Lymphoid Aggregates Research Articles

Abstract 4879: Delineating lymphocyte aggregates from tertiary lymphoid structures using spatial transcriptomics

Abstract Tertiary Lymphoid Structures (TLSs) are ectopic lymphoid structures that form in chronically inflamed non-lymphoid tissue. The presence of TLSs in tumors is largely associated with favorable outcomes among patients and also exhibit positive associations with response to immune checkpoint blockade. Although mainly comprised of lymphocytes (B cells and T cells), they are considered a separate entity from lymphocyte aggregates within tissue. Lymphocyte aggregates are fairly routine to identify in H&E stained tissue sections, but delineating TLSs from simple lymphocyte aggregates is subjective and not clearly defined. Additionally, the definition of TLSs is only recently being elucidated, so a clear molecular definition of both TLSs and lymphocyte aggregates is needed. To address this problem, we leveraged open-source spatial transcriptomics (ST) data from 17 samples across 7 cancer types. High resolution H&E images were manually evaluated for lymphocyte aggregates by identifying dense pockets of lymphocytic-appearing nuclei. Suspected aggregates were detected in 10 of 17 samples. We then applied a visium spot-level signature scoring method for 4 well published TLS signatures. Of the spots identified, 62% had enhanced signature expression for at least 2 of the signatures, although most of the incorrect annotations came from the lone gastric cancer sample. Differential expression between a TLS-sig- suspected lymphoid aggregate and a TLS-sig+ region not identified by H&E enriched primarily for B cell identity genes, suggesting B-cell-rich TLSs may not always appear as lymphoid aggregates in histology. [AG1] Strikingly, ST revealed strongly positive enhancements in the TLS signatures, where no annotations were made, and no obvious lymphoid aggregation was present, suggesting manual annotation from H&E is inadequate for identifying TLSs. Together, these results demonstrate that literature derived TLS signatures can correctly identify lymphoid aggregates as TLSs and that cellular and molecular differences between standard lymphoid aggregates and TLSs may be smaller than is currently accepted. Citation Format: Jordan Krull, Anjali Byappanahalli, Yi Jiang, Andrew Gunderson, Qin Ma. Delineating lymphocyte aggregates from tertiary lymphoid structures using spatial transcriptomics [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 4879.

Abstract 1152: Pan-cancer single-cell and subcellular characterization of lymphoid aggregates and tertiary lymphoid structures using high-plex spatial molecular imaging

Abstract Tertiary lymphoid structures (TLSs), clusters of immune cells that form in non-lymphoid tissues, are frequently identified in a wide range of solid tumors. They function as immunological “hubs”, driving anti-tumor responses. Previous studies have demonstrated that the presence of TLSs is often associated with improved responses to immunotherapy and superior clinical outcomes. Despite these insights, our understanding of their phenotypic diversity, spatial organization, intercellular communication, and interactions with cancer cells and the tumor microenvironment remains incomplete. Existing methods lack the resolution, depth, and scale required for a thorough understanding of these complex structures. We have obtained in-house and public spatial multi-omics data generated on various tissues across 9 different cancer types using the cutting-edge single-cell spatial transcriptomics and high-plex spatial molecular imaging platforms. We have developed innovative computational approaches for the detailed characterization of TLSs at single-cell and subcellular resolution and carried out an extensive analysis of lymphoid aggregates (LAs) and TLSs in tissue sections across these different solid cancer types. We defined the phenotypic state of each individual cell within TLSs including B/plasma cells, follicular helper/regulatory/stressed/exhausted T cells, follicular dendritic cells, fibroblasts and high endothelial venules. Then, we performed unbiased phenotyping and classification of TLSs based on their cellular states, compositions and organization patterns across various types of tissues. Additionally, we performed spatial neighborhood analysis, characterized the neighboring cells of each TLS and analyzed their cell-cell communication networks. Our approach allowed an unprecedented examination of the spatial phenotypes of TLSs in large, complex tissues at super resolution, in ways that were not previously described. We comprehensively characterized TLSs across different solid tumor types. Our analysis revealed a high degree of spatial and phenotypic heterogeneity in LAs and TLSs. We created a comprehensive spatial atlas of LAs and TLSs across solid tumor types, profiled TLSs at various maturation states with distinct cellular composition and organization patterns, and discovered new TLS states. Notably, we observed distinct communication networks associated with TLS phenotypic states. Our innovative methods and analytical approaches offer a more comprehensive way to study LAs and TLSs, enhancing detection capabilities and providing deeper insights into their spatial interaction and functional phenotypes. This research holds the potential to significantly advance our understanding of TLSs and their roles in immunobiology and immunotherapy responses across a broad range of cancers and diseases. Citation Format: Yunhe Liu, Yuanyuan Zhang, Guangchun Han, Guangsheng Pei, Kyung Serk Cho, Enyu Dai, Yanshuo Chu, Jiahui Jiang, Ansam Sinjab, Luisa M. Soto, Alejandra Serrano, Jianjun Gao, Humam Kadara, Catherine Fridman, Wolf Herve Fridman, Linghua Wang. Pan-cancer single-cell and subcellular characterization of lymphoid aggregates and tertiary lymphoid structures using high-plex spatial molecular imaging [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 1152.

Abstract 5135: Tertiary lymphoid structures and T-cell aggregates signal a robust anti-tumor immune response in a subset of leiomyosarcoma patients

Abstract Cancer immunotherapy has ushered in a new era for treatment of many difficult to treat cancers such as Leiomyosarcoma (LMS), one of the most common subtypes of sarcomas. There are few biomarkers which can be used to identify patients who might benefit from immunotherapy. There is an urgent need to discover novel biomarkers which might be prognostic of a strong anti-tumor response. We thus sought to comprehensively profile the microenvironment of LMS tumors to identify features which might be suggestive of an active immune response and molecular features prognostic of response to immunotherapy. We began our analysis by classifying the LMS tumors from The Cancer Genome Atlas (TCGA) into 4 subtypes based on previously identified molecular features and comparing the enrichment of various immune gene signatures. Two of the subtypes had a high expression of various immune gene signatures including B Cells, M2 macrophages and Cytotoxic T cells. Visual inspection of the Hematoxylin and Eosin-stained tissue slides available with TCGA pointed to a presence of lymphoid aggregates or Tertiary lymphoid structures (TLS) in 10-15% of the tumors - predominantly belonging to one of the two subtypes. This subtype displayed features of smooth muscle differentiation and was also associated with improved survival. To validate our findings, we performed immunohistochemistry (IHC) and bulk RNA sequencing of LMS specimens (n=66) obtained from surgical resections from the Johns Hopkins Hospital. We identified mature CD21+ TLS in 15% of these cases and non-germinal center T cell aggregates present in more than 30% of the cases and these features were associated with improved survival. We digitally merged the different IHC sections to study if the markers were spatially correlated. Interestingly, spatial analysis showed that these CD8 T cells aggregates co-localized with neighborhoods of high density of PD-L1+ cells, hinting at the possibility of immune mediated upregulation of PD-L1 as means of immune suppression. The deconvolution of the transcriptome of these samples showed a marked presence of cytotoxic CD8 T cells and plasma B cells in this subset of patients, an enrichment of pathways related to T cell trafficking, activation and proliferation. Single cell RNA sequencing of tumor infiltrating immune cells showed an oligoclonal expansion of B and T cells. Taken together, this study shows that a subset of LMS patients have a strong anti-tumor immune response which is characterized by the presence of Tertiary Lymphoid Structures and patches of high density of PD-L1 expressing cells. These features might serve as biomarkers in screening LMS patients which might benefit from immunotherapy. Citation Format: Aditya Suru, Alexandre Maalouf, Lingling Chen, Ada Tam, William Villasi, Kaushal Sharma, Lindy Zhang, Christian Meyer, Jonathan Greer, Fabian Johnston, Carol Morris, Sophia Strike, Adam Levin, Daniel Rhee, Brian Ladle, Kornel Schuebel, Yan Zhang, Rulin Wang, Srinivasan Yegnasubramanian, John Gross, Robert Anders, Andrew Pardoll, Nicolas Llosa. Tertiary lymphoid structures and T-cell aggregates signal a robust anti-tumor immune response in a subset of leiomyosarcoma patients [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 5135.

Abstract 3988: Deep spatial immunophenotyping of lymphoid aggregates in pancreatic cancer using multi-omic integration of ultra high-plex proteomics and transcriptomics

Abstract The most well-described mechanism of immune evasion in PDAC is exclusion of immune cells by a desmoplastic stroma, but such immune exclusion is not uniform. Through single-nucleus RNA-seq and whole-transcriptome digital spatial profiling of patient tumors, we previously reported marked heterogeneity in the immune composition of PDAC. Here, we developed a novel multi-omics workflow integrating single cell spatial proteomics (96-plex, Akoya Phenocycler®-Fusion) and transcriptomics (990-plex, Nanostring CosMx) in primary patient PDAC tumors to systematically study spatial relationships and functional states of malignant, stromal, and immune cells. Consecutive sections from four independent tumors enabled whole slide proteomic assessment of >3.5M cells, and transcriptomic assessment of >200K cells. Strikingly, we observed an abundance of immune cell aggregates in all tissue sections, up to 57 within a 1 cm2 tumor section. Aggregates were diverse in composition with representation of myeloid cells, CD8+ T cells, and stromal cells, including fibroblasts and nerves. B/T lymphoid aggregates were well vascularized with co-localized CD31+CD34+ endothelial cells and appeared at various phases of maturation towards putative tertiary lymphoid structures (TLSs) containing CD38+ B cells, CD4+ T cells, and CD21+ follicular DCs. These tissues were enriched for interferon stimulated genes in T cells and type 1 interferon response signatures in malignant cells and cancer associated fibroblasts (CAFs) forming inflammatory multicellular hubs that have been associated with improved response to immune checkpoint blockade. Surprisingly, B cells, in addition to macrophages and CD8+ T cells, were major producers of interferon gamma (B cells: 13.7%, macrophages: 36.6%, CD8+ T cells: 12.1% of IFNG+ cells). Finally, to gain insight into the necessary factors for TLS formation in PDAC, we quantified the expression of chemokines and adhesion molecules associated with TLS initiation. Activated dendritic cells expressed CCL19 and CCL21, endothelial cells expressed CCL21, and in concert with inflammatory CAFs (iCAFs), CXCL12. Malignant cells expressed CXCL13 and CCL20 and displayed high levels of cell adhesion molecules ICAM1, VCAM1, and ICAM2. Together, our data suggests that the functions of lymphoid tissue organizer cells can be substituted by endothelial cells, dendritic cells, iCAFs, and, surprisingly, malignant cells. Our spatial immunophenotyping study of pancreatic cancer thus highlights the marked immune heterogeneity of the disease and suggests a central role for malignant and stromal cells in mediating diverse mechanisms of immune evasion and engagement. Functional studies will be conducted to investigate these mechanisms of tumor-immune crosstalk and identify novel immunotherapeutic vulnerabilities for this deadly disease. Citation Format: Dennis Gong, Jingyi Cao, Peter Wang, Niyati Jhaveri, Yue Hou, Bassem B. Cheikh, HaYeun Ji, Mark Gregory, Jason Reeves, Michael Patrick, Carina Shiau, Jennifer Su, Jimmy A. Guo, Joseph M. Beechem, Martin Hemberg, William L. Hwang, Ryan Park. Deep spatial immunophenotyping of lymphoid aggregates in pancreatic cancer using multi-omic integration of ultra high-plex proteomics and transcriptomics [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 3988.

Abstract 1148: Multimodal spatial transcriptomics and proteomics analysis of the resectable NSCLC ecosystem following neoadjuvant chemoimmunotherapy

Abstract Neoadjuvant and perioperative immunotherapy with checkpoint inhibitors (ICIs), combined with chemotherapy (CT), have improved outcomes for patients with resectable non-small cell lung cancer (NSCLC). Pathologic tumor response has been utilized in clinical trials as a surrogate of clinical efficacy. Yet, over half of treated patients experience non-response, highlighting the need for novel markers to identify those who are most likely to derive therapeutic benefit. To dissect the spatial complexity of the tumor-associated immune landscape following neoadjuvant chemoimmunotherapy (CT+ICI), we selected patients (n =3) with a range of residual viable tumor (%RVT) - pathological complete response (pCR; 0% RVT), partial response (20% RVT), and no-response (100% RVT). We performed multimodal spatial transcriptomics (ST) and proteomics (using a 35-plex protein panel) on the same tissue sections using the Visium CytAssist from 10X Genomics. We also examined spatial patterns of 20-plex panel of immune and stromal markers at single-cell resolution in the same samples using the COMET Lunaphore platform. In the pCR patient, we studied two tumor areas to assess the impact of tumor response heterogeneity on the immune landscape. Our analysis identified distinct immune profiles across three patients with varying %RVT, reflecting the heterogeneity of the immune response. We found robust gene-protein correlation, such as MS4A1 gene expression and CD20 protein levels, in our multimodal spatial-omics analysis. Intriguingly, the arrangement of B cells and their interaction with surrounding plasma cells (PCs) varied notably across patients. Unlike in the patients with partial or no response, pronounced tertiary lymphoid structure (TLS) formation and signatures were observed in the pCR patient, with the CXCL13-CXCR5 axis overlapping with the TLS profile, suggesting a functional interplay that may be crucial in mediating response to therapy. Intriguingly, in the pCR patient, PCs uniquely arrayed around dense B cell aggregates, marked by MS4A1, CR2, FCER1, and CXCL13. PCs were not only less abundant in non-PCR patients but they also did not array around lymphoid aggregates. Our high-resolution and multimodal analysis identified spatially-resolved expression and patterns for B lineage cells that may be associated, and thus, underlie response of resectable NSCLC to neoadjuvant CT+ICI. Ongoing efforts on this expanding cohort will shed further light on transcriptional states and immunogenomic roles of B lineage cells in neoadjuvant CT+ICI-treated NSCLC. Citation Format: Zahraa Rahal, Tieling Zhou, Sujuan Yang, Alejandra Serrano, Jiping Feng, Ansam Sinjab, John T. Le, Xin Sun, Michael Wang, Wei Hu, Jianjun Zang, Tullia Bruno, Hai T. Tran, Stephen G. Swisher, Cheuk Hong Leung, Heather Y. Lin, J. Jack Lee, Jing Wang, John Victor Heymach, Don Lynn Gibbons, Ignacio Ivan Wistuba, Annikka Weissferdt, Jared K. Burks, Luisa Maren Solis Soto, Humam Kadara, Tina Cascone. Multimodal spatial transcriptomics and proteomics analysis of the resectable NSCLC ecosystem following neoadjuvant chemoimmunotherapy [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 1148.

Abstract 7466: Exploring tertiary lymphoid structures and immune cell interactions in gastric cancer prognosis

Abstract Tertiary lymphoid structures (TLSs) are ectopic lymphoid aggregates found in chronically inflamed, infected, and tumor tissues. Prior research indicates that the presence of TLS and tumor-infiltrating lymphocytes (TILs) correlates with a more favorable prognosis in several cancers. Our study investigates the role of TLSs and TILs in gastric cancer, the 4th leading cause of cancer-related death worldwide. We analyzed tumor samples from 107 late-stage gastric cancer patients from mainland China, with a 5-year follow-up period. We performed H&E and multiplex immunofluorescence staining to assess the tumor area and infiltrating immune cells, and we evaluated potential relationships between TILs, clinicopathological features, and patient prognosis. Immunohistological analysis showed that CD4 T cells may have a more significant impact on patient prognosis than CD8 T cells. Notably, the presence of PDL1 and CD4 co-expressing cells was associated with better disease outcomes. We observed PDL1 expressing cells surrounding the tumor margin in a subset of cases, a distribution pattern that could lead to delayed recurrence or complete remission in gastric cancer. In the context of cancer-related TLS, we found that CD20 and CD86 co-expression in the center of the structures may indicate a higher maturation level. Additionally, the ratio and spatial distribution of CD86 and CD163 macrophages appear to play a crucial role in prognosis. We extracted the expression levels of markers from representative samples to examine the spatial relationships between cancer cells and each immune cell type. We then visualized these cellular interactions in the tumor microenvironment using topographic plots. This study broadens our understanding of tumor and immune cell interactions in gastric cancer, with the potential to inform future directions in immunotherapy treatments. Citation Format: Zheng Zeng, Yee Man Au-Yeung, Jiandong Huang. Exploring tertiary lymphoid structures and immune cell interactions in gastric cancer prognosis [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 7466.

Clinical features of inflammatory bowel disease unclassified: a case-control study

BackgroundApproximately 10-15% of inflammatory bowel disease (IBD) patients with overlapping features of ulcerative colitis (UC) and Crohn’s disease (CD) are termed as inflammatory bowel disease unclassified (IBDU). This study aimed to describe the clinical features of IBDU and evaluate the potential associated factors of reclassification.MethodsThe clinical data of 37 IBDU patients were retrospectively analyzed from November 2012 to November 2020. 74 UC and 74 CD patients were randomly selected and age- and sex-matched with the 37 IBDU patients. Clinical characteristics were compared between the three patient groups. Potential factors associated with the IBDU reclassification were evaluated.Results60% of IBDU patients displayed rectal-sparing disease, and 70% of them displayed segmental disease. In comparison to UC and CD, the IBDU group demonstrated higher rates of gastrointestinal bleeding (32.4%), intestinal perforation (13.5%), spontaneous blood on endoscopy (51.4%), and progression (56.8%). The inflammation proceeded relatively slowly, manifesting as chronic alterations like pseudopolyps (78.4%) and haustra blunt or disappearance (56.8%). 60% of IBDU patients exhibited crypt abscess, and 16.7% of them exhibited fissuring ulcers or transmural lymphoid inflammation. The proportions of IBDU patients receiving immunosuppressants, surgery, and infliximab were basically the same as those of CD patients. During the 79 (66, 91) months of follow-up, 24.3% of IBDU patients were reclassified as UC, while 21.6% were reclassified as CD. The presence of intestinal hemorrhaging was associated with CD reclassification, while hypoalbuminemia was associated with UC reclassification.ConclusionsIBDU may evolve into UC or CD during follow-up, and hemorrhage was associated with CD reclassification. Different from the other two groups, IBDU exhibited a more acute onset and a gradual progression. When an IBD patient presents with transmural inflammation or crypt abscess but lacks transmural lymphoid aggregates or fissuring ulcers, the diagnosis of IBDU should be considered.

Histological criteria for selecting patients who need clonality test for non-gastric MALT lymphoma diagnosis

The histological diagnosis of extranodal marginal zone lymphoma of the mucosa-associated lymphoid tissue (MALT lymphoma) is difficult for pathologists. Recently, digital pathology systems have been widely used to provide tools that can objectively measure lesions on slides. In this study, we measured the extent of marginal zone expansion in suspected MALT lymphoma cases and compared the results with those of a molecular clonality test. In total, 115 patients who underwent an IGH gene rearrangement test for suspected MALT lymphoma were included in this study. All cases were histologically classified into three patterns; “small lymphoid aggregates with no germinal center (Pattern 1),” “lymphoid follicles with germinal center (Pattern 2)” and " fused marginal zone or diffuse small lymphocytic proliferation (Pattern 3).” The proportions of monoclonality in Pattern 1, 2, and 3 were 25.0%, 55.0%, and 97.9%, respectively. The ratios of marginal zone thickness to germinal center diameter and entire lymphoid follicle area to germinal center area were measured in Pattern 2 cases using a digital pathology system. Combining the width cutoff of 1.5 and the areal cutoff of 3.5, the sensitivity, specificity, positive predictive value, and negative predictive value for MALT lymphoma were 96.97%, 70.37%, 80.00%, and 95.00%, respectively. In conclusion, through objective measurement of the marginal zone, suspected cases of MALT lymphoma requiring a molecular clonality test can be effectively selected.

Abstract B091: Mechanisms underlying the omental support of ovarian cancer peritoneal metastasis

Abstract More than 80% of ovarian cancer (OC) cases have metastasized to the peritoneal cavity at diagnosis. The five-year survival rate for these patients is 25%. Within the peritoneal cavity, the most common and early site of metastasis is the omentum, a well-vascularized, specialized adipose tissue that arises off the greater curvature of the stomach. Because OC homes to the omentum, it is removed as a standard of care for patients with peritoneal metastases. In mouse models, the omentum is also an early metastatic site, and omentectomy reduces tumor expansion. The mechanism by which the omentum promotes OC growth is unknown, perhaps due to the complex omental microenvironment. The omentum contains many features that could impact OC seeding. It is covered by a mesothelial lining that extends in a sheet anchoring the omentum to surrounding tissues and is highly perfused by blood vessels. It contains lymphoid aggregates or milky spots that may contribute to pro- and anti-tumor immunity. This study explores the association of OC metastases with specific omentum features. We intraperitoneally injected luciferase-expressing ID8 OC cells to mimic high-grade serous OC metastasis. Using high-resolution, spatial ex vivo bioluminescence imaging (BLI), we found that tumor nodules preferentially formed in adipocyte-rich regions of the omentum and the mesentery, another peritoneal tissue favorable for OC seeding. To determine whether adipocytes, the most abundant omental cell type, are the primary support of OC, we crossed Adiponectin Cre+ and DTA mice to generate mice lacking white and brown adipocytes from birth. To overcome the metabolic syndrome the fatless mice develop, they receive subcutaneous fat transplants dorsally, referred to as distal adipocyte rescue of lipodystrophy (DARL) mice. Littermate controls receive sham surgeries. The blood triglyceride, glucose, and insulin levels are normalized post-transplant. Thus, the peritoneal cavity lacks mature adipocytes, but the mice have typical metabolic profiles, allowing us to study the role of omental adipocytes in peritoneal metastasis. Once the mice recovered from surgery, they were intraperitoneally injected with luciferase-expressing ID8s. While it was hypothesized that OC metastatic seeding within the adipocyte-free omentum and associated peritoneum would be impaired without local mature adipocytes, BLI revealed that the tumor burden was not decreased in DARL mice. Notably, specific regions of OC seeding, where mature adipocytes would have localized if present, such as the omentum and mesentery, retained tumor nodules in DARL mice. This conserved OC localization suggests that other cells in this specific microenvironment might be responsible for metastatic spread. To identify these cells, studies are ongoing to profile these regions in the DARL and control mice via flow cytometry and single-cell RNA sequencing. By unraveling the support for OC cells in the omentum, therapeutic strategies can be identified to repolarize the metastatic milieu and halt further spread to improve patient outcomes. Citation Format: Rachel Mintz, Alexandre Gallerand, Jichang Han, Sarah Ning, Wei Zou, Mary Wohltmann, Bernd Zinselmeyer, Gwendalyn Randolph. Mechanisms underlying the omental support of ovarian cancer peritoneal metastasis [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B091.

Abstract B005: Lymphoid aggregates dictate immune activity in melanoma and lung brain metastases

Abstract B005: Lymphoid aggregates dictate immune activity in melanoma and lung brain metastases

Digital spatial proteomic profiling reveals immune checkpoints as biomarkers in lymphoid aggregates and tumor microenvironment of desmoplastic melanoma

BackgroundDesmoplastic melanoma (DM) is a rare melanoma subtype characterized by dense fibrous stroma, a propensity for local recurrence, and a high response rate to programmed cell death protein 1 (PD-1)...

A comprehensive exploration of diverse skin cell types in the limb of the desert tortoise (Testudo graeca) through light, transmission, scanning electron microscopy, and immunofluorescence techniques

The Greek tortoise, inhabiting harsh desert environments, provides a compelling case for investigating skin adaptations to extreme conditions. We have utilized light microscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and immunofluorescence analysis to describe the structure of the arid-adapted limb skin in the Greek tortoise. Our aim was to identify the cell types that reflect the skin adaptation of this tortoise to arid conditions. Utilizing seven antibodies, we localized and elucidated the functions of various skin cells, shedding light on how the tortoise adapts to adverse environmental conditions. Our findings unveiled numerous scales on the limbs, varying in size and color, acting as protective armor against abrasions, bites, and other potential threats in their rocky habitats. The epidermis comprises four layers: stratum basalis, stratum spinosum, peri-corneous layer, and stratum corneum. Cytokeratin 14 (CK14) was explicitly detected in the basal layer of the epidermis, suggesting a role in maintaining epidermal integrity and cellular function. Langerhans cells were observed between epidermal cells filled with ribosomes and Birbeck granules. Numerous dendritic-shaped Langerhans cells revealed through E-Cadherin signify strong immunity in tortoises' skin. Melanophores were identified using the Melan-A antibody, labeling the cytoplasm, and the SOX10 antibody, labeling the nucleus, providing comprehensive insights into melanophores morphology and distribution. Two types of melanophores were found: dendritic below the stratum basalis of the epidermis and clustered oval melanophores in the deep dermal layer. Varied melanophores distribution resulted in a spotted skin pattern, potentially offering adaptive camouflage and protection against environmental challenges. Numerous myofibroblasts were discerned through alpha-smooth actin (α-SMA) expression, indicating that the Greek tortoise's skin possesses a robust tissue repair and remodeling capacity. B-cell lymphocytes detected via CD20 immunostaining exhibited sporadic distribution in the dermis, concentrating in lymphoid aggregates and around vessels, implying potential roles in local immune responses and inflammation modulation. Employing Tom20 to identify skin cells with abundant mitochondria revealed a notable presence in melanophores and the basal layer of the epidermis, suggesting high metabolic activity in these cell types and potentially influencing cellular functions. These findings contribute to our comprehension of tortoise skin anatomy and physiology, offering insights into the remarkable adaptations of this species finely tuned to their specific environmental habitats.

Persistent Buccal Ulcer with Underlying Non-Neoplastic Ectopic Lymphoid Aggregates: A Tonsillar Tissue?

The online version contains supplementary material available at 10.1007/s12070-024-04536-8.

Machine-Learning-Based Classification Model to Address Diagnostic Challenges in Transbronchial Lung Biopsy.

When obtaining specimens from pulmonary nodules in TBLB, distinguishing between benign samples and mis-sampling from a tumor presents a challenge. Our objective is to develop a machine-learning-based classifier for TBLB specimens. Three pathologists assessed six pathological findings, including interface bronchitis/bronchiolitis (IB/B), plasma cell infiltration (PLC), eosinophil infiltration (Eo), lymphoid aggregation (Ly), fibroelastosis (FE), and organizing pneumonia (OP), as potential histologic markers to distinguish between benign and malignant conditions. A total of 251 TBLB cases with defined benign and malignant outcomes based on clinical follow-up were collected and a gradient-boosted decision-tree-based machine learning model (XGBoost) was trained and tested on randomly split training and test sets. Five pathological changes showed independent, mild-to-moderate associations (AUC ranging from 0.58 to 0.75) with benign conditions, with IB/B being the strongest predictor. On the other hand, FE emerged to be the sole indicator of malignant conditions with a mild association (AUC = 0.66). Our model was trained on 200 cases and tested on 51 cases, achieving an AUC of 0.78 for the binary classification of benign vs. malignant on the test set. The machine-learning model developed has the potential to distinguish between benign and malignant conditions in TBLB samples excluding the presence or absence of tumor cells, thereby improving diagnostic accuracy and reducing the burden of repeated sampling procedures for patients.

Immune mapping of human tuberculosis and sarcoidosis lung granulomas.

Tuberculosis (TB) and sarcoidosis are both granulomatous diseases. Here, we compared the immunological microenvironments of granulomas from TB and sarcoidosis patients using in situ sequencing (ISS) transcriptomic analysis and multiplexed immunolabeling of tissue sections. TB lesions consisted of large necrotic and cellular granulomas, whereas "multifocal" granulomas with macrophages or epitheloid cell core and a T-cell rim were observed in sarcoidosis samples. The necrotic core in TB lesions was surrounded by macrophages and encircled by a dense T-cell layer. Within the T-cell layer, compact B-cell aggregates were observed in most TB samples. These B-cell clusters were vascularized and could contain defined B-/T-cell and macrophage-rich areas. The ISS of 40-60 immune transcripts revealed the enriched expression of transcripts involved in homing or migration to lymph nodes, which formed networks at single-cell distances in lymphoid areas of the TB lesions. Instead, myeloid-annotated regions were enriched in CD68, CD14, ITGAM, ITGAX, and CD4 mRNA. CXCL8 and IL1B mRNA were observed in granulocytic areas in which M. tuberculosis was also detected. In line with ISS data indicating tertiary lymphoid structures, immune labeling of TB sections expressed markers of high endothelial venules, follicular dendritic cells, follicular helper T cells, and lymph-node homing receptors on T cells. Neither ISS nor immunolabeling showed evidence of tertiary lymphoid aggregates in sarcoidosis samples. Together, our finding suggests that despite their heterogeneity, the formation of tertiary immune structures is a common feature in granulomas from TB patients.

Association of Total Hip Arthroplasty Flexural Rigidity With Magnetic Resonance Imaging and Histological Findings

BackgroundModular connections in total hip arthroplasty (THA) offer surgical advantages, but can contribute to implant fretting and corrosion due to micromotion at the head–stem interface. Previous studies implicated lower flexural rigidity as a key contributing factor to THA corrosion and fretting, but none associated flexural rigidity with direct histological evaluation or magnetic resonance imaging (MRI) outcomes. The purpose of this study was to determine how implant flexural rigidity is associated with MRI imaging metrics and histopathological outcomes in patients who have a failed THA. MethodsPatients requiring revision THA surgery underwent preoperative MRIs with 3-dimensional multispectral imaging techniques to suppress metal artifacts. The MRI images were graded for adverse local tissue reactions. For each hip, trunnion flexural rigidity was measured from the retrieved femoral stem, and a periprosthetic tissue sample was retrieved and evaluated using semiquantitative histology. Generalized linear models and analyses of variance were used to assess associations between flexural rigidity and MRI and histology outcomes. ResultsA total of 106 THA stems were retrieved (46 women and 60 men, age: 68 years (range, 60 to 73 years). After adjustment for length of implantation, flexural rigidity was negatively correlated with histologic aseptic lymphocyte-dominant vasculitis-associated lesion severity (β = −26.27, P = .018), Fujishiro lymphocyte grading (β = −13.4, P = .039), perivascular lymphocyte layers (β = −17.8, P = .022), the grade of tissue organization (β = −22.5, P = .009), the presence of diffuse synovitis (β = −66.5, P = .003), and the presence of lymphoid aggregates (β = −75.9, P = .022). No association was found between MRI metrics and flexural rigidity. ConclusionsAmong these implants, decreased trunnion stiffness was associated with increased histologic features of adverse host-mediated soft tissue reactions.

Quantitative multiplex immunohistochemistry reveals inter-patient lymphovascular and immune heterogeneity in primary cutaneous melanoma.

Quantitative, multiplexed imaging is revealing complex spatial relationships between phenotypically diverse tumor infiltrating leukocyte populations and their prognostic implications. The underlying mechanisms and tissue structures that determine leukocyte distribution within and around tumor nests, however, remain poorly understood. While presumed players in metastatic dissemination, new preclinical data demonstrates that blood and lymphatic vessels (lymphovasculature) also dictate leukocyte trafficking within tumor microenvironments and thereby impact anti-tumor immunity. Here we interrogate these relationships in primary human cutaneous melanoma. We established a quantitative, multiplexed imaging platform to simultaneously detect immune infiltrates and tumor-associated vessels in formalin-fixed paraffin embedded patient samples. We performed a discovery, retrospective analysis of 28 treatment-naïve, primary cutaneous melanomas. Here we find that the lymphvasculature and immune infiltrate is heterogenous across patients in treatment naïve, primary melanoma. We categorized five lymphovascular subtypes that differ by functionality and morphology and mapped their localization in and around primary tumors. Interestingly, the localization of specific vessel subtypes, but not overall vessel density, significantly associated with the presence of lymphoid aggregates, regional progression, and intratumoral T cell infiltrates. We describe a quantitative platform to enable simultaneous lymphovascular and immune infiltrate analysis and map their spatial relationships in primary melanoma. Our data indicate that tumor-associated vessels exist in different states and that their localization may determine potential for metastasis or immune infiltration. This platform will support future efforts to map tumor-associated lymphovascular evolution across stage, assess its prognostic value, and stratify patients for adjuvant therapy.

HERMANSKY-PUDLAK SYNDROME-ASSOCIATED INFLAMMATORY BOWEL DISEASE: A SYSTEMATIC REVIEW ON CLINICAL MANIFESTATIONS AND INVESTIGATIONS INTO OPTIMAL MANAGEMENT

Abstract Hermansky-Pudlak Syndrome (HPS) is a rare autosomal condition resulting from a mutation in 1 of at least 7 different genes leading to the triad of tyrosine-positive oculocutaneous albinism, platelet dysfunction leading to prolonged bleeding time, and ceroid lipofuscin within the reticuloendothelial system. The latter of these can result in systemic derangements including renal failure, pulmonary fibrosis, cardiomyopathy, and a disabling granulomatous colitis. This colitis is a unique type of inflammatory bowel disease (IBD) with complications such as intestinal fistulization, ileitis, enterocolitis and perianal disease. There is data to suggest that HPS colitis is due to the development of classical IBD. However, optimal management remains undetermined. Our systematic review aims to analyze the clinical presentation behind HPS colitis, endoscopic versus histopathological findings, and ultimate treatment plans required for optimal management of this disease among HPS genotypes. A comprehensive literature review was conducted on patients with HPS and its associated colitis manifestations from inception to August 2023. Analyses on patient demographics, clinical presentation, HPS genotype, endoscopic versus histopathological findings, medical versus surgical interventions were thoroughly performed. Six cohort studies on HPS patients with coexisting colitis were identified. A total of 84 patients comprised these studies with mean age 32 +/- 9.65. Racial breakdowns were 68% Puerto Rican, 23% white, 4% Indian, 2% Mexican, 1% Honduran, and 1% African American. 59 patients had the HPS-1 genotype, 8 with HPS-3, 3 with HPS-4, and 1 with HPS-6. The most common presenting symptoms were hematochezia (37%), diarrhea (23%), abdominal pain (16%), and constipation (10%). Endoscopic evaluation showed normal gross findings in 65% of cases and abnormalities in only 35%. However, IBD-associated histopathological positivity was more prevalent with the most common findings being granulomas (33%), macrophages without colitis (19%), macrophages with colitis (15%), and lymphoid aggregates (10%). Medical therapy was initiated in 48 cases with the most common medications being steroids (46%), followed by anti-TNFa agents (40%), and immunomodulators (35%). 28 cases required surgical intervention with the most common operation performed being a (subtotal) colectomy and ileostomy. There were scattered cases of laparoscopic loop ileostomies, total colectomies, and proctocolectomies too. Interestingly, 10 patients received no intervention whatsoever. HPS-associated colitis is a debilitating condition that remarkably resembles IBD. There is no consensus on optimal therapy and many patients ultimately require operative intervention. The HPS-1 gene appears to be highly prevalent in this condition and younger patients are more affected, however more research is needed.

Epigenetic targets to enhance antitumor immune response through the induction of tertiary lymphoid structures.

Tertiary lymphoid structures (TLS) are ectopic lymphoid aggregates found in sites of chronic inflammation such as tumors and autoimmune diseases. The discovery that TLS formation at tumor sites correlated with good patient prognosis has triggered extensive research into various techniques to induce their formation at the tumor microenvironment (TME). One strategy is the exogenous induction of specific cytokines and chemokine expression in murine models. However, applying such systemic chemokine expression can result in significant toxicity and damage to healthy tissues. Also, the TLS formed from exogenous chemokine induction is heterogeneous and different from the ones associated with favorable prognosis. Therefore, there is a need to optimize additional approaches like immune cell engineering with lentiviral transduction to improve the TLS formation in vivo. Similarly, the genetic and epigenetic regulation of the different phases of TLS neogenesis are still unknown. Understanding these molecular regulations could help identify novel targets to induce tissue-specific TLS in the TME. This review offers a unique insight into the molecular checkpoints of the different stages and mechanisms involved in TLS formation. This review also highlights potential epigenetic targets to induce TLS neogenesis. The review further explores epigenetic therapies (epi-therapy) and ongoing clinical trials using epi-therapy in cancers. In addition, it builds upon the current knowledge of tools to generate TLS and TLS phenotyping biomarkers with predictive and prognostic clinical potential.

OP39 Vedolizumab response in ulcerative colitis associates with reduced IgG+ plasma cells and FcγR signaling

Abstract Background Vedolizumab (VDZ) targets the gut-homing receptor α4β7. Despite its widespread use as a frontline therapy for ulcerative colitis (UC), its mode of action (MOA) remains unclear. Previously, we documented that response to VDZ is associated with size loss of gut-associated lymphoid tissues (GALT). To further understand drug MOA, here we aimed at defining the relationship between GALT attrition and resolution of inflammation. Methods Tissue immunofluorescence (IF) was performed on two independent UC cohorts (n=60, n=21) studied longitudinally pre- and post-VDZ. Peripheral blood mononuclear cells from UC patients (n=56) were examined at week 0 (pre-VDZ) and 14 (post-VDZ) by flow cytometry, while intestinal biopsies were analyzed in a subset of patients. Transcriptomic analyses were performed in a third cohort of 401 UC patients and 243 controls where bulk RNA-sequencing was performed on intestinal biopsies. Finally, data from a fourth validation cohort (n=31) was examined pre- and post-VDZ. Results Using IF, we examined follicular naïve B (FB) cells (IgD+), germinal center (GC) B cells (BCL6+CD3-) and total T cells (CD3+), and identified three GALT types: organized aggregates (demarcated B/T cell zones) with GC, organized aggregates without GC, and poorly organized aggregates (Fig1A). When comparing pre- and post-VDZ, Responders (R) had a significant reduction in GALT organization, while no change was seen in Non-Responders (NR). The number of FB cells was significantly reduced post-VDZ in R but not in NR (Fig1B), with no change in T cells (Fig1C). The reduction in FB was validated using transcriptomic data (Fig1D). To explore potential consequences of GALT attrition, we analyzed circulating β7+ (GALT-generated) and β7- plasmablasts (PB). The frequencies of β7+IgG+ and β7+IgA+ PB were significantly reduced post-VDZ in R, but not in NR (Fig1E). No change was observed in β7- PB post-VDZ (Fig1F). Among β7+ PB, there was a greater reduction in IgG+ compared to IgA+ (Fig1G). Furthermore, colonic IgG+ plasma cells (PC) were significantly reduced post-VDZ in R but not in NR (Fig1H). To evaluate IgG-mediated proinflammatory function, we studied the expression of FcγR-pathway genes in colonic biopsies. Importantly, the FcγR-pathway was enhanced in inflamed tissues of UC patients (Fig1I-J) and was significantly reduced in R (but not in NR) post-VDZ (Fig1K). Conclusion Our work demonstrates a novel effect of anti-α4β7 blockade that results in intestinal lymphoid aggregate attrition through impaired recruitment of naïve B cells, leading to the suppression of IgG-driven immune responses and proinflammatory FcγR signaling. These data highlight the targeting of GALT as a novel therapeutic paradigm in IBD.