UGRs carry tissue-specific Information that significantly alters pre- and post-prostatectomy in the prostate cancer patients. Although further validation in randomized clinical trials is required, this new class of EV-RNAs hold promise in liquid-biopsy by avoiding highly invasive biopsy procedures in prostate cancer.

Application value of folate receptor-positive circulating tumor cells in the diagnosis of head and neck squamous cell carcinoma

A total of 82 patients and healthy subjects in the First Affiliated Hospital of Sun Yat-sen University from March to August 2023 were recruited. The cohort consisted of 43 patients with head and neck squamous cell carcinoma (HNSCC) and 39 non-cancer patients or healthy subjects. There were 63 males and 19 females, with a median age of 62 (46, 67) years. The levels of folate receptor-positive circulating tumor cells (FR+CTCs) in the blood of HNSCC patients and non-cancer/healthy subjects were 12.4 (8.5, 17.8) floate unit (FU)/3 ml and 5.0 (3.8, 6.6) FU/3 ml, respectively, with a statistically significant difference (P<0.001). The area under the receiver operating characteristic (ROC) curve for FR+CTCs levels was 0.937 (95%CI: 0.888-0.986, P<0.001), with a cut-off value of 7.4 FU/3 ml determined by the maximum Youden index. At this cut-off value, the sensitivity and specificity of FR+CTCs for diagnosing HNSCC were 90.70% and 89.74%, respectively. The current study suggests that FR+CTCs could be used as a liquid biopsy marker for the screening and diagnosis of HNSCC.

Synchronized Glioma Insights: Trends, Blood Group Correlations, Staging Dynamics, and the Vanguard of Liquid Biopsy Advancements.

Gliomas are the most frequent, heterogeneous group of tumors arising from glial cells, characterized by difficult monitoring, poor prognosis, and fatality. Tissue biopsy is an established procedure for tumor cell sampling that aids diagnosis, tumor grading, and prediction of prognosis. We studied and compared the levels of liquid biopsy markers in patients with different grades of glioma. Also, we tried to prove the potential association between glioma and specific blood group antigens. 78 patients were found, among whom the maximum percentage with glioblastoma had blood group O+ (53.8%). The second highest frequency had blood group A+ (20.4%), followed by B+ (9.0%) and A- (5.1%), and the least with O-. Liquid biopsy biomarkers included Alanine Aminotransferase (ALT), Lactate Dehydrogenase (LDH), lymphocytes, Urea, Alkaline phosphatase (AST), Neutrophils, and C-Reactive Protein (CRP). The levels of all the components increased significantly with the severity of the glioma, with maximum levels seen in glioblastoma (grade IV), followed by grade III and grade II, respectively. Gliomas have significant clinical challenges due to their progression with heterogeneous nature and aggressive behavior. A liquid biopsy is a non-invasive approach that aids in setting up the status of the patient and figuring out the tumor grade; therefore, it may show diagnostic and prognostic utility. Additionally, our study provides evidence to prove the role of ABO blood group antigens in the development of glioma. However, future clinical research on liquid biopsy will improve the sensitivity and specificity of these tests and confirm their clinical usefulness to guide treatment approaches. </p>.

Identification of exosomal circSLC26A4 as a liquid biopsy marker for cervical cancer.

Circular RNA SLC26A4 (circSLC26A4) functions as an oncogene in the initiation and progression of cervical cancer (CC). However, the clinical role of plasma exosomal circSLC26A4 in CC is poorly known. This study aims to develop an accurate diagnostic method based on circulating exosomal circSLC26A4. In this study, exosomal circSLC26A4 derived from CC cell lines (CaSki, SiHa, and HeLa) and human cervical epithelial cells (HcerEpic) was measured and compared using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Additionally, 56 volunteers, including 18 CC patients, 18 cervical high-grade squamous intraepithelial lesion (HSIL) patients, and 20 healthy volunteers, were enrolled. qRT-PCR was also performed to measure the plasma exosomal circSLC26A4 levels in all participants. The exosomal circSLC26A4 expression level derived from CC cells was significantly elevated compared to it derived from HcerEpic cells. Plasma exosomal circSLC26A4 levels in CC patients were significantly higher than in healthy women and HSIL patients (P < 0.05). In addition, high plasma exosomal circSLC26A4 expression was positively associated with lymph node metastasis and FIGO stage (all P < 0.05). However, no significant correlation was found between plasma exosomal circSLC26A4 expression and age, intravascular cancerous embolus, and perineural invasion (P > 0.05). The high exosomal circSLC26A4 expression is closely related to the occurrence of CC. Plasma exosomal circSLC26A4 can be used as a diagnostic marker for CC.

Emerging Prognostic Markers in Patients Undergoing Liver Resection for Hepatocellular Carcinoma: A Narrative Review.

In patients with hepatocellular carcinoma (HCC), liver resection is potentially curative. Nevertheless, post-operative recurrence is common, occurring in up to 70% of patients. Factors traditionally recognized to predict recurrence and survival after liver resection for HCC include pathologic factors (i.e., microvascular and capsular invasion) and an increase in alpha-fetoprotein level. During the past decade, many new markers have been reported to correlate with prognosis after resection of HCC: liquid biopsy markers, gene signatures, inflammation markers, and other biomarkers, including PIVKA-II, immune checkpoint molecules, and proteins in urinary exosomes. However, not all of these new markers are readily available in clinical practice, and their reproducibility is unclear. Liquid biopsy is a powerful and established tool for predicting long-term outcomes after resection of HCC; the main limitation of liquid biopsy is represented by the cost related to its technical implementation. Numerous patterns of genetic expression capable of predicting survival after curative-intent hepatectomy for HCC have been identified, but published findings regarding these markers are heterogenous. Inflammation markers in the form of prognostic nutritional index and different blood cell ratios seem more easily reproducible and more affordable on a large scale than other emerging markers. To select the most effective treatment for patients with HCC, it is crucial that the scientific community validate new predictive markers for recurrence and survival after resection that are reliable and widely reproducible. More reports from Western countries are necessary to corroborate the evidence.

Advances in Liquid Biopsy for Diagnosis of Bladder Cancer.

Bladder cancer (BCa) is the most common malignancy of the urinary system. It has a high recurrence rate and requires longterm follow-up. Significant advances in BCa research have been made in recent years; however, the initial diagnosis and follow-up of BCa relies on cystoscopy, which is an invasive and expensive procedure. Over the past decade, liquid biopsies (e.g., blood and urine) have proven to be highly efficient methods for the discovery of BCa biomarkers. This noninvasive sampling method is used to analyze unique tumor components released into body fluids and enables serial sampling and longitudinal monitoring of tumor progression. Several liquid biopsy biomarkers have been studied extensively and have shown promising results in the clinical applications of BCa, including early detection, microscopic residual disease detection, recurrence prediction, and treatment response. Therefore, this review aims to provide an update on various new liquid biopsy markers and the advantages and current limitations of liquid biopsy in the diagnosis of BCa.

The SURVIVE study: Liquid biopsy guided surveillance after intermediate- to high-risk early breast cancer.

TPS620 Background: Current guidelines limit routine breast cancer (BC) 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). Methods: Funded by the German Federal Ministry of Education and Research, the SURVIVE study is the first large, randomized BC 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. Eligible patients are adults with confirmed primary invasive intermediate to high-risk early BC, defined as an indication for (neo-)adjuvant chemotherapy, and/or large tumor size (&gt; 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 previously. Patient enrollment during adjuvant endocrine, antibody- or targeted therapy is allowed. 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, and the intensified surveillance arm will be tested for standard tumor markers, CTCs and ctDNA via tumor-informed approach (RaDaR assay). 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. Statistics: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. 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 IDFS, DDFS, DRFS, BCSS and quality of life (QoL). Aims: 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. Clinical trial information: NCT05658172 .

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 ( &amp;gt; 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.

From biology to the clinic - exploring liver metastasis in prostate cancer.

Liver metastases from prostate cancer are associated with an aggressive disease course and poor prognosis. Results from autopsy studies indicate a liver metastasis prevalence of up to 25% in patients with advanced prostate cancer. Population data estimate that ~3-10% of patients with metastatic castration-resistant prostate cancer harbour liver metastases at the baseline, rising to 20-30% in post-treatment cohorts, suggesting that selective pressure imposed by novel therapies might promote metastatic spread to the liver. Liver metastases are associated with more aggressive tumour biology than lung metastases. Molecular profiling of liver lesions showed an enrichment of low androgen receptor, neuroendocrine phenotypes and high genomic instability. Despite advancements in molecular imaging modalities such as prostate-specific membrane antigen PET-CT, and liquid biopsy markers such as circulating tumour DNA, early detection of liver metastases from prostate cancer remains challenging, as both approaches are hampered by false positive and false negativeresults, impeding the accurate identification of early liver lesions. Current therapeutic strategies showed limited efficacy in this patient population. Emerging targeted radionuclide therapies, metastasis-directed therapy, and novel systemic agents have shown preliminary activity against liver metastases, but require further validation. Treatment with various novel prostate cancer therapies might lead to an increase in the prevalence of liver metastasis, underscoring the urgent need for coordinated efforts across preclinical and clinical researchers to improve characterization, monitoring, and management of liver metastases from prostate cancer. Elucidating molecular drivers of liver tropism and interactions with the liver microenvironment might ultimately help to identify actionable targets to enhance survival in this high-risk patient group.

Liquid Biopsy: A Game Changer for Type 2 Diabetes.

As the burden of type 2 diabetes (T2D) continues to escalate globally, there is a growing need for novel, less-invasive biomarkers capable of early diabetes detection and monitoring of disease progression. Liquid biopsy, recognized for its minimally invasive nature, is increasingly being applied beyond oncology, and nevertheless shows its potential when the collection of the tissue biopsy is not possible. This diagnostic approach involves utilizing liquid biopsy markers such as cell-free nucleic acids, extracellular vesicles, and diverse metabolites for the molecular diagnosis of T2D and its related complications. In this context, we thoroughly examine recent developments in T2D liquid biopsy research. Additionally, we discuss the primary challenges and future prospects of employing liquid biopsy in the management of T2D. Prognosis, diagnosis and monitoring of T2D through liquid biopsy could be a game-changing technique for personalized diabetes management.

Tumor-Educated Platelet RNA and Circulating Free RNA: Emerging Liquid Biopsy Markers for Different Tumor Types.

The incidence and mortality from malignant tumors continue to rise each year. Consequently, early diagnosis and intervention are vital for improving patient' prognosis and survival. The traditional pathological tissue biopsy is currently considered the gold standard for cancer diagnosis. However, it suffers from several limitations including invasiveness, sometimes not repeatable or unsuitable, and the inability to capture the dynamic nature of tumors in terms of space and time. Consequently, these limit the application of tissue biopsies for the diagnosis of early-stage tumors and have redirected the research focus towards liquid biopsies. Blood-based liquid biopsies have thus emerged as a promising option for non-invasive assessment of tumor-specific biomarkers. These minimally invasive, easily accessible, and reproducible tests offer several advantages, such as being mostly complication-free and efficient at monitoring tumor progression and tracing drug resistance. Liquid biopsies show great potential for cancer prediction, diagnosis, and prognostic assessment. Circulating tumor-educated platelets (TEPs) possess the unique ability to absorb nucleic acids from the bloodstream and to modify transcripts derived from megakaryocytes in response to external signals. In addition, circulating free RNA (cfRNA) constitutes a significant portion of the biomolecules present in the bloodstream. This paper aims to provide a comprehensive overview of the current research status regarding TEP RNA and cfRNA in liquid biopsies from various tumor types. Our analysis includes cancers of the lung, liver, pancreas, breast, nasopharynx, ovary and colon, as well as multiple myeloma and sarcoma. By synthesizing this information, we intend to establish a solid theoretical foundation for exploring potential applications of circulating RNA as a reliable biomarker for tumor diagnosis and monitoring.

Negative Charge-Carrying Glycans Attached to Exosomes as Novel Liquid Biopsy Marker.

Prostate cancer (PCa) is the second most common cancer. In this paper, the isolation and properties of exosomes as potential novel liquid biopsy markers for early PCa liquid biopsy diagnosis are investigated using two prostate human cell lines, i.e., benign (control) cell line RWPE1 and carcinoma cell line 22Rv1. Exosomes produced by both cell lines are characterised by various methods including nanoparticle-tracking analysis, dynamic light scattering, scanning electron microscopy and atomic force microscopy. In addition, surface plasmon resonance (SPR) is used to study three different receptors on the exosomal surface (CD63, CD81 and prostate-specific membrane antigen-PMSA), implementing monoclonal antibodies and identifying the type of glycans present on the surface of exosomes using lectins (glycan-recognising proteins). Electrochemical analysis is used to understand the interfacial properties of exosomes. The results indicate that cancerous exosomes are smaller, are produced at higher concentrations, and exhibit more nega tive zeta potential than the control exosomes. The SPR experiments confirm that negatively charged α-2,3- and α-2,6-sialic acid-containing glycans are found in greater abundance on carcinoma exosomes, whereas bisecting and branched glycans are more abundant in the control exosomes. The SPR results also show that a sandwich antibody/exosomes/lectins configuration could be constructed for effective glycoprofiling of exosomes as a novel liquid biopsy marker.

Clinical algorithm model based on cfDNA to predict SLE disease activity.

Circulating cell-free DNA (cfDNA) has been widely used as a new liquid-biopsy marker. Dysregulation of cfDNA has been found in patients with systemic lupus erythematosus (SLE). However, the detailed association between cfDNA and SLE has not been thoroughly studied. Plasma samples were collected from 88 patients with active SLE and 39 patients with inactive SLE. The cfDNA concentration was determined, and the length and distribution of cfDNA fragments were verified. cfDNA concentrations were significantly higher in patients with active SLE than in patients with inactive SLE (0.4 [0.18-0.897] ng/µL vs 0.249 [0.144-0.431] ng/µL; p = .043). cfDNA fragments were enriched in the ranges of 153-198 bp and 300-599 bp. cfDNA concentrations were associated with the reduction of the anti-double-stranded DNA (dsDNA) antibodies titer (r = -0.301, p = .034). The presence of anti-U1 ribonucleoprotein (p = .012), anti-Sjogren syndrome A (p = .024), anti-dsDNA (p = .0208), and anti-nucleosome antibodies (p = .0382) might associate to the variation of cfDNA concentration. Reduced cfDNA concentration was associated with renal damage in active SLE patients (0.31 [0.11-0.73] ng/µL vs 0.65 [0.27-1.53] ng/µL; p = .009). The Active index, a combination model including cfDNA concentration and other clinical indices, had an area of 0.886 under the receiver operating characteristics curve for distinguishing active SLE. The Active index was positively correlated with the SLE disease activity index score (r = 0.6724, p < .0001). Through systematic stratified analysis and clinical algorithm model, this study found that plasma cfDNA concentration is closely related to SLE disease severity, which has guiding significance for the future clinical application of cfDNA in SLE.

Enhancing the cancer metastasis diagnosis: Ultrasensitive nano sensors exploiting Cancer stem cell associated DNA methylation as a liquid biopsy marker

Cancer metastasis presents a significant clinical challenge due to its impact on patient survival rates. Early detection of metastasis is pivotal for effective intervention and improved patient outcomes. Liquid biopsy, a non-invasive diagnostic approach, holds promise for cancer detection, yet its utility in metastasis detection remains underexplored. Stable and accurate, DNA methylation reflects early tumor stages and enables molecular characterization, providing valuable longitudinal information. Despite its potential, clinical translation of DNA methylation biomarkers is limited, partially due to technology constraints and tumor heterogeneity. To enable clinical translation, an assay is needed that 1) quantifies DNA methylation, 2) integrates seamlessly into clinical workflows, and 3) ensures accuracy and sensitivity. In this study, we introduce an ultrasensitive nanosensor, Methylprobe, for DNA methylation-based liquid biopsy of metastasis. Methylprobe, synthesized via ultrashort pulsed ionization, provides signal amplification, enabling direct detection of methylated DNA in plasma. The technology is scalable and adaptable for clinical settings. The assay comprises the Methylprobe, metastasis-specific biomarkers, and a machine-learning algorithm. Tumor heterogeneity is addressed by integrating DNA-methylation signatures from distinct cancer stem cell phenotypes, enhancing assay reliability. The Methylprobe assay is validated on breast, lung, and colorectal cancer samples, demonstrating 99.8% accuracy in metastasis detection. This innovative approach offers potential as a reliable, sensitive, and accurate platform for assessing metastasis-associated DNA methylation patterns in blood plasma. Integrating Methylprobe into clinical practice could revolutionize metastasis detection, providing comprehensive insights into metastatic phenotypes and informing tailored therapeutic strategies.