Needle Biopsy Samples Research Articles

Histopathological-Based Analysis of Human Kidney Spatial Transcriptomics Data: Toward Precision Pathology

The application of spatial transcriptomics (ST) technologies is booming and has already yielded important insights across many different tissues and disease models. In nephrology, ST technologies have helped to decipher the cellular and molecular mechanisms at work in kidney diseases and have allowed the recent creation of spatially anchored human kidney atlases in healthy and diseased kidney tissues. During ST data analysis, the obtained computationally annotated clusters are often superimposed on a histologic image without their initial identification being based on the morphologic and spatial analyses of the tissues and lesions. In this study, we conduct a histopathologic-based analysis of ST data on a human kidney sample corresponding as closely as possible to the reality of the interpretation of a kidney biopsy sample in a health care or research context. This study shows the feasibility of a morphology-based approach to interpreting ST data, helping to improve our understanding of the lesion phenomena at work in chronic kidney disease at both the cellular and the molecular level. Finally, we show that our newly identified pathology-based clusters can be accurately projected onto other slides from nephrectomy or needle biopsy samples. They thus serve as a reference for analyzing other kidney tissues, paving the way for the future of molecular microscopy and precision pathology.

DEPDC1 as a metabolic target regulates glycolysis in renal cell carcinoma through AKT/mTOR/HIF1α pathway

Renal cell carcinoma (RCC) is considered a “metabolic disease” characterized by elevated glycolysis in patients with advanced RCC. Tyrosine kinase inhibitor (TKI) therapy is currently an important treatment option for advanced RCC, but drug resistance may develop in some patients. Combining TKI with targeted metabolic therapy may provide a more effective approach for patients with advanced RCC. An analysis of 14 RCC patients (including three needle biopsy samples with TKI resistance) revealed by sing-cell RNA sequencing (scRNA-seq) that glycolysis played a crucial role in poor prognosis and drug resistance in RCC. TCGA-KIRC and glycolysis gene set analysis identified DEPDC1 as a target associated with malignant progression and drug resistance in KIRC. Subsequent experiments demonstrated that DEPDC1 promoted malignant progression and glycolysis of RCC, and knockdown DEPDC1 could reverse TKI resistance in RCC cell lines. Bulk RNA sequencing (RNA-seq) and non-targeted metabolomics sequencing suggested that DEPDC1 may regulate RCC glycolysis via AKT/mTOR/HIF1α pathway, a finding supported by protein-level analysis. Clinical tissue samples from 98 RCC patients demonstrated that DEPDC1 was associated with poor prognosis and predicted RCC metastasis. In conclusion, this multi-omics analysis suggests that DEPDC1 could serve as a novel target for TKI combined with targeted metabolic therapy in advanced RCC patients with TKI resistance.

3D engineered scaffold for large-scale Vigil immunotherapy production

Previously, we reported successful cellular expansion of a murine colorectal carcinoma cell line (CT-26) using a three-dimensional (3D) engineered extracellular matrix (EECM) fibrillar scaffold structure. CCL-247 were grown over a limited time period of 8 days on 3D EECM or tissue culture polystyrene (TCPS). Cells were then assayed for growth, electroporation efficiency and Vigil manufacturing release criteria. Using EECM scaffolds, we report an expansion of CCL-247 (HCT116), a colorectal carcinoma cell line, from a starting concentration of 2.45 × 105 cells to 1.9 × 106 cells per scaffold. Following expansion, 3D EECM-derived cells were assessed based on clinical release criteria of the Vigil manufacturing process utilized for Phase IIb trial operation with the FDA. 3D EECM-derived cells passed all Vigil manufacturing release criteria including cytokine expression. Here, we demonstrate successful Vigil product manufacture achieving the specifications necessary for the clinical trial product release of Vigil treatment. Our results confirm that 3D EECM can be utilized for the expansion of human cancer cell CCL-247, justifying further clinical development involving human tissue sample manufacturing including core needle biopsy and minimal ascites samples.

Abstract PO4-08-01: Methylsterol monooxygenase 1 (MSMO1) modulates endoplasmic reticulum stress through cholesterol metabolic reprogramming to alter chemotherapy sensitivity in breast cancer

Abstract Background Endoplasmic reticulum stress (ER stress) is a process in which cells activate unfolded protein response and other signaling pathways in response to misfolded and unfolded protein aggregation and calcium ion balance disturbance in the endoplasmic reticulum cavity. Depending on the intensity of endoplasmic reticulum stress, it can not only induce chaperone expression to play a protective effect, but also induce apoptosis independently. Metabolic reprogramming is one of the characteristics of tumor, which refers to the metabolic changes that cells make in response to various stimuli. Here, we demonstrate that methylsterol monooxygenase 1 (MSMO1) regulates cellular cholesterol metabolism and influences breast cancer susceptibility to chemotherapy by regulating the relative intracellular T-MAS content. Methods RNA-seq was performed on core needle biopsy samples from 26 patients receiving neoadjuvant chemotherapy for breast cancer to screen for genes that may influence neoadjuvant chemotherapy sensitivity. Cell proliferation, CCK8 assay and apoptosis assay were performed for MSMO1 phenotype study. Transmission electron microscopy (TEM) was conducted to visualize the structure of endoplasmic reticulum. Western Blot was used to detect the activation level of the unfolded protein response pathway. The relative content of sterols in MSMO1 knockdown cells was detected by targeted lipid mass spectrometry. AO/PI staining of breast cancer organoids was performed to verify the effect of MSMO1 metabolic substrate T-MAS on chemotherapy drug sensitivity. Results RNA-seq of core needle biopsy samples from breast cancer patients receiving neoadjuvant chemotherapy suggested that MSMO1 was highly expressed in non-pCR patients, and clinical survival data suggested that MSMO1 was associated with poor prognosis in patients with all types of breast cancer, suggesting that MSMO1 might be related to chemotherapy sensitivity of breast cancer. Phenotypic experiments revealed that MSMO1 regulates chemotherapy sensitivity of breast cancer both in vivo and in vitro. Ectopic overexpression of MSMO1 promoted cancer cell resistance to chemotherapy. To uncover the underlying mechanisms, RNA-seq of MSMO1 knock-down cells were carried out and identified that MSMO1 was associated with ER stress. TEM and western blot assay indicated that downregulation of MSMO1 expression leads to endoplasmic reticulum swelling and activation of the unfolded protein response. Considering that MSMO1 is a cholesterol metabolizing enzyme, targeted lipids mass spectrometry was performed to detect the changes in the contents of various intermediate metabolites in the cholesterol pathway, which indicated that the relative content of T-MAS, a metabolic substrate of MSMO1, was significantly up-regulated in MSMO1 knockdown cells. Then, in order to verify whether this metabolite can independently regulate ER stress, breast cancer cells were given T-MAS externally, and TEM and Western Blot analysis indicated that T-MAS could induce ER stress and activate the unfolded protein response independently. Apoptosis assay and AO/PI staining of breast cancer organoids revealed that T-MAS can improve the sensitivity of breast cancer to chemotherapy drugs. Conclusion In summary, these results shed light on the role of MSMO1 in cholesterol metabolism reprogramming, which adjust the endoplasmic reticulum stress state of cells by changing the relative content of intracellular T-MAS, and thereby influencing the sensitivity of breast cancer chemotherapy. As an intermediate metabolite of cholesterol metabolism, T-MAS has potential clinical application value of chemotherapy sensitization. Citation Format: Hengyu Ren, Liren Wangxu, Yayun Chi, Jiong Wu. Methylsterol monooxygenase 1 (MSMO1) modulates endoplasmic reticulum stress through cholesterol metabolic reprogramming to alter chemotherapy sensitivity in breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-08-01.

Abstract 7391: Beyond traditional staining: A virtual-IHC digital assay for HER2 score prediction from routine H&E images

Abstract Background: In breast cancer diagnostics, the accurate assessment of HER2 status is essential for treatment stratification. Conventionally, HER2 immunohistochemistry (IHC) scoring necessitates specialized staining protocols, which are resource- and time-intensive. This study presents a virtual IHC (vIHC) digital assay to predict HER2 scores directly from hematoxylin and eosin (H&E) stained images, which are routinely acquired for all specimens. This methodology aims to accelerate standard practices by reducing the need for additional IHC staining, thereby decreasing laboratory workload, costs, and turnaround time. It also presents an opportunity to screen a wider array of samples promptly, potentially identifying candidates for HER2-targeted therapy that might otherwise be delayed or overlooked. Methods: Serially sectioned breast core needle biopsy samples were stained with H&E and HER2-IHC following Leica-Bond staining protocols. Stained slides were imaged using the Hologic GeniusTM Digital Diagnostics System with Research Use Only software for Whole Slide Imaging. From this initial image dataset, 240 regions of interest (ROIs) were manually selected in order to create a dataset that uniformly covers: (i) all HER2-IHC scores (0-3), (ii) a wide range of HER2 stain intensities, and (iii) expression patterns. The IHC scores were assigned to each selected ROI by a pathology expert, following the scoring recommendations of ASCO/CAP American Society of Clinical Oncology and College of American Pathologists, with the caveat that ductal carcinoma in situ was also given a score for the purpose of training the model. These ROIs were then extracted from the images of the serially sectioned H&E-stained slides using Reveal Bio’s proprietary co-registration digital assay. A predictive digital assay was developed at Reveal Biosciences to classify the 240 H&E-stained breast tissue ROIs into four HER2 IHC score categories: 0, 1, 2, and 3. For model evaluation, cross-validation was performed 10 times by randomly splitting the dataset of H&E ROIs into 80% for training MIL and 20% for testing the model, followed by averaging the performance metrics across the 10 dataset splits. The performance metrics included classification accuracy, precision, recall, and F1 scores for each HER2 score category. Results: The vIHC digital assay for predicting the HER2 scores directly from H&E stained breast tissue images achieved the average classification accuracy of 93% across the four HER2 score categories on the test set of ROIs. This performance demonstrates that the model is capable of successfully distinguishing between the HER2 score categories directly from H&E images without the need for IHC staining. The results suggest that the vIHC digital assay could be a reliable and cost-effective tool for preliminary breast cancer screening, providing a valuable adjunct to traditional histopathological analysis. Citation Format: Misagh Naderi, Nikola Mitic, Nikola Spasic, Djordje Cikic, Kristen Ruf, Nikolaus Wagner, Igor Mihajlovic, Sinisa Todorovic, Ray Jenoski, Sid Mayer, Michael Quick, Claire Weston. Beyond traditional staining: A virtual-IHC digital assay for HER2 score prediction from routine H&E images [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7391.

Abstract 5261: Immunologic signatures of peripheral blood cells in patients with advanced pancreatic cancer under lenvatinib-pembrolizumab maintenance therapy

Abstract Background: The treatment of pancreatic cancer is limited by chemotherapy toxicities which impacts quality of life. This is a phase 2 clinical trial of lenvatinib and pembrolizumab as maintenance therapy in patients with advanced pancreatic cancer. We observed preclinical activity of this combination in pancreatic mouse models. We hypothesize that this combination may address unmet needs to minimize toxicities associated with standard treatments, stimulate anti-tumor T cell immunity, and improve the progression free and overall survival. Exploratory studies aim to characterize immune-related systemic changes in peripheral blood over the course of therapy and to identify global changes in expression of tumor-related genes in needle biopsy samples of liver metastatic lesions. Methods: Multiplex gene expression analysis of unsorted peripheral blood mononuclear cells (PBMC) was performed using PanCancer Immune Profiling Panel on NanoString nCounter platform. PBMC samples from pre- through week 32 of treatment were assessed for differential expression of 730 immune profiling genes defining T cell functions and associated immune categories. Results: PBMC immune profiling from 4/5 patients show a distinctive pattern of increased activation of T cells during the first 9 weeks of treatment followed by a sharp inhibition starting at weeks 9-12 after 3-4 cycles of treatment (CD3E, CD247, LCK, TBX1, TCF7, STAT4, IL2RG). Subsequent upregulation of macrophage-derived scavenger, inflammatory, and immunosuppressive gene expression patterns continued for the remainder of treatment (CD14, MSR1, MARCO, CD68, TGFB1, IL10RA). The median PFS of first 10 patients was 21.5 (9-32) weeks. Conclusions: Immunotherapy resistance may be related to rapidly developing terminal differentiation and unresponsiveness of T cells in parallel with upregulation of macrophage-derived inflammatory and immunosuppressive processes. Studies are on-going to prevent inhibition of the T-cells. Citation Format: Vincent Chung, Ferdynand Kos, Edwin Manual, Shannon Dempsey, Don J. Diamond. Immunologic signatures of peripheral blood cells in patients with advanced pancreatic cancer under lenvatinib-pembrolizumab maintenance therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5261.

Abstract 324: Comparison of xGen Exome Hyb Panel v2 (IDT) kit and Twist Exome 2.0 (TWIST) kit for whole exome sequencing optimization from low input and degraded FFPE samples

Abstract FFPE is the most common method used to store precious clinical samples at room temperature for extended periods (sometimes>15 years). FFPE samples are therefore one of the most commonly available samples that can be used for discovery and diagnosis. Whole Exome Sequencing (WES) is one of the most used NGS assays that is used for profiling somatic and tumor mutations. With future clinical trials moving towards less invasive collection methodology, core needle biopsy samples are becoming a method of choice. The FFPE fixation process leads to the degradation of nucleic acids due to cross-linkage and the availability of sufficient nucleic acid from CNB fixed samples for downstream NGS assays is becoming limited. Here we report a comparison of two Whole Exome capture kits from several degraded FFPE samples, NA12878, OncoSpan gDNA HD827 and SeraSeq Breast CNV mix gDNA samples using the xGen Exome Hyb Panel v2 (IDT) Kit and the Twist Exome 2.0 (TWIST) Kit. Libraries were prepared in triplicate at 50ng, and 10ng using the Ultra™ II DNA Library Prep (NEB) kit. Pre-capture libraries were evaluated to determine the total DNA library available for capture using the KAPA Library Quantification Kit (Roche) kit. The recommendations for input amount vary between the kits, with IDT recommending 500ng, and TWIST recommending 200ng per library. KAPA Library Quantification Kit kit was used to assess the nanomolar amounts of ligated libraries. Final libraries were pooled equimolar and then sequenced as 2 × 100bp Paired-end sequencing on the NovaSeq 6000 instrument to achieve a target of 150-300X overall coverage. The performance of each capture kit was evaluated on the number of libraries eligible for capture, percent duplication rate, median insert size, and mean target coverage. IDT capture had 49% of libraries eligible at recommended input of 500ng, with 34% of libraries generating between 100-499ng input and the remaining 17% failing to generate 100ng input for hybridization. TWIST capture had 83% of libraries eligible at recommended input of 200ng, with 4% of libraries generating between 50-199ng input and the remaining 13% failing to generate 50ng input for hybridization. Most notably, 96% of libraries at 50ng DNA input were eligible for TWIST capture, while only 46% of libraries at 50 ng DNA input were eligible for IDT capture. The average Q30 for IDT and TWIST capture were 93-94% respectively. Both IDT and TWIST capture kits produced similar duplication rates and average mean target coverage was comparable. Taken together, our testing shows that TWIST and IDT kits generated comparable sequencing metrics. However, the TWIST Kit outperforms the IDT Kit in percent eligible libraries qualifying for hybridization capture. TWIST Kit is a great qualifier for highly degraded FFPE samples, especially those with limited input DNA material. Citation Format: Rachel Rock, Nripesh Prasad, Melanie Robinson, Dinnen Wildman, Olivia Montoya, Michael Sykes, Boris Umylyn, Thomas Halsey. Comparison of xGen Exome Hyb Panel v2 (IDT) kit and Twist Exome 2.0 (TWIST) kit for whole exome sequencing optimization from low input and degraded FFPE samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 324.

Digital Validation in Breast Cancer Needle Biopsies: Comparison of Histological Grade and Biomarker Expression Assessment Using Conventional Light Microscopy, Whole Slide Imaging, and Digital Image Analysis.

Given the widespread use of whole slide imaging (WSI) for primary pathological diagnosis, we evaluated its utility in assessing histological grade and biomarker expression (ER, PR, HER2, and Ki67) compared to conventional light microscopy (CLM). In addition, we explored the utility of digital image analysis (DIA) for assessing biomarker expression. Three breast pathologists assessed the Nottingham combined histological grade, its components, and biomarker expression through the immunohistochemistry of core needle biopsy samples obtained from 101 patients with breast cancer using CLM, WSI, and DIA. There was no significant difference in variance between the WSI and CLM agreement rates for the Nottingham grade and its components and biomarker expression. Nuclear pleomorphism emerged as the most variable histologic component in intra- and inter-observer agreement (kappa ≤ 0.577 and kappa ≤ 0.394, respectively). The assessment of biomarker expression using DIA achieved an enhanced kappa compared to the inter-observer agreement. Compared to each observer's assessment, DIA exhibited an improved kappa coefficient for the expression of most biomarkers with CLM and WSI. Using WSI to assess prognostic and predictive factors, including histological grade and biomarker expression in breast cancer, is acceptable. Furthermore, incorporating DIA to assess biomarker expression shows promise for substantially enhancing scoring reproducibility.

Histopathological Spectrum of Liver Allograft Biopsies and Association with Clinical and Pathological Parameters in Transplant Patients: A Cross-sectional Study

Introduction: Transplantation is vital for acute and terminal irreversible liver conditions. While imaging and functional measurements are valuable for evaluating post-transplant hepatocellular or biliary issues, liver allograft biopsies are used to determine the underlying causes of these changes. Long-term Immunosuppression (IMS), complex clinical circumstances, and de novo complications present challenges in transplant pathology, necessitating a multidisciplinary approach. Aim: To conduct a histopathological assessment of allograft liver biopsies for differential diagnosis, timing (postoperative day), and prevalence of post-transplantation complications, including identifying the causes of graft damage. Materials and Methods: This retrospective cross-sectional study was conducted between January 2019 and June 2022 at Vayalil Parambath Shamsheer (VPS) Lakeshore Hospital, Kochi, Kerala, India. A total of 45 post-transplant needle biopsy samples were analysed, examining histological characteristics and clinical data extracted from hospital records. Over 3.5 years, 45 post-liver transplant biopsies were performed. All clinical records and biopsy findings were examined using staining, and immunohistochemical analysis was performed. The Banff Working Group’s criteria were used to grade rejection based on a semiquantitative index, the Rejection Activity Index (RAI), into indeterminate (Score 1, 2); mild (Score 3, 4); moderate (Score 5, 6); and severe (>6). The classic histologic findings are characterised by predominant portal-based lesions, including a classical triad of mixed inflammatory cell infiltrates, venous endothelial inflammation, and inflammatory infiltration of bile ducts. Results: Among 38 patients, a total of 45 needle biopsies were performed. The first specimen was collected within a few hours of transplantation, and the final specimen was collected after 770 days. Notably, T-Cell-Mediated Rejection (TCMR) was diagnosed in 9 out of 45 (20.00%) specimens. Other complications included Intrahepatic Cholestasis (IHC) in 11 out of 45 (24.44%) cases, biliary obstruction in 5 out of 45 (11.11%) patients, Herpes Simplex Virus (HSV) hepatitis in 3 out of 45 (6.67%) specimens, Plasma Cell-Rich Rejection (PCRR) in 2 out of 45 (4.44%), and Isolated Central Perivenulitis (ICP), Dengue Haemorrhagic Fever (DHF), and veno-occlusive-like disease in 2 out of 45 (4.44% each) specimens. One patient had ethanolinduced liver injury (1 out of 45, 2.22%). Conclusion: Post-transplant liver biopsies are essential for accurate and timely diagnosis of rejection and other complications, guiding therapeutic interventions. This study offers insights into the types, prevalence, and timing of critical complications following liver transplantation (LTx).

Binary semantic segmentation for detection of prostate adenocarcinoma using an ensemble with attention and residual U-Net architectures.

An accurate determination of the Gleason Score (GS) or Gleason Pattern (GP) is crucial in the diagnosis of prostate cancer (PCa) because it is one of the criterion used to guide treatment decisions for prognostic-risk groups. However, the manually designation of GP by a pathologist using a microscope is prone to error and subject to significant inter-observer variability. Deep learning has been used to automatically differentiate GP on digitized slides, aiding pathologists and reducing inter-observer variability, especially in the early GP of cancer. This article presents a binary semantic segmentation for the GP of prostate adenocarcinoma. The segmentation separates benign and malignant tissues, with the malignant class consisting of adenocarcinoma GP3 and GP4 tissues annotated from 50 unique digitized whole slide images (WSIs) of prostate needle core biopsy specimens stained with hematoxylin and eosin. The pyramidal digitized WSIs were extracted into image patches with a size of 256 × 256 pixels at a magnification of 20×. An ensemble approach is proposed combining U-Net-based architectures, including traditional U-Net, attention-based U-Net, and residual attention-based U-Net. This work initially considers a PCa tissue analysis using a combination of attention gate units with residual convolution units. The performance evaluation revealed a mean Intersection-over-Union of 0.79 for the two classes, 0.88 for the benign class, and 0.70 for the malignant class. The proposed method was then used to produce pixel-level segmentation maps of PCa adenocarcinoma tissue slides in the testing set. We developed a screening tool to discriminate between benign and malignant prostate tissue in digitized images of needle biopsy samples using an AI approach. We aimed to identify malignant adenocarcinoma tissues from our own collected, annotated, and organized dataset. Our approach returned the performance which was accepted by the pathologists.

High-dimensional deconstruction of pancreatic cancer identifies tumor microenvironmental and developmental stemness features that predict survival

Numerous cell states are known to comprise the pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME). However, the developmental stemness and co-occurrence of these cell states remain poorly defined. Here, we performed single-cell RNA sequencing (scRNA-seq) on a cohort of treatment-naive PDAC time-of-diagnosis endoscopic ultrasound-guided fine needle biopsy (EUS-FNB) samples (n = 25). We then combined these samples with surgical resection (n = 6) and publicly available samples to increase statistical power (n = 80). Following annotation into 25 distinct cell states, cells were scored for developmental stemness, and a customized version of the Ecotyper tool was used to identify communities of co-occurring cell states in bulk RNA-seq samples (n = 268). We discovered a tumor microenvironmental community comprised of aggressive basal-like malignant cells, tumor-promoting SPP1+ macrophages, and myofibroblastic cancer-associated fibroblasts associated with especially poor prognosis. We also found a developmental stemness continuum with implications for survival that is present in both malignant cells and cancer-associated fibroblasts (CAFs). We further demonstrated that high-dimensional analyses predictive of survival are feasible using standard-of-care, time-of-diagnosis EUS-FNB specimens. In summary, we identified tumor microenvironmental and developmental stemness characteristics from a high-dimensional gene expression analysis of PDAC using human tissue specimens, including time-of-diagnosis EUS-FNB samples. These reveal new connections between tumor microenvironmental composition, CAF and malignant cell stemness, and patient survival that could lead to better upfront risk stratification and more personalized upfront clinical decision-making.

Automatic Screening System to Distinguish Benign/Malignant Breast-Cancer Histology Images Using Optimized Deep and Handcrafted Features

Breast Cancer (BC) has been increasing in incidence among women for a variety of reasons, and prompt detection and management are essential to reducing mortality rates. In the context of clinical-level breast cancer screening, the needle biopsy sample is used to generate Breast Histology Images (BHIs), which will then be used to confirm the results. Using a novel Deep-Learning Plan (DLP), the proposed work identifies BHI accurately and confirms the severity of BC by confirming its severity. As part of the proposed DLP implementation, four phases are involved: (i) the collection and enhancement of images, (ii) the extraction of features, (iii) the reduction of features and their integration, and (iv) binary classification and validation. The purpose of this study is to optimize deep features and machine features using particle swarm algorithms. To evaluate the performance of the proposed scheme, we compare the results obtained using individual deep features, dual deep features, and hybrid features. Using the hybrid image features in the classifier, this study has determined that ResNet18 with k-nearest neighbor provides superior classification accuracy (> 94%).

WTP8.12 Measurement of a panel of biological markers in breast cancer, a comparison between core needle biopsy and surgical excision samples

Abstract Aims ER, PR and HER2 are routinely measured in breast cancer in clinical practice. Numerous other biomarkers exist which are only utilised in a research setting, but may have a potential role in breast cancer. The majority of research in breast cancer is conducted in surgical excision (SE) specimens due to the availability of tissue; however, it is important markers can be measured in core needle biopsy (CNB) samples in patients undergoing neoadjuvant or non-operative treatment. Methods A panel of 15 biomarkers was measured in 202 CNB and paired SE samples in a series of older women with primary breast cancer using immunohistochemistry and H-scores: ER, PR, EGFR, HER2, HER3, HER4, VEGF, p53, BRCA1, LKB1, Ki67, BCL2, CK5/6, CK 7/8, MUC1. Concordance rates were calculated between positive/negative cut-offs for each marker measured in the two samples. Results The percentage concordance rates between positive/negative cut-offs were >60% for ER, PR, EGFR, HER2, VEGF, p53, Ki67, BCL2, CK 7/8, MUC1 and <45% for HER3, HER4, CK 5/6, BRCA1 and LKB1. In general, cases were more likely to be positive in the SE sample. This could suggest SE result is an overestimation of the true value and CNB is more accurate, or at least not likely to overestimate. Conclusions The biomarkers most frequently studied in breast cancer (ER, PR, EGFR, HER2, VEGF, p53, Ki67) did show good concordance between SE and CNB samples. This may suggest that for markers less frequently studied, there is disagreement with interpretation of staining, leading to greater variation.

Dedifferentiated Chondrosarcoma: Diagnostic Controversies and Emerging Therapeutic Targets.

The pathogenesis of dedifferentiated chondrosarcoma is controversial, and no genetic abnormality has consistently been identified in the disease. Focusing on the diagnostic challenges encountered in dedifferentiated chondrosarcoma, the following review aims at summarizing the tumor's active neoplastic pathways while highlighting therapeutic modalities that could potentially be explored to enhance patient survivorship. Owing to the challenging examination of small needle biopsy sampling as well as the disease's overlapping morphological and immunohistochemical features with other bone and soft-tissue sarcomas, the diagnosis of dedifferentiated chondrosarcoma can be problematic. While combined doxorubicin- and cisplatin-based regimens remain the first-line systemic chemotherapy in the disease, ~50% of tumors carry EXT1/2 or IDH1/2 mutations, advancing EXT or IDH inhibitors as potential alternative therapies, respectively. Despite systemic chemotherapy, dedifferentiated chondrosarcoma remains an aggressive tumor with dismal prognosis and limited survival. A multidisciplinary collaboration across multiple cancer centers is warranted to yield an accurate diagnosis, understand the disease's underlying pathogenesis, develop adequate treatment, and improve patient survivorship.

Differences in Cell Death and BMP-2 Expression in Core Biopsy Specimens of Malignant Bone Tumors Given Hydrogen Peroxide Compared to Liquid Nitrogen

Introduction: Malignant bone tumors have a global prevalence of 1% of the overall malignancy case, with a very low 5-year survival rate and high local recurrence. The limb-salvage surgery with bone recycling is widely developed in the management of malignant bone tumors. Hydrogen peroxide and liquid nitrogen are bone recycling mediator agents destroying the malignant tumor cells while preserving healthy bones as much as possible. The purpose of this study was to compare the effectiveness of hydrogen peroxide and liquid nitrogen with the number of tumor cell deaths using the Huvos score and BMP-2 expression in malignant bone tumors. Materials and Method: In vitro experimental research was performed on 30 core needle biopsy samples of bone malignant tumors divided into 2 groups of hydrogen peroxide and liquid nitrogen. The parameters measured are Huvos score and BMP-2 level. Results: Based on this study, the characteristics of malignant bone tumors were dominated by primary tumors (83.9%), in female patients (53.3%) with an average age of 30 years. Liquid nitrogen gave better results than hydrogen peroxide, marked by a lower Huvos score (average 12.13 vs. 18.87, p=0.033) and a higher BMP-2 (average 39.53 ± 26.59 vs. 63.87 ± 27.61, p=0.020). Conclusion: Liquid nitrogen is an effective bone recycling agent for the management of malignant bone tumors.

Hepatic Pin1 Expression, Particularly in Nuclei, Is Increased in NASH Patients in Accordance with Evidence of the Role of Pin1 in Lipid Accumulation Shown in Hepatoma Cell Lines.

Our previous studies using rodent models have suggested an essential role for Pin1 in the pathogenesis of non-alcoholic steatohepatitis (NASH). In addition, interestingly, serum Pin1 elevation has been reported in NASH patients. However, no studies have as yet examined the Pin1 expression level in human NASH livers. To clarify this issue, we investigated the expression level and subcellular distribution of Pin1 in liver specimens obtained using needle-biopsy samples from patients with NASH and healthy liver donors. Immunostaining using anti-Pin1 antibody revealed the Pin1 expression level to be significantly higher, particularly in nuclei, in the livers of NASH patients than those of healthy donors. In the samples from patients with NASH, the amount of nuclear Pin1 was revealed to be negatively related to serum alanine aminotransferase (ALT), while tendencies to be associated with other serum parameters such as aspartate aminotransferase (AST) and platelet number were noted but did not reach statistical significance. Such unclear results and the lack of a significant relationship might well be attributable to our small number of NASH liver samples (n = 8). Moreover, in vitro, it was shown that addition of free fatty acids to medium induced lipid accumulation in human hepatoma HepG2 and Huh7 cells, accompanied with marked increases in nuclear Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1), in accordance with the aforementioned observations in human NASH livers. In contrast, suppression of Pin1 gene expression using siRNAs attenuated the free fatty acid-induced lipid accumulation in Huh7 cells. Taken together, these observations strongly suggest that increased expression of Pin1, particularly in hepatic nuclei, contributes to the pathogenesis of NASH with lipid accumulation.

Abstract 4651: Comparison of the tumor and lymph node immune microenvironment in early non-small cell lung cancer through multimodal single cell sequencing

Abstract Background/Purpose: Previous studies have analyzed the tumor and local immune microenvironments in lung cancers and suggest immune modulation is associated with worse clinical outcome. However, the tumor-immune microenvironment in early stage lung tumors and lymph nodes (LNs) have not been fully described. We aim to compare cell states in the immune microenvironments between lung tumors and LNs through multi-modal profiling of the transcriptome and surface proteins. Methods: Needle biopsy samples were taken from 10 treatment-naive early stage lung cancer patients undergoing lung cancer resections. Tissues were obtained from normal lung, lung tumor, and multiple mediastinal LNs, and processed for scRNA-seq including labeling with Total-Seq C CITE-seq panel to quantify the levels of 130 cell surface proteins. In total, 76,721 cells (4,462 normal lung; 39,019 tumor; 33,240 LN) were identified with a median of 1,673 genes and 92 protein features detected per cell. Protein expression was decontaminated through the decontX algorithm. Weighted-Nearest Neighbor analysis from the Seurat R package was applied to integrate the CITE-seq and RNA-seq level data for clustering cells into subpopulations. Results: Six broad cell populations were identified including T/NK, myeloid (CD14+), B (CD19+), mast (TPSAB1+), pDC (IRF8+), and epithelial (EPCAM+) cells. Among 8 CD4+ T lymphocyte subpopulations and 11 CD8+ T lymphocyte subpopulations observed through clustering, a naïve CD4+ and a CD8+ T subpopulation (LEF1+, TCF7+) was observed respectively. These naïve T lymphocyte populations displayed increased proportions in LNs in comparison to tumors. In addition, 5 of the 8 observed CD4+ T lymphocyte populations were enriched in LNs. Immune populations enriched in LNs were largely shared and uniform across different patients. In contrast, a single CD4+ and CD8+ T lymphocyte subpopulation displayed expression of T lymphocyte exhaustion markers (TIGIT+, LAG3+, PD-1+) and were enriched in tumors. 6 of the 11 observed CD8+ T lymphocyte populations were enriched in tumor samples in comparison to LNs. Two alveolar macrophage populations (MARCO+) were enriched in normal lung tissue, in which one showed a heightened stress response. Conclusion: Single-cell profiling reveals diversity in immune cell populations between LNs, tumor, and adjacent normal tissue in early-stage LUAD. The results suggest the composition of immune cell type is fairly consistent across LNs but more heterogeneous in the tumor and adjacent normal tissue in early-stage patients. In the future, we aim to determine if these immune subpopulations are associated with survival, recurrence, aggressiveness, and predict responses for neoadjuvant treatments, which could improve prognosis and patient quality of life. Citation Format: Zhanhao Xi, Yusuke Koga, Shannon McDermott, Jennifer Beane, Sarah A. Mazzilli, Kei Suzuki, Joshua D. Campbell. Comparison of the tumor and lymph node immune microenvironment in early non-small cell lung cancer through multimodal single cell sequencing. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4651.

Abstract 4353: Utilizing scRNA sequencing to understand biomarkers of response and resistance to Sacitizumab Govetican in localized TNBC

Abstract Triple-Negative Breast Cancer (TNBC) is an aggressive breast cancer subset, which lacks expression of estrogen receptors, progesterone receptors, and human epidermal growth factor receptor-2. This subset of breast cancer disproportionately affects Black and African American women and improving TNBC treatment options is vital to reducing breast cancer mortality. The novel antibody-drug conjugate, Sacitizumab Govetican (SG), which targets TROP2 at the cell surface, has shown promising clinical results from the NeoSTAR trial (NCT04230109), a phase II study evaluating neoadjuvant SG therapy in a localized TNBC setting. As part of the NeoSTAR clinical trial, we have collected and processed pre- and post- SG treatment patient samples, with the aim of understanding response to this monotherapy. Herein, we identify biomarkers of response and resistance to SG monotherapy through the use of single cell RNA sequencing of matched pre- and post-treatment patient biopsies combined with exome sequencing these patient samples. Pre-treatment core needle biopsy samples, and if applicable, post-treatment residual disease biopsies were dissociated into single cell suspensions and subjected to single cell RNA sequencing. Additionally, fixed patient tissue samples were processed accordingly for exome sequencing analyses. Overall, we analyzed over 144,000 cells from 37 total scRNA seq libraries with an average of 3800 cells per biopsy sample, demonstrating the feasibility of this method. From these analyses, we observed several cell-type differences between patients who achieved a pathological complete response (pCR) and patients who had residual disease (RD). Specifically, our data shows that tumor infiltrating lymphocytes are a potential prognostic biomarker of response to SG. Furthermore, we detected alterations in among stromal and immune cell subsets, among non-responders, indicating that these cell types maybe indicative of SG resistance. Taken together, we outline biomarkers of response to SG treatment for an improved understanding of resistance mechanisms in the neoadjuvant setting to improve TNBC outcomes among patients. Citation Format: Nicole Peiris, Simona Cristea, Mengran Zhang, Siang Koh, James Coates, Ilze Smidt, Veerle Bossuyt, Laura Spring, Aditya Bardia, Leif Ellisen. Utilizing scRNA sequencing to understand biomarkers of response and resistance to Sacitizumab Govetican in localized TNBC. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4353.

Abstract 5461: Gain early insights from single cell RNA-sequencing of clinical trial needle biopsy cores

Abstract Single cell RNA-sequencing (scRNA-seq) yields valuable insights into the molecular heterogeneity of multiple cell types in normal and cancer tissues. scRNA-seq approach has the potential to not only answer basic research questions but also enhance clinical studies and clinical trials and accelerate drug development and testing. However, a consistent and reproducible method for isolating viable single cells and generating high quality single cell data from needle biopsy cores has been challenging to develop. Based on our experience isolating single cells from a large number of surgical tumor tissues, we set out to optimize a dissociation and processing protocol to generate viable single cell suspensions in adequate quantity for scRNA-seq from needle biopsy cores. A total of 17 de-identified 18-gauge needle biopsy samples were collected in collaboration with MD Anderson Cancer Center investigators. Samples were from liver, lymph node, pelvis, abdominal, neck, and lung tissues and included matched pre- and post-treatment samples. Patient biopsies were kept in RPMI medium on ice and transferred to EMPIRI (time to capture ranged from 3 to 6.5 hours after biopsy collection). Samples were dissociated using mechanical and enzymatic methods to achieve a single cell suspension. Cells were washed and viability and cell counts were verified before capture via 10x Chromium Connect. Captured cells were processed through the 10x Genomics Chromium Next GEM Single Cell workflow before sequencing on Illumina NovaSeq 6000 sequencer. Raw sequencing data were processed through EMPIRI’s computational pipeline that includes QC steps, doublet removal, and Seurat and other downstream analyses. Recovery of viable cells from individual 18-gauge biopsy cores ranged from 2,000-300,000 cells per core, depending on the tissue type and cellularity. Cell viability was strictly monitored and was maintained above 80% for 16 of 17 samples (most samples >90%). Cell counts after sequencing and pipeline analysis averaged 6,466 cells per sample. The median number of UMI counts per cell was 5,580, and an average of 1,699 genes were identified per cell. Average sequencing saturation was 73%. Here, we demonstrate the feasibility of integrating scRNA-seq analysis into clinical trials or clinical studies to obtain rapid insights into on-target drug effects and anticipated cellular responses to therapies, including immunotherapies, within 3 weeks of biopsy collection. We have established a method for isolating high quality single cells for scRNA-seq from tissue biopsy core, as small as a single 18-gauge needle core. We tested our protocol on 17 human samples and showed successful isolation of highly viable cells from lung, lymph node, pelvis, abdominal, neck, and lung tissues. We were able to generate scRNA-seq data sets from all samples for analysis. Acknowledgements: We thank our MDACC collaborators, particularly Drs. Scott Kopetz and Van Morris. Citation Format: Thomas Gallup, Sang Yun, Dave Gallup, Kyuson Yun. Gain early insights from single cell RNA-sequencing of clinical trial needle biopsy cores. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5461.

Clinical Genetic Features and Neoadjuvant Chemotherapy Response in HER2-Low Breast Cancers: A Retrospective, Multicenter Cohort Study.

The objective of this work is to reveal differences in clinical and genetic features, as well as neoadjuvant chemotherapy (NAC) response, between HER2-low and HER2-zero or HER2-positive breast cancers. A total of 245 female patients with breast cancer were retrospectively enrolled from seven hospitals. Core needle biopsy (CNB) samples were collected before NAC and used for next-generation sequencing by a commercial gene panel. Clinical and genetic features, as well as NAC response, were compared between HER2-low and HER2-zero or HER2-positive breast cancers. The nonnegative matrix factorization (NMF) method was applied to cluster the C-Score of enrolled cases to reveal the intrinsic features of each HER2 subgroup. A total of 68 (27.8%) cases are HER2-positive, 117 (47.8%) cases are HER2-low, and 60 (24.5%) cases are HER2-zero. HER2-low breast cancers have a significantly lower pathologic complete response (pCR) rate than HER2-positive and HER2-zero breast cancers (p < 0.050 for all comparisons). Compared with HER2-low breast cancers, HER2-positive cases have higher rates of TP53 mutation, TOP2A amplification, and ERBB2 amplification, as well as lower rates of MAP2K4 mutation, ESR1 amplification, FGFR1 amplification, and MAPK pathway alteration (p < 0.050 for all comparisons). After clustering HER2-low cases by the NMF method, 56/117 (47.9%) are in cluster 1, 51/117 (43.6%) are in cluster 2, and 10/117 (8.5%) are in cluster 3. HER2-low cases in cluster 2 have the lowest pCR rate among the three clusters (p < 0.050). HER2-low breast cancers have significant genetic differences from HER2-positive cases. Genetic heterogeneity exists in HER2-low breast cancers and impacts on NAC response in this subgroup.