Biopsy Approach Research Articles

Rapid Detection of Recurrent Non-Muscle Invasive Bladder Cancer in Urine Using ATR-FTIR Technology.

Non-muscle Invasive Bladder Cancer (NMIBC) accounts for 80% of all bladder cancers. Although it is mostly low-grade tumors, its high recurrence rate necessitates three-times-monthly follow-ups and cystoscopy examinations to detect and prevent its progression. A rapid liquid biopsy-based assay is needed to improve detection and reduce complications from invasive cystoscopy. Here, we present a rapid spectroscopic method to detect the recurrence of NMIBC in urine. Urine samples from previously-diagnosed NMIBC patients (n = 62) were collected during their follow-up visits before cystoscopy examination. Cystoscopy results were recorded (41 cancer-free and 21 recurrence) and attenuated total refraction Fourier transform infrared (ATR-FTIR) spectra were acquired from urine samples using direct application. Spectral processing and normalization were optimized using parameter grid searching. We assessed their technical variability through multivariate analysis and principal component analysis (PCA). We assessed 35 machine learning models on a training set (70%), and the performance was evaluated on a held-out test set (30%). A Regularized Random Forests (RRF) model achieved a 0.92 area under the receiver operating characteristic (AUROC) with 86% sensitivity and 77% specificity. In conclusion, our spectroscopic liquid biopsy approach provides a promising technique for the early identification of NMIBC with a less invasive examination.

Findings from an international survey of urology trainee experience with prostate biopsy.

To assess urology trainees' exposure to transperineal prostate biopsy (TP-Bx) and intent to use TP-Bx in practice. A 34-question survey about prostate biopsy was distributed to urology trainees in the United States and Europe. Primary outcomes were exposure to TP-Bx in training and intent to use TP-Bx post training. Exposure to transrectal prostate biopsy (TR-Bx) and magnetic resonance imaging-targeted biopsy (MRI-Bx) was also assessed. Survey answers were compared between groups as categorical variables using Fisher's exact test. Multivariable logistic regression was used to identify factors associated with intent of performing TP-Bx post training. A total of 658 trainees from 19 countries completed the survey. Of these, 313 trainees (48%) reported exposure to TP-Bx, 370 (56%) reported exposure to MRI-Bx, and 572 (87%) reported exposure to TR-Bx. There was significant heterogeneity in TP-Bx exposure among countries (P< 0.001), with the highest prevalence in Italy (72%) and the lowest prevalence in Greece (4%). Intent to perform TP-Bx post training was higher in those exposed to TP-Bx during training (89% vs 58%; P< 0.001) and did not differ between trainees in postgraduate year (PGY) 1-3 vs those in PGY ≥4 (73% vs 72%; P= 0.7). On multivariable regression, exposure to TP-Bx in training was independently associated with increased intent to perform TP-Bx post training (odds ratio 5.09, 95% confidence interval 3.29-8.03; P< 0.001). Fewer than half of 658 surveyed urology trainees reported exposure to TP-Bx, with significant heterogeneity among countries. Greater experience with TP-Bx in training was associated with greater intent to perform TP-Bx post training. A minimum requirement of TP-Bx cases during urological training may increase resident familiarity and adoption of this guideline-endorsed prostate biopsy approach.

Biomarkers for the Detection and Risk Stratification of Aggressive Prostate Cancer.

Current strategies for the clinical management of prostate cancer are inadequate for a precise risk stratification between indolent and aggressive tumors. Recently developed tissue-based molecular biomarkers have refined the risk assessment of the disease. The characterization of tissue biopsy components and subsequent identification of relevant tissue-based molecular alterations have the potential to improve the clinical decision making and patient outcomes. However, tissue biopsies are invasive and spatially restricted due to tumor heterogeneity. Therefore, there is an urgent need for complementary diagnostic and prognostic options. Liquid biopsy approaches are minimally invasive with potential utility for the early detection, risk stratification, and monitoring of tumors. In this review, we focus on tissue and liquid biopsy biomarkers for early diagnosis and risk stratification of prostate cancer, including modifications on the genomic, epigenomic, transcriptomic, and proteomic levels. High-risk molecular alterations combined with orthogonal clinical parameters can improve the identification of aggressive tumors and increase patient survival.

Dual NGS Comparative Analysis of Liquid Biopsy (LB) and Formalin-Fixed Paraffin-Embedded (FFPE) Samples of Non-Small Cell Lung Carcinoma (NSCLC).

Lung cancer ranks second worldwide after breast cancer and third in Europe after breast and colorectal cancers when both sexes and all ages are considered. In this context, the aim of this study was to emphasize the power of dual analysis of the molecular profile both in tumor tissue and plasma by NGS assay as a liquid biopsy approach with impact on prognosis and therapy modulation in NSCLC patients. NGS analysis was performed both from tissue biopsies and from cfNAs isolated from peripheral blood samples. Out of all 29 different mutations detectable by both NGS panels (plasma and tumor tissue), seven different variants (24.13%; EGFR L858R in two patients, KRAS G13D and Q61H and TP53 G244D, V197M, R213P, and R273H) were detected only in plasma and not in the tumor itself. These mutations were detected in seven different patients, two of them having known distant organ metastasis. Our data show that NGS analysis of cfDNA could identify actionable mutations in advanced NSCLC and, therefore, this analysis could be used to monitor the disease progression and the treatment response and even to modulate the therapy in real time.

Leukocyte Depletion and Size-Based Enrichment of Circulating Tumor Cells Using a Pressure-Sensing Microfiltration Device.

Considering the challenges in isolating circulating tumor cells (CTCs) pertaining to cellular stress and purity, we report the application of a blood microfiltration device as an optimal approach for noninvasive liquid biopsy to target CTCs. We experimentally analyzed the filtration behavior of the microfilter using pressure sensing to separate tumor cells from leukocytes in whole blood. This approach achieved an average recovery of >96% of the spiked tumor cells and depletion of >99% of total leukocytes. Furthermore, we carried out genomic profiling of the CTCs using the blood microfiltration device. The method was also applied in a clinical setting; DNA amplification was performed using a small number of microfiltered CTCs and epidermal growth factor receptor mutations were successfully detected to characterize the efficacy of molecularly targeted drugs against lung cancer. Overall, the proposed method can provide a tool for evaluating efficient filtration pressure to concentrate CTCs from whole blood.

Abstract B028: Changes in exosomal RNA expression associate with treatment response to BRAF + EGFR + PD-1 blockade in MSS, BRAFV600E colorectal cancer: A liquid biopsy approach

Abstract Introduction: BRAF + EGFR inhibition induces decreased expression of mismatch repair (MMR) genes in preclinical models of microsatellite stable (MSS), BRAFV600E metastatic colorectal cancer (mCRC) but has not been reported in patients. We evaluated exosomal RNA (exoRNA) isolated from serial blood specimens collected from patients (pts) with MSS, BRAFV600E mCRC treated with encorafenib (E) + cetuximab (C) + nivolumab (N) as a novel blood-based approach to compare differences in gene expression according to treatment response. Methods: Pts with refractory MSS, BRAFV600E mCRC were treated with E (300 mg daily), C (500 mg/m2 q2 weeks), and N (480 mg q4 weeks) on an IRB-approved clinical trial at MD Anderson. Responses to treatment were assessed every 8 weeks (RECIST v1.1). ExoRNA isolated from pretreatment and on-treatment (week 8) blood samples were sequenced (SMART-seq) and deconvoluted (DeMix) in order to estimate cancer-specific gene expression. Previously validated MAPK and interferon (IFN)-γ gene expression signatures were used for measuring transcriptomic changes following treatment with E+C+N. Scores between responders and non-responders were compared by unpaired t-tests. For Gene Set Enrichment Analysis (GSEA), an adjusted false discovery rate (FDR) &amp;lt; 0.25 was applied to find significantly enriched pathways between on-treatment and pretreatment samples separately for responders and non-responders. Results: 26 pts were treated with E+C+N, and 24 were evaluable for response. Overall response rate was 50% (95% confidence interval (CI), 29-71), and median progression-free survival was 7.4 months (95% CI, 6.0-NA). Four pts remained on study &amp;gt; 12 months. Between responders vs non-responders (N=12 each), mean changes in MAPK signature score was decreased (-0.63 vs 0.15, respectively; p=.13). Both patients with response &amp;gt; 18 months demonstrated dramatic increases in IFN-γ score following treatment with E+C+N (+13.1 and +14.6), a trend not observed in all other patients (range, -18.0 to 3.6). Among responders to E+C+N, GSEA analysis demonstrated dynamic pathway changes in normalized enrichment score (NES) for DNA repair (NES -2.3; p-adj=.008), IL6/JAK/STAT3 (NES -2.1, p-adj=.02), and IFN-α response (NES -2.0, p-adj=.03). For non-responders, relative increases in NES were observed for angiogenesis (NES 2.1, p-adj=.02) and IL-2/STAT5 (NES 2.0, p-adj= .02). Conclusions: Transcriptome analysis using exoRNA isolated from serial blood samples is feasible for assessing treatment response in pts with mCRC. Decreased MAPK score in exoRNA may be associated with treatment response to E+C+N in pts with MSS, BRAFV600E mCRC. Changes in immune NES scores following treatment may distinguish responders from non-responders to MAPK + PD-1 blockade and highlight targets for novel immune-mediated combinations for MSS, BRAFV600E mCRC. Citation Format: Van Karlyle Morris, Kimal I. Rajapakshe, Vahid Bahrambeigi, Christine M. Parsehgian, Benny Johnson, Kanwal P. Raghav, Arvind Dasari, Ryan W. Huey, Michael J. Overman, Jason Willis, Michael S. Lee, Robert A. Wolff, Bryan K. Kee, Phat Le, Paola A. Guerrero, Scott Kopetz, Anirban Maitra. Changes in exosomal RNA expression associate with treatment response to BRAF + EGFR + PD-1 blockade in MSS, BRAFV600E colorectal cancer: A liquid biopsy approach [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr B028.

Abstract PR006: Single-molecule and single-cell epigenetics: Decoding the epigenome for cancer research and diagnostics

Abstract Genes and genomic elements are packaged by chromatin structures that regulate their activity. We developed a novel high-throughput single-molecule imaging technology to decode combinatorial modifications on millions of individual nucleosomes. We apply this technology to image nucleosomes and delineate their combinatorial epigenetic patterns, and how these patterns are deregulated in cancer. In addition, we adapt single-cell technologies based on CyTOF to profile the global levels of multiple histone modifications in single cells, thus revealing epigenetic heterogeneity in cancer. Our research focuses on pediatric gliomas harboring lysine to methionine substitution of residue 27 on histone H3 (K27M). We provide evidence for widespread effects of the H3-K27M oncohistone on multiple core epigenetic pathways, and highlight the capability of single-molecule and single-cell tools to reveal mechanisms of chromatin deregulation and heterogeneity in cancer. We also harness the single-molecule technology as a novel liquid biopsy approach, by comprehensively profiling combinatorial epigenetic marks of plasma-isolated nucleosomes. Applying this analysis to a cohort of plasma samples detect colorectal cancer at high accuracy and sensitivity, even at early stages. Finally, combining this proteomic analysis with single-molecule DNA sequencing reveals the tissue-of-origin of the tumor. Citation Format: Efrat Shema. Single-molecule and single-cell epigenetics: Decoding the epigenome for cancer research and diagnostics. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr PR006.

Results of Open Incisional Biopsy and Core Needle Biopsy of Bone Tumors

Objective: The goal of this research was to assess the diagnostic efficacy of open vs percutaneous core needle biopsy (CNB) for musculoskeletal malignancies. Study Design: Retrospective study Place and Duration: This retrospective study was conducted at Department of Orthopaedic, Khyber Teaching Hospital Peshawar during the period from April, 2022 to September, 2022. Methods: There were 96 patients were presented in this study. All patients with a primary bone or soft tissue tumour suspected of being malignant who receive a percutaneous CNB or open biopsy and subsequently develop a tumour were included. Patients were equally divided in two groups. Group I had 48 patients and received open incisional biopsy and group II received core needle biopsy in 48 cases. Fischer’s test was used to compared outcomes among both groups. SPSS 23.0 was used to analyze all data. Results: We found that 28 patients in group I and 30 patients in group II were males. Mean age of the cases in group I was 51.6±7.48 years and in group II mean age was 54.7±10.34 years. As per anatomical distribution, lower extremity was the most common in both groups followed by trunk and upper extremity. In our study favorable outcomes were obtained by CNB in bone sarcoma in terms of diagnostic accuracy, NPV, PPV, sensitivity and specificity as compared to open biopsy but difference was insignificant and in soft tissue sarcoma open biopsy showed a significant good result with p value &lt;0.005. Conclusion: Results from our study showed that the percutaneous biopsy approach was only slightly less effective than open biopsy for soft tissue tumours, whereas the open biopsy technique was more effective for bone tumours. Keywords: Soft tissue sarcoma, Bone sarcoma, Open Biopsy, Core Needle Biopsy

Liquid Biopsy Versus Tissue Biopsy to Determine Front Line Therapy in Metastatic Non-Small Cell Lung Cancer (NSCLC)

In the last decade, non–small-cell lung cancer (NSCLC) treatment has improved with the approval of multiple therapies to target specific genetic alterations. Though, next generation sequencing (NGS) has traditionally been conducted from tissue biopsy samples, developing data supports the use of plasma-based circulating tumor DNA (ctDNA), also known as “liquid biopsy,” to complement tissue biopsy approaches in guiding front-line therapy. This study is a retrospective analysis of 170 new NSCLC patients treated at 2 cancer centers within a 5-year period who received both tissue and liquid biopsy NGS as standard of care. Based on a treatment schema defined by testing sufficiency, biomarker detection, and turnaround time (TAT), physicians based the majority of their treatments on liquid biopsy results (73.5%) versus tissue biopsy (25.9%). Liquid biopsy NGS returned results on average 26.8 days faster than tissue and reported higher testing success. For guideline-recommended biomarkers, liquid biopsy was 94.8% to 100% concordant with tissue. In comparing testing modalities, a liquid-first approach identified guideline-recommended biomarkers in 76.5% of patients versus 54.9% in a tissue-first approach. There was no significant difference in time-to-treatment, or survival outcomes (overall survival and progression free survival) based on liquid versus tissue biopsy findings. This research demonstrates that liquid biopsy NGS is an effective tool to capture actionable genetic alterations in NSCLC. Due to its high concordance to tissue, faster TAT, and similarity in outcomes and time-to-treatment, liquid biopsy can be used either as a first-line test or concordantly with tissue biopsy to guide treatment decisions in NSCLC.

BIOM-06. IDENTIFICATION OF AN INFLAMMATORY BIOMARKER SIGNATURE IN PLASMA-DERIVED EXTRACELLULAR VESICLES OF GLIOBLASTOMA PATIENTS

Abstract Diagnosis of Glioblastoma (GBM) remains a clinical challenge, currently relying on symptomatic presentation of the tumour, brain imaging and invasive biopsy. Description of effective biomarkers in biofluids could prove invaluable in GBM diagnosis. Extracellular vesicles (EVs) are essential to intercellular crosstalk in the tumour bulk and circulating EVs have been described as a potential reservoir of GBM biomarkers. Our work focuses on the: (i) isolation of EVs from blood liquid biopsies of GBM patients. (ii) Characterisation of their transcriptomic and proteomic cargoes to identify/validate novel candidate GBM biomarkers that could improve GBM diagnosis/prognosis. (iii) For cases where the original GBM tumour tissues are available, we will examine the expression of the identified biomarker signatures (derived from blood-EVs) to see if the content of EVs mirrors the transcriptomic/proteomic profile of the original tissue. Similar comparisons will be performed in GBM cohorts available in TCGA. Ultimately, in future studies, transcriptomic/proteomic analyses will be assessed during patients’ follow-ups to correlate the observed biomarker profiles with MRI data,treatment,recurrence, including molecular and clinical features.Our preliminary data comparing the proteomic cargoes of EVs derived from GBM patients (n=15) and those from healthy volunteers (n=10) indicated the presence of a GBM inflammatory biomarker signature comprising members of the complement and regulators of inflammation and coagulation. Bioinformatic analysis highlighted that all potential markers exclusively identified in patient samples have been linked with either GBM diagnosis,prognosis or associated signalling, suggesting that sEVs protein cargo could mirror the landscape of the original tumour and that selective circulating sEV-derived proteins might be used as hallmarks for GBM patients.Overall, this study is a step forward in the development of a non-invasive liquid biopsy approach for the identification of valuable biomarkers that could significantly improve GBM diagnosis and, consequently, patients’ prognosis and quality of life.

Suitability of conventional systematic vs. MRI-guided targeted biopsy approaches to assess surgical treatment delay for radical prostatectomy.

To assess if systematic (SBx) vs. transrectal or transperineal mpMRI-ultrasound targeted combined with systematic (TBx + SBx) biopsy confer different effects on treatment delay to radical prostatectomy measured as Gleason grade group (GGG) upgrade of prostate cancer (PCa). We relied on a multi-institutional cohort of localized PCa patients who underwent RP in Martini-Klinik, Hamburg, or Prostate Center Northwest, Gronau, between 2014 and 2022. Analyses were restricted to PCa GGG 1-3 diagnosed at SBx (n = 4475) or TBx + SBx (n = 1282). Multivariable logistic regression modeling (MVA) predicting RP GGG upgrade of ≥ 1 was performed separately for SBx and TBx + SBx. Treatment delay to RP of < 90, 90-180 and 180-365days was reported in 59%, 35% and 6.2% of SBx and in 60%, 34% and 5.9% of the TBx + SBx patients, respectively. Upgrade to GGG ≥ 4 at RP was detected in 15% of SBx patients and 0.86% of TBx patients. In MVA performed for SBx, treatment delay yielded independent predictor status (OR 1.17 95% CI 1.02-1.39, p = 0.028), whereas for TBx + SBx MVA, statistical significance was not achieved. Treatment delay remained independently associated with radical prostatectomy GGG upgrade after adjustment for clinical variables in the patients diagnosed with SBx alone, but not in those who received combined TBx + SBx. These findings can be explained through inherent misclassification rates of SBx, potentially obfuscating historical observations of natural PCa progression and potential dangers of treatment delay. Thus, mpMRI-guided combined TBx + SBx appears mandatory for prospective delay-based examinations of PCa.

Pitfalls and Rewards of Setting Up a Liquid Biopsy Approach for the Detection of Driver Mutations in Circulating Tumor DNAs: Our Institutional Experience

We describe our institutional experience of developing a liquid biopsy approach using circulating tumor DNA (ctDNA) analysis for personalized medicine in cancer patients, focusing on the hurdles encountered during the multistep process in order to benefit other investigators wishing to set up this type of study in their institution. Blood samples were collected at the time of cancer surgery from 209 patients with one of nine different cancer types. Extracted tumor DNA and circulating cell-free DNA were sequenced using cancer-specific panels and the Illumina MiSeq machine. Almost half of the pairs investigated were uninformative, mostly because there was no trackable pathogenic mutation detected in the original tumor. The pairs with interpretable data corresponded to 107 patients. Analysis of 48 gene sequences common to both panels was performed and revealed that about 40% of these pairs contained at least one driver mutation detected in the DNA extracted from plasma. Here, we describe the choice of our overall approach, the selection of the cancer panels, and the difficulties encountered during the multistep process, including the use of several tumor types and in the data analysis. We also describe some case reports using longitudinal samples, illustrating the potential advantages and rewards in performing ctDNA sequencing to monitor tumor burden or guide treatment for cancer patients.

Characterization of BCMA Expression in Circulating Rare Single Cells of Patients with Plasma Cell Neoplasms.

B-cell maturation antigen (BCMA), a key regulator of B-cell proliferation and survival, is highly expressed in almost all cases of plasma cell neoplasms and B-lymphoproliferative malignancies. BCMA is a robust biomarker of plasma cells and a therapeutic target with substantial clinical significance. However, the expression of BCMA in circulating tumor cells of patients with hematological malignancies has not been validated for the detection of circulating plasma and B cells. The application of BCMA as a biomarker in single-cell detection and profiling of circulating tumor cells in patients' blood could enable early disease profiling and therapy response monitoring. Here, we report the development and validation of a slide-based immunofluorescence assay (i.e., CD138, BCMA, CD45, DAPI) for enrichment-free detection, quantification, and morphogenomic characterization of BCMA-expressing cells in patients (N = 9) with plasma cell neoplasms. Varying morphological subtypes of circulating BCMA-expressing cells were detected across the CD138(+/-) and CD45(+/-) compartments, representing candidate clonotypic post-germinal center B cells, plasmablasts, and both normal and malignant plasma cells. Genomic analysis by single-cell sequencing and correlation to clinical FISH cytogenetics provides validation, with data showing that patients across the different neoplastic states carry both normal and altered BCMA-expressing cells. Furthermore, altered cells harbor cytogenetic events detected by clinical FISH. The reported enrichment-free liquid biopsy approach has potential applications as a single-cell methodology for the early detection of BCMA+ B-lymphoid malignancies and in monitoring therapy response for patients undergoing anti-BCMA treatments.

Circulating cell-free messenger RNA enables non-invasive pan-tumour monitoring of melanoma therapy independent of the mutational genotype.

Plasma-derived tumour-specific cell-free nucleic acids are increasingly utilized as a minimally invasive, real-time biomarker approach in many solid tumours. Circulating tumour DNA of melanoma-specific mutations is currently the best studied liquid biopsy biomarker for melanoma. However, the combination of hotspot genetic alterations covers only around 80% of all melanoma patients. Therefore, alternative approaches are needed to enable the follow-up of all genotypes, including wild-type. We identified KPNA2, DTL, BACE2 and DTYMK messenger RNA (mRNA) upregulated in melanoma versus nevi tissues by unsupervised data mining (N=175 melanoma, N=20 normal skin, N=6 benign nevi) and experimentally confirmed differential mRNA expression in vitro (N=18 melanoma, N=8 benign nevi). Circulating cell-free RNA (cfRNA) was analysed in 361 plasma samples (collected before and during therapy) from 100 melanoma patients and 18 healthy donors. Absolute cfRNA copies were quantified on droplet digital PCR. KPNA2, DTL, BACE2 and DTYMK cfRNA demonstrated high diagnostic accuracy between melanoma patients' and healthy donors' plasma (AUC > 86%, p<.0001). cfRNA copies increased proportionally with increasing tumour burden independently of demographic variables and even remained elevated in individuals with radiological absence of disease. Re-analysis of single-cell transcriptomes revealed a pan-tumour origin of cfRNA, including endothelial, cancer-associated fibroblasts, macrophages and B cells beyond melanoma cells as cellular sources. Low baseline cfRNA levels were associated with significantly longer progression-free survival (PFS) (KPNA2 HR = .54, p=.0362; DTL HR = .60, p=.0349) and overall survival (KPNA2 HR = .52, p=.0237; BACE2 HR = .55, p=.0419; DTYMK HR = .43, p=.0393). Lastly, we found that cfRNA copies significantly increased during therapy in non-responders compared to responders regardless of therapy and mutational subtypes and that the increase of KPNA2 (HR = 1.73, p=.0441) and DTYMK (HR = 1.82, p=.018) cfRNA during therapy was predictive of shorter PFS. In sum, we identified a new panel of cfRNAs for a pan-tumour liquid biopsy approach and demonstrated its utility as a prognostic, therapy-monitoring tool independent of the melanoma mutational genotype.

INTERMITTENT OBSTRUCTION: A CASE OF ENCAPSULATED ENDOBRONCHIAL HAMARTOMA EXHIBITING A CUP-IN-BALL EFFECT

INTERMITTENT OBSTRUCTION: A CASE OF ENCAPSULATED ENDOBRONCHIAL HAMARTOMA EXHIBITING A CUP-IN-BALL EFFECT

AN ALTERNATE APPROACH: TRANSESOPHAGEAL BRONCHOSCOPIC ULTRASOUND-GUIDED FINE NEEDLE ASPIRATION (EUS-B-FNA) TO ASSESS PLEURAL MESOTHELIOMA

AN ALTERNATE APPROACH: TRANSESOPHAGEAL BRONCHOSCOPIC ULTRASOUND-GUIDED FINE NEEDLE ASPIRATION (EUS-B-FNA) TO ASSESS PLEURAL MESOTHELIOMA

Identification of an Inflammatory Biomarker Signature in Plasma-Derived Extracellular Vesicles of Glioblastoma Patients

Abstract AIMS Description of effective biomarkers present in biofluids could prove invaluable in GBM diagnosis. Extracellular vesicles (EVs) are essential to intercellular crosstalk in the tumour bulk and circulating EVs have been described as a potential reservoir of GBM biomarkers. Therefore, EV-based liquid biopsies have been suggested as a promising tool for GBM METHOD Ethical approval was obtained for a prospective study of healthy donors and consenting GBM patients at the University Hospitals Sussex (Brighton). To identify GBM specific proteins, small EVs (sEVs) were isolated from plasma samples using differential ultracentrifugation and validated through Nanoparticles tracking analysis, transmission electron microscopy and detection of known sEVs markers such as CD9, CD63, CD81 and HSP70. sEVs content was characterised through mass spectrometry and bioinformatic tools. RESULTS Our data indicate the presence of a GBM inflammatory biomarker signature comprising members of the complement and regulators of inflammation and coagulation including VWF, FCGBP, C3, PROS1, and SERPINA1. Bioinformatic analysis highlighted that all potential markers exclusively identified in patient samples had already been linked with either GBM diagnosis, prognosis or associated signalling, suggesting that sEVs protein cargo could mirror the landscape of the original tumour and that selective circulating sEV-derived proteins might be used as hallmarks for GBM patients. CONCLUSION this study is a step forward in the development of a non-invasive liquid biopsy approach for the identification of valuable biomarkers that could significantly improve GBM diagnosis and, consequently, patients’ prognosis and quality of life.

Small extracellular vesicles as a multicomponent biomarker platform in urinary tract carcinomas.

Extracellular vesicles are a large group of nano-sized vesicles released by all cells. The variety of possible cargo (mRNAs, miRNAs, lncRNAs, proteins, and lipids) and the presence of surface proteins, signaling molecules, and receptor ligands make them a rich source of biomarkers for malignancy diagnosis. One of the groups gathering the most interest in cancer diagnostic applications is small extracellular vesicles (sEVs), with ≤200 nm diameter, mainly composed of exosomes. Many studies were conducted recently, evaluating the diagnostic potential of sEVs in urinary tract carcinomas (UTCs), discovering and clinically evaluating various classes of biomarkers. The amount of research concerning different types of UTCs understandably reflects their incidence. sEV cargos getting the most interest are non-coding RNAs (miRNA and lncRNA). However, implementation of other approaches such as metabolomic and proteomic analysis is also evaluated. The results of many studies indicate that sEVs have an essential role in the cancer process and possess many possible diagnostic and prognostic applications for UTC. The relative ease of obtaining biofluids rich in sEVs (urine and blood) confirms that sEVs are essential for UTC detection in the liquid biopsy approach. A noticeable rise in research quality is observed as more researchers are aware of the research standardization necessity, which is essential for considering the clinical application of their findings.

Detection of Clinically Significant Prostate Cancer Using Targeted Biopsy with Four Cores Versus Target Saturation Biopsy with Nine Cores in Transperineal Prostate Fusion Biopsy: A Prospective Randomized Trial

BackgroundMultiparametric magnetic resonance imaging (mpMRI) and targeted biopsy (TB) facilitate accurate detection of clinically significant prostate cancer (csPC). However, it remains unclear how targeted cores should be applied for accurate diagnosis of csPC. ObjectiveTo assess csPC detection rates for two target-directed MRI/transrectal ultrasonography (TRUS) fusion biopsy approaches, conventional TB and target saturation biopsy (TS). Design, setting, and participantsThis was a prospective single-center study of outcomes for transperineal MRI/TRUS fusion biopsies for 170 men. Half of the men (n = 85) were randomized to conventional TB with four cores per lesion and half (n = 85) to TS with nine cores. Biopsies were performed by three experienced board-certified urologists. Outcome measurements and statistical analysisPC and csPC (International Society of Urological Pathology grade group ≥2) detection rates for systematic biopsy (SB), TB, and TS were analyzed using McNemar’s test for intrapatient comparisons and Fisher’s exact test for TS versus TB. A combination of targeted biopsy (TS or TB) and SB served as the reference. Results and limitationsAccording to the reference, csPC was diagnosed for 57 men in the TS group and 36 men in the TB group. Of these, TS detected 57/57 csPC cases and TB detected 33/36 csPC cases (p = 0.058). Detection of Gleason grade group 1 disease was 10/12 cases with TS and 8/17 cases with TB (p = 0.055). In addition, TS detected 97% of 63 csPC lesions, compared to 86% with TB (p = 0.1). Limitations include the single-center design, the limited generalizability owing to the transperineal biopsy route, the lack of central review of pathology and radical prostatectomy correlation, and uneven distributions of csPC prevalence, Prostate Imaging-Reporting and Data System (PI-RADS) 5 lesions, men with two or more PI-RADS ≥3 lesions, and prostate-specific antigen density between the groups, which may have affected the results. ConclusionsIn our study, rates of csPC detection did not significantly differ between TS and TB. Patient summaryIn this study, we investigated two targeted approaches for taking prostate biopsy samples after observation of suspicious lesions on prostate scans. We found that the rates of detection of prostate cancer did not significantly differ between the two approaches.

Feasibility of Leukemia-Derived Exosome Enrichment and Co-isolated dsDNA Sequencing in Acute Myeloid Leukemia Patients: A Proof of Concept for New Leukemia Biomarkers Detection

Simple SummaryThe present pilot study aimed at investigating the feasibility of a leukemia-derived exosome enrichment approach followed by exosomal dsDNA target re-sequencing for adult Acute Myeloid Leukemias (AML) marker detection. To our knowledge, this is the first time that a proof-of-concept combining a leukemia-derived exosome enrichment strategy based on a commercial CE-IVD kit and next-generation sequencing was applied in a cohort of adult AML patients. The reported approach is easy, quick and user friendly and gives the possibility of obtaining a good quantity of exosomal dsDNA (composed of exosomal cargo and surrounding DNA) suitable for further analysis. The time-effective procedure opens up future effective clinical applications. This pilot study presents the potential of a proof-of-concept based on exosome analysis to be applied in clinical practice, as well as the feasibility of this kind of investigations using a certified kit, avoiding many additional analyses. It may encourage further studies regarding extracellular vesicles in myeloid neoplasia.Exosomes are extracellular vesicles playing a pivotal role in the intercellular communication. They shuttle different cargoes, including nucleic acids from their cell of origin. For this reason, they have been studied as carriers of tumor markers in different liquid biopsy approaches, in particular for solid tumors. Few data are available concerning exosomes as markers of myeloid neoplasia. To better understand their real potential and the best approach to investigate leukemic exosomes, we present the results of a pilot feasibility study evaluating the application of next-generation sequencing analysis of dsDNA derived from exosomes isolated in 14 adult patients affected by acute myeloid leukemias. In particular, leukemia-derived exosome fractions have been analyzed. The concentration of dsDNA co-extracted with exosomes and the number and types of mutations detected were considered and compared with ones identified in the Bone Marrow (BM) and Peripheral Blood (PB) cells. Exosomal DNA concentration, both considering the cargo and the DNA surrounding the lipid membrane resulted in a linear correlation with leukemic burden. Moreover, exosomal DNA mutation status presented 86.5% of homology with BM and 75% with PB. The results of this pilot study confirmed the feasibility of a leukemia-derived exosome enrichment approach followed by exosomal dsDNA NGS analysis for AML biomarker detection. These data point to the use of liquid biopsy in myeloid neoplasia for the detection of active leukemic cells resident in the BM via a painless procedure.