Cancer Biopsies Research Articles

Abstract PR014: DNA associated with EVs is uniquely chromatinized and prevents metastasis by enhancing anti-tumor immunity

Abstract Cells release extracellular vesicles (EVs) loaded with functional biomolecules that facilitate intercellular communication in health and disease. These EVs contain DNA (EV-DNA), mirroring the mutational profile of parental cells, yet the mechanism of DNA packaging onto EVs and its role in diseases like cancer remain unclear. Using advanced imaging and biochemical techniques, we discovered that EV-DNA predominantly resides on the vesicle surface, bound to modified histones. Through genome-wide CRISPR knockout screens in cancer cell lines, we identified immune-related pathways and genes crucial for EV-DNA loading, such as APAF1 and NCF1. In several cancer models, loss of APAF1 reduced EV-DNA packaging, and enhancing metastasis, thus implicating EV-DNA in immune surveillance against cancer spread. We demonstrated that EV-DNA uptake by liver macrophages triggers DNA damage response, altering cytokine production and promoting immune activation in pre-metastatic sites. Moreover, quantifying EV-DNA in colorectal cancer biopsies revealed its potential as a predictive metastatic risk biomarker. Our findings shed light on EV-DNA packaging mechanisms and its significance, suggesting that elevated levels of tumor-derived EV-DNA bolster anti-tumor immunity and inhibit metastasis. Citation Format: Inbal Wortzel, Han-Sang Kim, David C. Lyden. DNA associated with EVs is uniquely chromatinized and prevents metastasis by enhancing anti-tumor immunity [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr PR014.

Comprehensive characterization of invasive mammary carcinoma with lobular features: integrating morphology and E-cadherin immunohistochemistry patterns.

Breast cancer treatment prioritizes molecular subtypes over histologic types. However, considering the unique biological behavior of invasive lobular carcinoma (ILC), its diagnosis is crucial for patient management. Therefore, this study aimed to review breast cancer cases, focusing on the E-cadherin patterns and lobular morphology of cases misclassified in the original reports. A comprehensive review was conducted on 481 breast cancer biopsy cases diagnosed as invasive breast carcinoma of no special type (IBC-NST) or ILC with E-cadherin staining. These cases were categorized into six groups based on tumor morphology (ductal/lobular) and E-cadherin expression pattern (membranous/loss/aberrant): (1) ductal/membranous, (2) lobular/loss, (3) lobular/aberrant, (4) mixed, (5) ductal/loss or aberrant, and (6) lobular/membranous. In 211 cases (43.8%), an E-cadherin pattern indicating ILC (loss and aberrant) was observed alongside lobular morphology, representing 5.52% of all breast cancer biopsies during the relevant period. Moreover, 181 cases (37.6%) showed a membranous pattern with ductal morphology, 4 (0.8%) were mixed IBC-NST and ILC, and 85 (17.7%) exhibited discordance between morphology and E-cadherin expression. Notably, only 25.9% (15/58) of cases in group 3, characterized by aberrant E-cadherin patterns, were initially diagnosed as ILC, highlighting a significant diagnostic discrepancy. In group 6, where membranous E-cadherin pattern was present with lobular morphology, only 3.4% (2/58) were diagnosed as ILC in the original reports, indicating diagnostic challenges in morphology and immunohistochemistry discordance. Similarly, in group 5, which had ductal morphology with loss or aberrant E-cadherin expression, the initial diagnosis rate of IBC-NST was 33.3% (9/27), reflecting the complexities in interpreting discordant cases. In real-world practice, diagnosing ILC often heavily depends on E-cadherin results. This study emphasizes the need for diagnostic clarification in cases with discordance between morphology and E-cadherin patterns.

Modern Endoscopic Approaches to Video-Assisted Sentinel Lymph Node Biopsy and Axillary Lymph Node Dissection: A Review

This article aims to analyze modern endoscopic techniques of sentinel lymph node biopsy and axillary lymph node dissection in breast cancer surgery.Owing to more accurate diagnosis and minimized patient morbidity and risks, endoscopic techniques have significantly expanded capabilities of breast cancer surgery.We review the main endoscopic techniques of sentinel lymph node biopsy and axillary lymph node dissection, analyze their efficacy and safety, and compare their benefits with those of conventional open techniques.The article discusses results of recent studies regarding diagnostic accuracy and complication rates. Furthermore, we discuss the impact of endoscopic axillary lymph node dissection on the prognosis and quality of life in patients with breast cancer.

Highly sensitive deep panel sequencing of 27 HPV genotypes in prostate cancer biopsies results in very low detection rates and indicates that HPV is not a major etiological driver of this malignancy.

Human papillomavirus (HPV) has been proposed to contribute to the carcinogenesis of prostate cancer. However, previous studies have yielded conflicting results. This study aims to add useful information to the ongoing discussion concerning the association between HPV infection and prostate cancer. We used two high-throughput next-generation sequencing (NGS) approaches to detect HPV RNA in malignant and adjacent normal (AN) prostate tissue (cohorts 1 and 2) and HPV DNA from carcinogenic and probably/possibly carcinogenic-classified HPV types (cohort 3) in malignant prostate, AN prostate, and benign prostatic hyperplasia (BPH) tissues. In total, 0% (cohort 1: 0/83, cohort 2: 0/16) of the malignant prostate tissue samples and 0% (cohort 1: 0/23, cohort 2: 0/8) of the AN prostate tissue samples were positive for HPV RNA. A total of 8.3% (1/12) of the BPH samples, 0% (0/28) of the AN samples, and 0.8% (1/132) of the malignant prostate samples were positive for HPV16 DNA. However, the normalized read count of the HPV16-positive malignant sample was close to the cut-off. In addition, no other carcinogenic-classified HPV types were detected in any of the BPH, AN, or malignant prostate tissue samples. Our study does not support HPV infection as a major contributor to the etiology of prostate cancer.

Abstract IA018: Liquid biopsy in head and neck cancers: A model for tissue-agnostic MRD detection

Abstract Head and neck cancer (HNC) is a molecularly heterogenous disease with a variable clinical course. Current treatment decisions rely primarily on pre-treatment prognostic factors without consideration of treatment response. This presentation will introduce liquid biopsy biomarkers that can be repeated in HNC patients for the purpose of dynamic risk stratification, treatment adaptation, and post-treatment surveillance. Novel approaches to measuring circulating tumor DNA (ctDNA) will be described, with particular attention to viral-associated and non-viral- associated subtypes of HNC. Detection of molecular residual disease (MRD) using ctDNA will be discussed. As HNC is often treated by non-surgical means, there is a pressing need for tissue- agnostic methods for ctDNA-based MRD detection. Analysis of tumor-specific genetic and epigenetic aberrations within ctDNA each present distinct opportunities and challenges for tissue-agnostic characterization of ctDNA. Viral ctDNA has emerged as a convenient target for MRD detection in viral-associated HNC, while ctDNA methylome profiling offers a versatile approach for tissue-agnostic MRD detection across HNC subtypes. Future applications of these methods may improve individualized management of HNC patients without requiring access to precious tumor tissue samples. Citation Format: Scott V Bratman. Liquid biopsy in head and neck cancers: A model for tissue-agnostic MRD detection [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr IA018.

Unveiling the influence of tumor and immune signatures on immune checkpoint therapy in advanced lung cancer.

This study investigates the variability among patients with non-small cell lung cancer (NSCLC) in their responses to immune checkpoint inhibitors (ICIs). Recognizing that patients with advanced-stage NSCLC rarely qualify for surgical interventions, it becomes crucial to identify biomarkers that influence responses to ICI therapy. We conducted an analysis of single-cell transcriptomes from 33 lung cancer biopsy samples, with a particular focus on 14 core samples taken before the initiation of palliative ICI treatment. Our objective was to link tumor and immune cell profiles with patient responses to ICI. We discovered that ICI non-responders exhibited a higher presence of CD4+ regulatory T cells, resident memory T cells, and TH17 cells. This contrasts with the diverse activated CD8+ T cells found in responders. Furthermore, tumor cells in non-responders frequently showed heightened transcriptional activity in the NF-kB and STAT3 pathways, suggesting a potential inherent resistance to ICI therapy. Through the integration of immune cell profiles and tumor molecular signatures, we achieved an discriminative power (area under the curve [AUC]) exceeding 95% in identifying patient responses to ICI treatment. These results underscore the crucial importance of the interplay between tumor and immune microenvironment, including within metastatic sites, in affecting the effectiveness of ICIs in NSCLC.

Unveiling the influence of tumor and immune signatures on immune checkpoint therapy in advanced lung cancer

This study investigates the variability among patients with non-small cell lung cancer (NSCLC) in their responses to immune checkpoint inhibitors (ICIs). Recognizing that patients with advanced-stage NSCLC rarely qualify for surgical interventions, it becomes crucial to identify biomarkers that influence responses to ICI therapy. We conducted an analysis of single-cell transcriptomes from 33 lung cancer biopsy samples, with a particular focus on 14 core samples taken before the initiation of palliative ICI treatment. Our objective was to link tumor and immune cell profiles with patient responses to ICI. We discovered that ICI non-responders exhibited a higher presence of CD4+ regulatory T cells, resident memory T cells, and TH17 cells. This contrasts with the diverse activated CD8+ T cells found in responders. Furthermore, tumor cells in non-responders frequently showed heightened transcriptional activity in the NF-kB and STAT3 pathways, suggesting a potential inherent resistance to ICI therapy. Through the integration of immune cell profiles and tumor molecular signatures, we achieved an discriminative power (area under the curve [AUC]) exceeding 95% in identifying patient responses to ICI treatment. These results underscore the crucial importance of the interplay between tumor and immune microenvironment, including within metastatic sites, in affecting the effectiveness of ICIs in NSCLC.

Sentinel lymph node biopsy in cT1-2N0 minor salivary gland cancer in oral cavity

ObjectiveTo evaluate the efficacy of sentinel lymph node biopsy (SLNB) in cT1/2N0 minor salivary gland cancer (mSGC) located within the oral cavity.MethodsA retrospective analysis was conducted on patients diagnosed with cT1/2N0 oral mSGC, who were categorized into two groups based on neck management approaches. The impact of SLNB versus observation on regional control and overall survival was assessed using a Cox model.ResultsA total of 177 patients were included in the study, with 53 cases undergoing SLNB. All patients had at least one sentinel lymph node, with the majority having two sentinel lymph nodes. The sentinel lymph nodes were predominantly situated in level I, followed by level II. Four patients had positive sentinel lymph nodes, all of whom had primary tumors in the tongue or the floor of the mouth, and were classified as cT2 stage. This yielded a sensitivity and specificity of 100%, a false negative rate of 0%, and a negative predictive value of 100% for SLNB in predicting occult metastasis. In terms of regional control, SLNB exhibited a reduced hazard ratio of 0.90 (95% confidence interval: 0.64–0.96) compared to observation. However, SLNB did not confer a superior overall survival benefit compared to observation.ConclusionIn patients with cT1/2N0 oral mSGC, SLNB proved to be both technically feasible and oncologically safe. When contrasted with observation, SLNB was associated with enhanced regional control, particularly recommending its use for cases of cT2 mSGC arising from the tongue or the floor of the mouth.

079. The Diagnostic Duel: Single Immunohistochemistry vs. in Combination with Hematoxylin Eosin in Sentinel Lymph Node Biopsy for Breast Cancer—Insights from a Meta-Analysis

Background: The timing of sentinel lymph node biopsy (SLNB) has gained significance with the increased use of neoadjuvant chemotherapy (NAC) in breast cancer. Hematoxylin and eosin (H&E) is the standard histological stain, yet the optimal histopathological protocol for SLNB remains unclear. The role of immunohistochemistry (IHC) in enhancing diagnostic accuracy is still debated. This study evaluates the false-negative rate (FNR) of SLNB in clinically node-negative breast cancer patients after NAC, comparing IHC alone versus IHC combined with H&E staining through a systematic review and meta-analysis. Methods: We conducted a comprehensive search of PubMed, Embase, and the Cochrane Library from January 2002 to August 2024, focusing on patients undergoing SLNB after NAC followed by axillary lymph node dissection (ALND). Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using a random-effects model via Review Manager Software version 5.3. Results: A total of 1,456 patients across 16 studies were analyzed. The FNRs varied from 0% to 33%. The estimated FNR was 1.55 (95% CI: 0.71-3.38) for studies using IHC, while the pooled FNR for H&E alone was 11% (95% CI: 4%-18%) compared to 4% (95% CI: 1%-7%) for H&E with IHC. No significant difference in FNR was observed between the two staining methods (P = 0.28). Conclusion: IHC combined with H&E is technically feasible and offers comparable accuracy for axillary staging in initially clinically node-negative breast cancer patients post-NAC.

P042 Omission of contralateral systematic biopsies in unilateral suspicious prostate cancer on magnetic resonance imaging: Implications for radiotherapeutical treatment planning

P042 Omission of contralateral systematic biopsies in unilateral suspicious prostate cancer on magnetic resonance imaging: Implications for radiotherapeutical treatment planning

A machine learning-based analysis for the definition of an optimal renal biopsy for kidney cancer

ObjectiveRenal Tumor biopsy (RTB) can assist clinicians in determining the most suitable approach for treatment of renal cancer. However, RTB's limitations in accurately determining histology and grading have hindered its broader adoption and data on the concordance rate between RTB results and final pathology after surgery are unavailable. Therefore, we aimed to develop a machine learning algorithm to optimize RTB technique and to investigate the degree of concordance between RTB and surgical pathology reports. Materials and methodsWithin a prospectively maintained database, patients with indeterminate renal masses who underwent RTB at a single tertiary center were identified. We recorded and analyzed the approach (US vs. CT), the number of biopsy cores (NoC), and total core tissue length (LoC) to evaluate their impact on diagnostic outcomes. The K-Nearest Neighbors (KNN), a non-parametric supervised machine learning model, predicted the probability of obtaining pathological characterization and grading. In surgical patients, final pathology reports were compared with RTB results. ResultsOverall, 197 patients underwent RTB. Overall, 89.8% (n=177) and 44.7% (n=88) of biopsies were informative in terms of histology and grading, respectively. The discrepancy rate between the pathology results from renal tissue biopsy (RTB) and the final pathology report following surgery was 3.6% (n=7) for histology and 5.0% (n=10) for grading. According to the machine learning model, a minimum of 2 cores providing at least 0.8 cm of total tissue should be obtained to achieve the best accuracy in characterizing the cancer. Alternatively, in cases of RTB with more than two cores, no specific minimum tissue threshold is required. ConclusionsThe discordance rates between RTB pathology and final surgical pathology are notably minimal. We defined an optimal renal biopsy strategy based on at least 2 cores and at least 0.8 cm of tissue or at least 3 cores and no minimum tissue threshold. Patients summaryRTB is a useful test for kidney cancer, but it's not always perfect. Our study shows that it usually matches up well with what doctors find during surgery. Using machine learning can make RTB even better by helping doctors know how many samples to take. This helps doctors treat kidney cancer more accurately.

Exploring intratumoral budding in colorectal cancer using computational pathology: a biopsy- based evaluation

Due to insufficient evidence, tumor budding (TB) is not currently evaluated in colorectal cancer (CRC) biopsies. This study investigates TB in CRC by establishing the value of intratumoral budding (ITB) in resection specimens and assessing the feasibility and clinical value of TB in biopsies. TB was assessed using an algorithm in all cases. In a test cohort of 555 primarily surgically treated CRC patients, we assessed the prognostic impact of ITB compared to peritumoral budding (PTB). The distribution of ITB in the uppermost five millimeters of resection specimens was analyzed to validate TB counting in biopsies. We further validated the prognostic and predictive impact of TB in biopsies of 285 rectal cancer patients, focusing on overall survival and response to neoadjuvant therapy. High-grade TB, whether intratumoral or peritumoral and in biopsies or resections, was associated with advanced pathological stage, lymphatic invasion, infiltrative tumor border, and poor overall survival in the test cohort. Superficial ITBs (0-3 mm from the lumen) accurately predicted the final TB grade based on PTB in 87% of tumors, with 87% of tumors having at least one superficial ITB hotspot. ITB (hazard ratio 3.5, 95% CI 1.1-10.8) was an independent predictor of overall survival, unlike PTB. In the validation cohort, TB presence in biopsies significantly reduced the likelihood of achieving a pathological complete response (odds ratio 0.3, 95% CI 0.1-0.7, p=0.007). ITB is as prognostic as PTB, and evaluating both can improve risk stratification in CRC. TB assessment in biopsies can identify poor prognosis and predict response to neoadjuvant therapy.

Persistence of activated anti-mesothelin hYP218 chimeric antigen receptor T cells in the tumour is associated with efficacy in gastric and colorectal carcinomas.

Patients with advanced gastric and colorectal cancers have limited treatment options. Since mesothelin is highly expressed in these tumour types, we evaluated the therapeutic benefits of anti-mesothelin hYP218 CAR T cells alone, and in combination with anti-PD1 antibody, pembrolizumab. GEPIA analysis was performed using human gastric (n=408) and colon cancer tumours (n=275) in TCGA database, to evaluate mRNA expression of mesothelin, compared to normal tissues. Mesothelin expression in gastric and colorectal cancer cell-lines (n=5) was analysed using flow cytometry. In vitro efficacy by hYP218 CAR T cells was tested by cytotoxicity and cytokine release assays. In vivo anti-tumour efficacy of hYP218 CAR T cells alone, and in combination with pembrolizumab, was evaluated in NSG mice bearing human gastric (HGC27) and colorectal (SW48) tumour xenografts. Additionally, hYP218 CAR-T cell persistence, activation and exhaustion marker-expression were studied. Mesothelin expression was significantly higher in gastric and colon cancer biopsies compared to normal tissues (p<.005). Mesothelin expression in gastric and colon cancer cell lines ranged from 10000 to 70000 molecules per cell. hYP218 CAR T cells demonstrated strong cytotoxic activity at low effector to target ratio, ranging from 0.24 to 1.0. In NSG mouse-models, hYP218 CAR T cells demonstrated anti-tumour efficacy and persisted in the tumour microenvironment in a functional state at day 40 posttreatment with expression of activation markers CD39 and CD69, increased production of IFN-γ and TNF-α and ability to kill tumour cells in vitro when isolated from tumours. There was increased PD1 expression. In combination with pembrolizumab, hYP218 CAR T cells led to slower tumour growth in NSG mice bearing large but not small HGC27 tumours. Anti-tumour efficacy of hYP218 CAR T cells is due to increased accumulation of activated CAR T cells in the tumour and combination with pembrolizumab resulted in improvement in anti-tumour activity of large established tumours. HIGHLIGHTS: Mesothelin expression is significantly higher in gastric and colorectal cancers than normal tissues. hYP218 CAR T cells demonstrate strong anti-tumour activity against mesothelin-positive gastric and colorectal carcinomas. Activated hYP218 CAR T cells persist in the tumour microenvironment and retain their cytotoxic activity. Addition of pembrolizumab in larger tumours enhance CAR T cell efficacy.

Biomarker Study for Selecting Neoadjuvant Chemotherapy Regimens Based on Prognostic Prediction Using Gastric Cancer Biopsy Specimens from a Phase II Randomized Controlled Trial.

The randomized phase II COMPASS trial revealed that neither the regimen nor the number of courses of preoperative neoadjuvant chemotherapy (NAC) for locally advanced gastric cancer (GC) significantly influence overall survival (OS). However, the impact of NAC regimens on OS may vary from patient to patient. The aim of this study was to identify biomarkers that can predict more appropriate individualized NAC regimens for improved prognosis using biopsy specimens from the COMPASS trial. RNA was extracted from endoscopic biopsy specimens of primary tumors obtained prior to NAC and real-time PCR analysis of 127 genes was conducted to identify those significantly affecting survival in the context of specific NAC regimens. THBS1, MSI1, and IGF2BP3 were identified as significant factors for stratifying survival among different NAC regimens, with statistically significantly interaction p values. Immunohistochemical analysis confirmed that the protein levels of THBS1, MSI1, and IGF2BP3 strongly correlated with their gene expression levels, validating these proteins as reliable biomarkers. This study effectively identified THBS1, MSI1, and IGF2BP3 as promising biomarkers for personalizing NAC regimens in patients with locally advanced GC. By tailoring NAC based on these biomarkers, it is possible to enhance survival outcomes and advance personalized treatment strategies. The findings underscore the potential for incorporating biomarker-guided approaches into clinical trials, aiming to refine and optimize NAC regimens for improved patient-specific treatment efficacy.

Sentinel Lymph Node Biopsy in Breast Cancer Using Different Types of Tracers According to Molecular Subtypes and Breast Density-A Randomized Clinical Study.

Background: Sentinel lymph node biopsy (SLNB) has become a method more and more frequently used in loco-regional breast cancer in the initial stages. Starting from the first report on the technical feasibility of the sentinel node method in breast cancer, published by Krag (1993) and Giuliano (1994), the method underwent numerous improvements and was also largely used worldwide. Methods: This article is a prospective study that took place at the "SJUPBT Surgery Clinic Timisoara" over a period of 1 year between July 2023 and July 2024, during which 137 underwent sentinel lymph node biopsy (SLNB) based on the current guidelines. For the identification of sentinel lymph nodes, we used various methods, including single traces and also a dual tracer and triple tracer. Results: Breast density represents a predictive biomarker for the identification rate of a sentinel node, being directly correlated with BMI (above 30 kg/m2) and with an age of above 50 years. The classification of the patients according to breast density represents an important criterion given that an adipose breast density (Tabar-Gram I-II) represents a lower IR of SLN compared with a density of the fibro-nodular type (Tabar-Gram III-V). We did not obtain any statistically significant data for the linear correlations between IR and the molecular profile, whether referring to the luminal subtypes (Luminal A and Luminal B) or to the non-luminal ones (HER2+ and TNBC), with p > 0.05, 0.201 [0.88, 0.167]; z = 1.82.

Characterizing dynamic tumor-immune interactions in lung adenocarcinoma through orthotopic allograft modeling.

The major clinical challenge in lung cancer immunotherapy is drug resistance. Therefore, establishing efficient orthotopic lung cancer mouse models to explore the mechanisms of drug immunotherapy resistance is highly important. In this study, we generated multiple fluorescently labeled lung adenocarcinoma cell lines from agenetically engineered KPZ mice model. Orthotopic transplantation of the primary 1F3 cell line induced a strong immune response, causing many small tumors to disappear, but some tumors evaded the immune attack and eventually formed large tumors. Tumor microenvironment analysis demonstrated that M2 macrophages play key roles in the immune response. Further mechanistic studies revealed that the chemokine CCL7 promoted the infiltration of M2 macrophages to facilitate immune escape, thereby promoting tumor growth in the orthotopic mouse model. Moreover, CCL7 levels were elevated in human lung cancer biopsies and positively correlated with M2 macrophage infiltration, and high CCL7 levels predicted advanced pathological stage and poor survival in lung cancer patients. Overall, we established a visualized and orthotopic mouse model with fluorescently labeled cells to better dissect the tumor microenvironment of lung cancer and define the critical role of CCL7 in promoting M2 macrophage polarization and tumorigenesis, providing new preclinical tools and potential targets for lung cancer immunotherapy.

Prostate cancer lesions in transition zone exhibit a higher propensity for pathological upgrading in radical prostatectomy.

The varying malignancy and lethality of different grades of prostate cancer (PCa) highlight the importance of accurate diagnosis. This study aims to evaluate the upgrading of transition zone (TZ) prostate cancer biopsies and identify factors to improve TZ biopsy accuracy. This retrospective study included 217 patients who underwent laparoscopic radical prostatectomy after 12 + X cores transperineal transrectal ultrasound-magnetic resonance imaging (MRI)-guided targeted prostate biopsy from 2018 to 2021 in our center. Patients with TZ lesions showed a higher incidence of International Society of Urological Pathology (ISUP) grade upgrading from 1 to higher grade compared to peripheral zone lesions (16.9% vs. 5.0%, p = 0.005). Multivariate analysis confirmed TZ lesions as an independent risk factor (OR: 4.594, 97.5% CI: 1.569-15.238, p = 0.008) for upgrading from 1 to higher. Additionally, the number of positive biopsy cores (OR: 0.586, 97.5% CI: 0.336-0.891, p = 0.029) and anterior TZ lesion location (OR: 10.797, 97.5% CI: 1.503-248.727, p = 0.048) were independent factors for the upgrading in TZ patients. This study found that PCa lesions located in the TZ, particularly the anterior TZ, have a higher risk of ISUP grade upgrading. This elevated risk arises from the insufficient distribution of biopsy cores around the TZ lesion. The findings underscore the importance of having an adequate number of biopsy cores around the lesion area to improve the accuracy of ISUP grade assessments.

A multi-modal single-cell and spatial expression map of metastatic breast cancer biopsies across clinicopathological features.

Although metastatic disease is the leading cause of cancer-related deaths, its tumor microenvironment remains poorly characterized due to technical and biospecimen limitations. In this study, we assembled a multi-modal spatial and cellular map of 67 tumor biopsies from 60 patients with metastatic breast cancer across diverse clinicopathological features and nine anatomic sites with detailed clinical annotations. We combined single-cell or single-nucleus RNA sequencing for all biopsies with a panel of four spatial expression assays (Slide-seq, MERFISH, ExSeq and CODEX) and H&E staining of consecutive serial sections from up to 15 of these biopsies. We leveraged the coupled measurements to provide reference points for the utility and integration of different experimental techniques and used them to assess variability in cell type composition and expression as well as emerging spatial expression characteristics across clinicopathological and methodological diversity. Finally, we assessed spatial expression and co-localization features of macrophage populations, characterized three distinct spatial phenotypes of epithelial-to-mesenchymal transition and identified expression programs associated with local T cell infiltration versus exclusion, showcasing the potential of clinically relevant discovery in such maps.

Exploring methylene blue as an effective alternative to radiotracer for sentinel lymph node biopsy in early breast cancer in resource-limited settings

Background: Sentinel lymph node biopsy is the recommended approach in the evaluation of axilla during breast cancer surgery. This study evaluated the results of patients who underwent methylene blue sentinel lymph node biopsy. Methods: The study included 35 female patients with T1, T2 and T3 tumours. 5 ml of 1% methylene blue was injected into the periareolar region. The axillary sentinel lymph node was found and removed, and axillary dissection was performed. The sentinel lymph node and axillary dissection specimen were histopathologically examined, and the results were compared. Results: The sentinel lymph node was found in 33 (90%) patients. There were no complications associated with the use of methylene blue dye. Methylene blue-stained sentinel nodes were histologically negative for malignancy in 42% and positive in 58% of patients. Corresponding to sentinel nodes, the final axillary staging was negative in 60% and positive in 40% of patients. On diagnostic accuracy parameters, the sensitivity of the methylene blue method in early breast cancer (cT1-3N0M0) patients was 90.47%, and the specificity was 100%. The PPV of the technique under study was found to be 100% and NPV was 85.71%. The accuracy rate was 93%, and the false negative rate was identified as 9.5%. Conclusions: Sentinel lymph node biopsy by methylene blue is a method that can be applied with high accuracy. Methylene blue can be considered as an alternative to radio colloid dye in sentinel lymph node biopsy in a limited resource setting.

TRPC3-mediated NFATc1 calcium signaling promotes triple negative breast cancer migration through regulating glypican-6 and focal adhesion.

Canonical transient receptor potential isoform 3 (TRPC3), a calcium-permeable non-selective cation channel, has been reported to be upregulated in breast cancers and a modulator of cell migration. Calcium-sensitive transcription factor NFATc1, which is important for cell migration, was shown to be frequently activated in triple negative breast cancer (TNBC) biopsy tissues. However, whether TRPC3-mediated calcium influx would activate NFATc1 and affect the migration of TNBC cells, and, if yes, the underlying mechanisms involved, remain to be investigated. By immunostaining followed by confocal microscopy, TNBC lines MDA-MB-231 and BT-549 were both found to express TRPC3 on their plasma membrane while ER+ line MCF-7 and HER2+ line SK-BR3 do not. Blockade of TRPC3 by pharmacological inhibitor Pyr3 or stable knockdown of TRPC3 by lentiviral vector both inhibited cell migration as measured by wound healing assay. Importantly, blocking TRPC3 by Pyr3 or knockdown of TRPC3 both caused the translocation of NFATc1 from the nucleus to the cytosol as revealed by confocal microscopy. Interestingly, NFATc1 was found to bind to the promoter of glypican 6 (GPC6) as determined by chromatin immunoprecipitation assay. Consistently, knockdown of TRPC3 decreased the expression of GPC6 as revealed by western blotting. Moreover, long-term knockdown of GPC6 by lentiviral vector also consistently decreased the migration of TNBC cells. Intriguingly, GPC6 proteins physically interact with vinculin in MDA-MB-231 as determined by co-immunoprecipitation. Blockade of TRPC3, knockdown of TRPC3 or knockdown of GPC6 all induced larger, stabilized actin-bound peripheral focal adhesion (FA) formations in TNBC cells as determined by co-staining of actin and vinculin followed by confocal microscopy. These large, stabilized actin-bound peripheral FAs indicated a defective FA turnover, and were reported to be responsible for impairing directed cell migration. Our results suggest that, in TNBC cells, calcium influx through TRPC3 channel positively regulates NFATc1 nuclear translocation and GPC6 expression, which maintains the dynamics of FA turnover and optimal cell migration. Our study reveals a novel TRPC3-NFATc1-GPC6-vinculin signaling cascade in maintaining the migration of TNBC cells.