Protein profile of purified plasma- and aqueous humor-derived extracellular vesicles from patients with ocular toxoplasmosis

ABSTRACT Purpose: To characterize the extracellular vesicle protein cargo in the aqueous humor and plasma of patients with ocular toxoplasmosis. Methods: Aqueous humor and plasma were collected from six patients with active ocular toxoplasmosis and six patients with cataract. Extracellular vesicles were isolated, and western blotting and mass spectrometry were performed for protein analysis. Results: All plasma samples from patients with ocular toxoplasmosis and cataract were positive for the tetraspanins CD63 and TSG101. However, the aqueous humor from patients with ocular toxoplasmosis was positive only for CD63. Sixty-seven new unreported proteins were identified in the aqueous humor and plasma of patients with the ocular toxoplasmosis and cataract. Of the 67 proteins, 10 and 7 were found only in the cataract and ocular toxoplasmosis groups, respectively. In general, these proteins were involved in immune system activation and retina homeostasis and were related to infections and retina-associated diseases. Conclusion: The distinct protein signatures between ocular toxoplasmosis and cataract may be helpful in the differential diagnosis of ocular toxoplasmosis. However, more studies are needed to better understand the role of these proteins in the pathogenesis of ocular toxoplasmosis.

A novel bailout technique using myocardial biopsy forceps to grasp a dislodged angio-seal collagen with footplate.

BACKGROUND: Hemostatic devices are now frequently used in femoral artery punctures, and the Angio-Seal (Terumo, Tokyo, Japan) is one of the most commonly used devices for closure of the femoral artery because it provides rapid hemostasis. Although device failure rarely occurs, if the collagen falls into the femoral artery, it may lead to severe limb ischemia. Herein, we describe a case of a novel endovascular technique for the treatment of Angio-Seal arterial closure device failure. CASE PRESENTATION: The patient in Case 1 was a 75-year-old man with severe left limb claudication. We used a contralateral antegrade approach and used the Angio-Seal for hemostasis. However, the Angio-Seal collagen and footplate dropped and stopped at the bifurcation of the superficial femoral artery and deep femoral artery. The collagen with the footplate was caught with myocardial biotome forceps (MBF) and pulled into the external iliac artery (EIA). The distal common femoral artery (CFA) was punctured, and we delivered a 10.0- × 80-mm stent (SMART®; Cordis, USA) to the EIA from the ipsilateral sheath. The stent was deployed at the EIA and crushed the collagen. The patient in Case 2 was an 88-year-old man with rest pain in the right limb. The right CFA was punctured using an ipsilateral approach and the Angio-Seal was used for hemostasis. The Angio-Seal collagen with the footplate dropped into the bifurcation of the deep femoral artery. The collagen and footplate were caught with MBF and pulled up to the EIA. The right CFA was punctured and a 10.0- × 60-mm stent (SMART®; Cordis) was delivered from the ipsilateral sheath. The stent was deployed at the EIA and crushed the collagen with the footplate. CONCLUSIONS: MBF were used to grasp the dislodged collagen with the anchor and cover it with a stent at the iliac artery. This may be a useful bailout technique for Angio-Seal dislodgement.

Enhanced lymphangiogenesis in the left lateral segment of a biopsied liver during portoenterostomy for biliary atresia.

PURPOSE: We investigate the histopathology of the portal vein branches and lymphatic vessels to elucidate the mechanism of atrophy of the left lateral segment (LLS) of the liver in biliary atresia (BA). METHODS: LLS and right anterior segment (RAS) liver biopsy samples obtained during Kasai portoenterostomy (KPE) from ten consecutive patients with BA underwent histopathological investigation of the portal vein and lymphatic vessels using double chromogenic immunostaining for CD31/D2-40 and the hepatitis-like findings (HLF) score. Each parameter and clinical data were compared between prognostic groups. RESULTS: HLF scores in the LLS were always higher than those in the RAS. There was no difference in portal vein and lymphatic vascular morphology, whereas the number of lymphatic vessels was correlated with the fibrotic area of all specimen areas. Left-to-right ratio of the number of lymphatic vessels was correlated with the age at KPE (r = 0.784, p = 0.007) and the pre-KPE CRP value (r = 0.723, p = 0.018). CONCLUSIONS: Lymphangiogenesis on the LLS compared to the RAS was significantly correlated with the degree of fibrosis and the age at KPE. Further investigation is warranted to clarify the causes of LLS atrophy and lymphangiogenesis relevant to immune dysregulation.

MEST C Score and Treatment Response in IgA Nephropathy in a Tertiary Care Hospital: A Descriptive Cross-sectional Study.

INTRODUCTION: IgA nephropathy is the leading cause of primary glomerulonephritis worldwide. The Oxford classification can predict IgA nephropathy prognosis through renal biopsy however its applicability to the Nepalese population remains unexplored. This study aimed to evaluate the MEST-C score and treatment response in patients with IgA nephropathy. METHODS: This descriptive cross-sectional study was conducted at a tertiary care center from November 2021 to November 2022 after obtaining ethical approval [IRC-193(6-11)t2078/079]. Total population sampling was done. Fifty-two consenting patients aged 16 or older with confirmed IgA nephropathy were included, excluding those with liver disease or expected survival of less than six months. The study assessed the MEST-C score, demographic factors, and clinical parameters. Data analysis was done using Statistical Package of Social Sciences. RESULTS: Among 52 patients with segmental glomerulosclerosis (S1), 11 (24.44%) achieved complete remission, 30 (66.67%) partial remission, and 5 (11.11%) progressed to end-stage renal disease. In those with tubular atrophy/interstitial fibrosis (T1), 1 (5.88%) achieved complete remission, 13 (76.47%) partial remission, and 4 (23.53%) progressed to end-stage renal disease. For glomerular crescents (C1), 9 (47.37%) achieved complete remission, 9 (47.37%) partial remission, and 1 (5.26%) progressed to end-stage renal disease. IFTA% of 0-25% had complete remission in 15 (46.88%). Among the two patients with IFTA% ≥50%, one (50%) developed end-stage renal disease and the other achieved partial remission. CONCLUSIONS: The S1 and T1/2 components of the MEST-C score had higher rates of partial remission and progression to end-stage renal disease, while other indices showed mixed results. The risk of failing to achieve complete increased with an IFTA of more than 25%.

Osteoblastoma of the Mandible: A Case Report.

Osteoblastoma is a rare, benign bone tumor primarily affecting the spine but can occasionally involve the jaws, particularly the mandible. This report discusses the case of a 38-year-old female patient presenting with mild pain and swelling in the left mandibular region for the past few months. Clinical, radiographic, and histopathological evaluation confirmed the diagnosis of osteoblastoma. A conservative surgical excision was performed. No recurrence was observed on follow-up. Differential diagnoses included low-grade osteosarcoma, osteoid osteoma, and other bone-forming lesions. This case report emphasizes the importance of distinguishing osteoblastoma from similar lesions to ensure appropriate treatment and favorable prognosis.

Gastric Injury in Percutaneous Transhepatic Liver Biopsy After Living Donor Liver Transplantation: A Report of Two Cases.

Percutaneous transhepatic liver biopsy (PTLB) is essential for assessing liver function but carries risks such as bleeding, cholangitis, bowel injuries, and rare fatal complications. Gastric injury following PTLB is rare and not widely reported. This report describes two cases of gastric injury during ultrasound (US)-guided PTLB in patients following living donor liver transplantation. Gastric injury is uncommon, particularly when sampling from the left lobe due to its proximity to the stomach. Ensuring a clear field of vision, meticulous equipment preparation, and skilled technique are crucial for safe PTLB. When there is a risk of gastric injury, using smaller and shorter needles or alternative methods to US-guided PTLB is essential. Gastric injury should be promptly considered and treated if multiple punctures are required and if abdominal symptoms or gastrointestinal bleeding occur after PTLB.

Impact of blastocyst grading and blastocyst biopsy dates on the clinical outcomes of patients undergoing preimplantation genetic testing.

Background: Preimplantation genetic testing (PGT) allows for the evaluation of embryo genetic information prior to implantation, enabling the selection of normal embryos for transfer and ultimately leading to better pregnancy outcomes. In this study, we explored factors that influence clinical outcomes of patients undergoing PGT. The effects of blastocyst grading and biopsy dates on clinical outcomes were also analyzed. Methods: The clinical data and pregnancy outcomes of 428 PGT cycles performed in the Reproductive Medicine Department of the Northern Theater General Hospital between January 2017 and December 2022 were retrospectively analyzed. Multifactorial logistic regression analysis and nomograms were used to determine factors influencing pregnancy outcomes. The impact of D5 blastocysts (290 cycles) and D6 blastocysts (138 cycles) with different quality levels on clinical outcomes was also compared. Results: Multifactorial logistic regression analysis showed that age, BMI, endometrial thickness, and embryo quality of women affected PGT clinical outcomes. Women aged <40 years or with a body mass index (BMI) >18.5 and endometrial thickness>1.0 cm had a significantly higher pregnancy success rate. Compared to that of D6 blastocyst biopsy, D5 blastocyst biopsy was associated with a higher pregnancy success rate but a similar live birth rate. No significant differences were observed in the pregnancy and live birth rates of D5 and D6 high-quality blastocysts. Conclusion: To achieve better pregnancy outcomes after PGT, considering blastocyst grading and biopsy dates when transferring embryos is essential for improving pregnancy outcomes. Furthermore, patients should adjust their BMI, endometrial receptivity, and endometrial thickness and pattern.

THE IMPACT OF PATIENT ANXIETY AND PAIN PERCEPTION ON THE ADEQUACY OF THYROID FINE-NEEDLE ASPIRATION BIOPSY SAMPLES: A PROSPECTIVE STUDY.

Background: Fine-needle aspiration biopsy (FNAB) is the most accurate diagnostic method to assess the malignancy risk of thyroid nodules. However, non-diagnostic results may delay diagnosis, cause unnecessary interventions, and distress patients. Aim: We aimed to determine whether a correlation exists between patients' situational anxiety, pain perception and non-diagnostic cytology results. Methods: The prospective study included patients who underwent thyroid FNAB at the Endocrinology Clinic of Sultan Abdulhamid Training and Research Hospital between 11/2022 and 02/2023. The State-Trait Anxiety Inventory (STAI) questionnaire and visual analogue scale (VAS) assessed situational anxiety and pain in patients undergoing biopsy procedures. We evaluated whether the STAI-S and VAS score is related to non-diagnostic results. Results: Of the 119 patients included in the study, 98 were female, and 21 were male. 25 (21%) nodules were non-diagnostic. The patients' mean STAI-S score before the biopsy was 47.31±12.37, and the mean VAS score after the thyroid biopsy was 2.57±1.51. A statistically significant relation was found between the patient's STAI-S score and VAS score and the cytology result of non-diagnostic (p= 0.001 and p=0.008). In univariate logistic regression, high pre-procedural anxiety (OR:3.09, 95% CI:1.07-8.94, P =0.037) and VAS score (OR:1.57, 95% CI: 1.17-2.10, P =0.002) were associated with non-diagnostic cytology. In multivariate logistic regression analysis, VAS score (OR: 1.59, 95% CI: 1.07-2.34, p=0.019) was still an independent factor related to specimen adequacy. Conclusions: Anxiety level and pain perception during FNAB may be considered risk factors for non-diagnostic cytology. Thus, reducing anxiety and pain may decrease the incidence of non-diagnostic outcomes.

PRIMARY BILATERAL ADRENAL LYMPHOMA PRESENTING WITH IMPENDING ADRENAL CRISIS.

Primary bilateral adrenal lymphoma is a very rare cause of adrenal insufficiency. We report the case of a 63-year-old woman who presented with signs and symptoms of impending adrenal crisis when referred for evaluation of large bilateral adrenal masses diagnosed on a computed tomography scan two weeks prior. Based on a high clinical suspicion of adrenal insufficiency, patient was initiated on glucocorticoid and mineralocorticoid therapy prior to laboratory confirmation of adrenal insufficiency. After stabilizing the patient and excluding pheochromocytoma, we proceeded with adrenal biopsy that revealed a nongerminal center-type diffuse large B-cell lymphoma. Our patient was treated with R-CHOP chemotherapy, with good response after 3 cycles but eventually died after the fifth cycle from neurologic complications. This case highlights the notion that primary adrenal insufficiency should be considered in patients presenting with bilateral adrenal masses. Although primary adrenal lymphoma is a very rare adrenal malignancy it should be considered in patients presenting with bilateral rapidly growing adrenal tumors and primary adrenal insufficiency.

Utilization of tissue-free minimal residual disease testing in colorectal cancer patients from Asia and Middle East.

Introduction: The presence of minimal residual disease (MRD) after curative-intent surgery for early-stage cancers is associated with disease recurrence. Circulating tumour deoxyribonucleic acid (ctDNA) has emerged as a promising biomarker for MRD assessment in patients with colorectal cancer (CRC) who have undergone surgery or completed adjuvant therapy. MRD tests are already available for use in clinics; however, treatment decisions following MRD results obtained in routine practice are infrequently described. Methods: In this observational study, we report on the real-world clinical use of Guardant Reveal, a validated tissue-free MRD assay, in the first 215 consecutive patients (279 samples) with CRC tested in Asia and the Middle East. Results: Overall, 22% of patients had ctDNA detected in their first MRD test, and the frequency of ctDNA positivity increased with increasing tumour stage. 132 samples were tested with an earlier version of Guardant Reveal, one that assessed both genomic and epigenomic features. An updated version of the assay assesses only ctDNA methylation data and was used for the remaining 147 samples. In patients with stage II CRC, 71% of tests were ordered within 12 weeks after tumour resection, while for patients with stage III disease, 69% of tests were ordered after completion of all curative-intent treatment. Discussion: Clinical cases utilizing tissue-free MRD assessment are described.

Multiple Low-Level Viraemia Suggest Hindered Liver Fibrosis Regression in Chronic Hepatitis B Patients During Antiviral Therapy.

Low-level viraemia (LLV) occurs in chronic hepatitis B (CHB) patients despite antiviral treatment, which may cause failed histological regression. Our study aimed to investigate the impact of different LLV types on fibrosis regression. The prospective study enrolled CHB patients with paired liver biopsies before and after 260 weeks of entecavir treatment. Fibrosis regression was defined by the Ishak score or P-I-R system. Patients were grouped as the SVR (HBV DNA < 20 IU/mL persistently) or LLV (HBV DNA between 20 and 2000 IU/mL), which were further grouped as very low-level viraemia (VLLV, HBV DNA < 50 IU/mL), occasionally LLV (OLLV, HBV DNA ≥ 50 IU/mL only once) and multiple LLV (MLLV, HBV DNA ≥ 50 IU/mL more than once). Logistic regression models were used to calculate the adjusted odds ratios (aORs) and 95% confidence intervals (CIs). The analysis included 111 CHB patients. In the SVR group (n = 54), 39 (72.2%) patients had fibrosis regression, which was higher than the LLV (56.1%, p = 0.080). The fibrosis regression rates for VLLV (30 patients), OLLV (17 patients) and MLLV (10 patients) were 70.0%, 52.9% and 30.0%, respectively. Compared with SVR, VLLV (aOR = 0.78; 95% CI: 0.28-2.21; p = 0.644) was not associated with fibrosis regression, but patients with non-VLLV (aOR = 0.27; 95% CI: 0.09-0.85; p = 0.025), especially with MLLV (aOR = 0.19; 95% CI: 0.04-0.97; p = 0.046) is significantly associated with hindered fibrosis regression. Our study suggests that patients with detectable serum HBV DNA levels higher than 50 IU/mL need to be monitored carefully, especially in those with more than once. Trial Registration: ClinicalTrials.gov identifiers NCT01938781 and NCT01938820.

Cellular liquid biopsy provides unique chances for disease monitoring, preclinical model generation and therapy adjustment in rare salivary gland cancer patients.

While cell-free liquid biopsy (cfLB) approaches provide simple and inexpensive disease monitoring, cell-based liquid biopsy (cLB) may enable additional molecular genetic assessment of systemic disease heterogeneity and preclinical model development. We investigated 71 blood samples of 62 patients with various advanced cancer types and subjected enriched circulating tumor cells (CTCs) to organoid culture conditions. CTC-derived tumoroid models were characterized by DNA/RNA sequencing and immunohistochemistry, as well as functional drug testing. Results were linked to molecular features of primary tumors, metastases, and CTCs; CTC enumeration was linked to disease progression. Of 52 samples with positive CTC counts (≥1) from eight different cancer types, only CTCs from two salivary gland cancer (SGC) patients formed tumoroid cultures (P = 0.0005). Longitudinal CTC enumeration of one SGC patient closely reflected disease progression during treatment and revealed metastatic relapse earlier than clinical imaging. Multiomics analysis and functional in vitro drug testing identified potential resistance mechanisms and drug vulnerabilities. We conclude that cLB might add a functional dimension (to the genetic approaches) in the personalized management of rare, difficult-to-treat cancers such as SGC.

The nodule-pleura relationship affects pneumothorax in CT-guided percutaneous transthoracic needle biopsy: avoiding to cross pleural tail sign may reduce the incidence of pneumothorax.

OBJECTIVES: To explore the role of nodule-pleural relationship, including nodule with pleural tail sign (PTS), nodule with pleural contact and nodule with pleural unrelated in CT-guided percutaneous transthoracic needle biopsy (PTNB)-induced pneumothorax, and whether employing different puncture routes has an impact on the incidence of pneumothorax in PTNB of nodules with PTS. METHODS: Between April 1, 2019, to June 30, 2021, 775 consecutive PTNB procedures of pulmonary nodules in the Peking University Cancer Hospital were retrospectively reviewed. The univariate and multivariate regression analysis were used to identify the risk factors for pneumothorax in PTNB. RESULTS: The nodule with pleural contact group has a lower incidence of pneumothorax than the nodule with PTS group (p = 0.001) and the nodule with pleural unrelated group (p = 0.002). It was observed that a higher incidence of pneumothorax caused by crossing PTS compared with no crossing PTS (p < 0.001). Independent risk factors for pneumothorax included crossing PTS (p < 0.001), perifocal emphysema (p < 0.001), biopsy side up (p < 0.001), longer puncture time (p < 0.001), deeper needle insertion depth (intrapulmonary) (p < 0.001) and nodules in the middle or lower lobe (p = 0.007). CONCLUSION: Patients with crossing PTS, a nodule in the middle or lower lobe, longer puncture time, biopsy side up, deeper needle insertion depth (intrapulmonary), and perifocal emphysema were more likely to experience pneumothorax in PTNB. When performing the biopsy on a nodule with PTS, selecting a route that avoids crossing through the PTS may be advisable to reduce the risk of pneumothorax.

Impacts of double biopsy and double vitrification on the clinical outcomes following euploid blastocyst transfer: a systematic review and meta-analysis.

STUDY QUESTION: Compared to the 'single biopsy + single vitrification' approach, do 'double biopsy + double vitrification' or 'single biopsy + double vitrification' arrangements compromise subsequent clinical outcomes following euploidy blastocyst transfer? SUMMARY ANSWER: Both 'double biopsy + double vitrification' and 'single biopsy + double vitrification' led to reduced live birth/ongoing pregnancy rates and clinical pregnancy rates. WHAT IS KNOWN ALREADY?: It is not uncommon to receive inconclusive results following blastocyst biopsy and preimplantation genetic testing for aneuploidy (PGT-A). Often these blastocysts are warmed for re-test after a second biopsy, experiencing 'double biopsy + double vitrification'. Furthermore, to achieve better workflow, IVF laboratories may choose to routinely vitrify all blastocysts and schedule biopsy at a preferred timing, involving 'single biopsy + double vitrification'. However, in the current literature, there is a lack of systematic evaluation of both arrangements regarding their potential clinical risks in reference to the most common 'single biopsy + single vitrification' approach. STUDY DESIGN, SIZE, DURATION: A systematic review and meta-analysis were performed, with the protocol registered in PROSPERO (CRD42023469143). A search in PUBMED, EMBASE, and the Cochrane Library for relevant studies was carried out on 30 August 2023, using the keywords 'biopsy' and 'vitrification' and associated variations respectively. Only studies involving frozen transfers of PGT-A tested euploid blastocysts were included, with those involving PGT-M or PGT-SR excluded. PARTICIPANTS/MATERIALS, SETTING, METHODS: Study groups included blastocysts having undergone 'double biopsy + double vitrification' or 'single biopsy + double vitrification', with a 'single biopsy + single vitrification' group used as control. The primary outcome was clinical pregnancy, while secondary outcomes included live birth/ongoing pregnancy, miscarriage, and post-warming survival rates. Random effects meta-analysis was performed with risk ratios (RR) and 95% CIs were used to present outcome comparisons. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 607 records were identified through the initial search and nine studies (six full articles and three abstracts) were eventually included. Compared to 'single biopsy + single vitrification', 'double biopsy + double vitrification' was associated with reduced clinical pregnancy rates (six studies, n = 18 754; RR = 0.80, 95% CI = 0.71-0.89; I2 = 0%) and live birth/ongoing pregnancy rates (seven studies, n = 20 964; RR = 0.72, 95% CI = 0.63-0.82; I2 = 0%). However, no significant changes were seen in miscarriage rates (seven studies, n = 22 332; RR = 1.40, 95% CI = 0.92-2.11; I2 = 53%) and post-warming survival rates (three studies, n = 13 562; RR = 1.00, 95% CI = 0.99-1.01; I2 = 0%) following 'double biopsy + double vitrification'. Furthermore, 'single biopsy + double vitrification' was also linked with decreased clinical pregnancy rates (six studies, n = 13 284; RR = 0.84, 95% CI = 0.76-0.92; I2 = 39%) and live birth/ongoing pregnancy rates (seven studies, n = 16 800; RR = 0.79, 95% CI = 0.69-0.91; I2 = 70%), and increased miscarriage rates (five studies, n = 15 781; RR = 1.48, 95% CI = 1.31-1.67; I2 = 0%), but post-warming survival rates were not affected (three studies, n = 12 452; RR = 0.99, 95% CI = 0.97-1.01; I2 = 71%) by 'single biopsy + double vitrification'. LIMITATIONS, REASONS FOR CAUTION: All studies included in this meta-analysis were retrospective with varying levels of heterogeneity for different outcomes. Not all studies had accounted for potential confounding factors. Only one study reported neonatal outcomes. WIDER IMPLICATIONS OF THE FINDINGS: Our data indicated adverse impacts of 'double biopsy + double vitrification' and 'single biopsy + double vitrification' on clinical outcomes following euploid blastocyst transfers. Patients should be carefully consulted about the risks when offered such approaches. The biopsy process should be carried out as carefully and competently as possible to minimize an inconclusive diagnosis. STUDY FUNDING/COMPETING INTEREST(S): R.W. is supported by a National Health and Medical Research Council Emerging Leadership Investigator Grant (2009767). There is no other external funding to report. All authors report no conflict of interest. REGISTRATION NUMBER: CRD42023469143.

Intravascular large B-cell lymphoma with respiratory failure diagnosed by random skin biopsy: A case report.

Intravascular large B-cell lymphoma (IVLBCL) typically involves nonspecific symptoms that complicate diagnosis. This report discusses the case of a 70-year-old man, who presented with dyspnea, fatigue, and weight loss that evolved into severe respiratory failure, diagnosed with IVLBCL via random skin biopsy. The initial improvement in respiratory symptoms was followed by coronavirus disease. Response to steroid therapy and elevated lactate dehydrogenase levels suggested IVLBCL, confirmed by a random skin biopsy. The combination chemotherapy of rituximab, cyclophosphamide, vincristine, doxorubicin, and prednisolone improved the respiratory condition. This case highlights the diagnostic challenges associated with IVLBCL and the crucial role of random skin biopsy.

Celiac Disease Masking Duodenal Adenocarcinoma: A Case Report and Literature Review.

Small bowel adenocarcinoma (SBA) is an uncommon gastrointestinal malignancy, with the duodenum being the most commonly affected site. This report describes a 33-year-old woman who initially presented with abdominal pain and vomiting. Initial imaging revealed abnormalities of the proximal jejunum. Endoscopic evaluation initially revealed celiac disease (CD); however, with disease progression, the patient developed bowel obstruction that led to surgical intervention with an open duodenal biopsy. The open duodenal biopsy confirmed the presence of duodenal adenocarcinoma (DA). This case demonstrates the diagnostic complexity of DA in the presence of concurrent CD due to overlapping presentations. Physicians must maintain a high suspicion of DA in the setting of progressive and difficult-to-treat CD, as early diagnosis and management of DA can significantly improve patient outcomes.

A novel liquid biopsy assay for detection of ERBB2 (HER2) amplification in circulating tumor cells (CTCs).

Purpose: Circulating tumor cell (CTC)-based ERBB2 (HER2) assay is a laboratory test developed by Epic Sciences using single-cell genomics to detect ERBB2 (HER2) amplification in CTCs found in the peripheral blood of metastatic breast cancer (MBC) patients. Patients and methods: Peripheral blood was collected in Streck tubes and centrifugation was used to remove plasma and red blood cells. The remaining nucleated cells were deposited on glass slides, immunofluorescent-stained with proprietary antibodies, scanned by a high-definition digital scanner, and analyzed by a proprietary algorithm. In addition, single-cell genomics was performed on selected CTC. Analytical validation was performed using white blood cells from healthy donors and breast cancer cell lines with known levels of ERBB2 amplification. Clinical concordance was assessed on MBC patients whose blood was tested by the CTC ERBB2 (HER2) assay and those results are compared to results of matched metastatic tissue biopsy (immunohistochemistry [IHC] 3+ or IHC2+/in situ hybridization [ISH+]). Results: Epic's ERBB2 (HER2) assay detected 2-fold ERBB2 amplification with 85% sensitivity and 94% specificity. In the clinical concordance study, among the 50% of the cases that had ERBB2 status results from CTCs found to be chromosomally-unstable, the CTC ERBB2 (HER2) assay showed sensitivity of 69% and specificity of 78% when compared to HER2 status by metastatic tissue biopsy. Conclusions: The CTC ERBB2 (HER2) assay can consistently detect ERBB2 status in MBC cell lines and in the population of patients with MBC with detectable chromosomally unstable CTCs for whom tissue biopsy is not available or is infeasible.

Usefulness of multigene liquid biopsy of bile for identifying driver genes of biliary duct cancers.

Liquid biopsy (LB) is an essential tool for obtaining tumor-derived materials with minimum invasion. Bile has been shown to contain much higher free nucleic acid levels than blood plasma and can be collected through endoscopic procedures. Therefore, bile possesses high potential as a source of tumor derived cell-free DNA (cfDNA) for bile duct cancers. In this study, we show that a multigene panel for plasma LB can also be applied to bile cfDNA for comparing driver gene mutation detection in other sources (plasma and tumor tissues of the corresponding patients). We collected cfDNA samples from the bile of 24 biliary tract cancer cases. These included 17 cholangiocarcinomas, three ampullary carcinoma, two pancreatic cancers, one intraductal papillary mucinous carcinoma, and one insulinoma. Seventeen plasma samples were obtained from the corresponding patients before surgical resection and subjected to the LiquidPlex multigene panel LB system. We applied a machine learning approach to classify possible tumor-derived variants among the prefiltered variant calls by a LiquidPlex analytical package with high fidelity. Among the 17 cholangiocarcinomas, we could detect cancer driver mutations in the bile of 10 cases using the LiquidPlex system. Of the biliary tract cancer cases examined with this method, 13 (54%) and 4 (17%) resulted in positive cancer driver mutation detection in the bile and plasma cfDNAs, respectively. These results suggest that bile is a more reliable source for LB than plasma for multigene panel analyses of biliary tract cancers.

Trends and efficacy of omitting axillary lymph node dissection in early-stage male breast cancer with limited nodal involvement: A population-based cohort study.

BACKGROUND: The effectiveness of sentinel lymph node biopsy (SLNB) versus axillary lymph node dissection (ALND) in managing early-stage male breast cancer (MBC) patients with T1-2 tumors and limited lymph node metastasis, all receiving radiotherapy, remains uncertain. This study examines trends and survival outcomes for SLNB and ALND in the United States. METHODS: We conducted a retrospective analysis of the Surveillance, Epidemiology, and End Results (SEER) data from 2010 to 2020 for MBC patients with T1-2 tumors and 1-2 positive lymph nodes undergoing radiotherapy. Patients were classified by nodes removed (SLNB ≤5, ALND ≥10), comparing overall survival (OS) and breast cancer-specific survival (BCSS) between the groups before and after propensity score matching. RESULTS: Of 299 MBC patients analyzed, SLNB usage increased from 18.8% in 2010 to 61.0% in 2020. Multivariable logistic regression highlighted significant associations of SLNB use with diagnosis year, race, surgery type, positive lymph node count, and metastasis size. No significant differences in 5-year OS (77.98% SLNB vs. 85.85% ALND, p = 0.337) or BCSS (91.54% SLNB vs. 94.97% ALND, p = 0.214) were observed. Propensity score matching (96 patients per group) confirmed similar 5-year OS (83.9% for SLNB vs. 82.0% for ALND, p = 0.925) and BCSS (90.1% for SLNB vs. 96.9% for ALND, p = 0.167). CONCLUSION: SLNB and ALND provide comparable survival outcomes in early-stage MBC patients with limited lymph node metastasis undergoing radiotherapy. The increased utilization of SLNB supports its consideration to reduce surgical morbidity in selected MBC patients despite limited direct evidence.

Rapid Assessment of Biomarkers on Single Extracellular Vesicles Using "Catch and Display" on Ultrathin Nanoporous Silicon Nitride Membranes.

Extracellular vesicles (EVs) are particles released from cells that facilitate intercellular communication and have tremendous diagnostic and therapeutic potential. Bulk assays lack the sensitivity to detect rare EV subsets relevant to disease, and while single EV analysis techniques remedy this, they are often undermined by complicated detection schemes and prohibitive instrumentation. To address these issues, a microfluidic technique for EV characterization called "catch and display for liquid biopsy (CAD-LB)" is proposed. In this method, minimally processed samples are pipette-injected and fluorescently labeled EVs are captured in the nanopores of an ultrathin membrane.  This enables the rapid assessment of EV number and biomarker colocalization by light microscopy. Here, nanoparticles are used to define the accuracy and dynamic range for counting and colocalization. The same assessments are then made for purified EVs and for unpurified EVs in plasma. Biomarker detection is validated using CD9 and Western blot analysis to confirm that CAD-LB accurately reports relative protein expression levels. Using unprocessed conditioned media, CAD-LB captures the known increase in EV-associated ICAM-1 following endothelial cell cytokine stimulation. Finally, to demonstrate CAD-LB's clinical potential, EV biomarkers indicative of immunotherapy responsiveness are successfully detected in the plasma of bladder cancer patients treated with immune checkpoint blockade.