Additional Immunohistochemistry Research Articles

Poorly differentiated chordoma: recognising this complex and rare aggressive tumour with characteristic immunohistochemical profile.

Poorly differentiated chordoma (PDC) is an uncommon subtype of chordoma, distinct in its occurrence in paediatric age group, location, variable epithelioid/rhabdoid/spindled histomorphology and the lack of physaliphorous cells (classical of chordoma) and immunohistochemistry (INI-1 loss, brachyury positive). We describe two cases of PDC. A3-year-old male and 4-year-old female child presented with neck stiffness and infiltrating tumour involving the skull base and upper cervical vertebral segments. Histopathology showed a tumour with sheets of cells having epithelioid to rhabdoid morphology and absence of physaliphorous cells. The tumour cells were positive for pan-cytokeratin, EMA, CD99 and vimentin and showed loss of INI-1 suggesting differentials of epithelioid sarcoma and atypical teratoid/rhabdoid tumour. On careful review of the clinical, radiological and pathological features, the additional immunohistochemistry for brachyury was performed, and its positivity clinched the diagnosis of PDC. Both the patients succumbed within a short span post-surgery. The present case study helps in creating an awareness and attempts to expand our knowledge in relation to the spectrum of chordoma (clinico-histological) and its immunohistochemical profile.

C4d Is an Independent Predictor of the Kidney Failure in Primary IgA Nephropathy.

Background: C4d deposits are present in a substantial proportion of patients with IgA nephropathy (IgAN), indicating the activation of the lectin pathway (LP) of the complement system. It seems that patients with activated LP have worse renal prognosis. The aim of this study was to investigate the prevalence and prognostic significance of C4d in our cohort of patients with primary IgA nephropathy (pIgAN). Methods: Patients with pIgAN were recruited from a hospital register of kidney biopsies of the Department of Nephrology and Dialysis, Dubrava University Hospital, Zagreb. Additional immunohistochemistry staining for C4d was performed on paraffin-embedded kidney tissue, and patients were stratified into being C4d positive or C4d negative. The clinical and histologic features of patients were analyzed and compared regarding C4d positivity. The primary outcome was defined as kidney failure (KF), and predictor variables of KF and renal survival were analyzed. Results: Of a total of 95 patients with pIgAN included in the study, C4d was present in 43 (45.3%). C4d-positive patients had a higher value of systolic (p = 0.039) and diastolic (p = 0.006) blood pressure at diagnosis as well as higher 24 h proteinuria (p = 0.018), serum urate (p = 0.033), and lower eGFR (p < 0.001). C4d-positive patients had worse renal survival (p < 0.001), higher rates of disease progression to KF (p < 0.001), and higher proteinuria (p < 0.001) and lower eGFR (p < 0.001) at the last follow-up. Glomerular C4d was an independent predictor of disease progression to KF (HR = 5.87 [0.95 CI 1.06-32.44], p = 0.032). Conclusions: C4d is an independent predictor of disease progression in patients with pIgAN. C4d may be used as an additional marker of progressive disease course in IgAN. The therapeutic implications of C4d status in IgAN, particularly in terms of complement inhibitors application, are not yet known.

Female Adnexal Tumor of Probable Wolffian Origin (Wolffian Tumor): A Potential Mimic of Peritoneal Mesothelioma.

Wolffian tumor and its nosologic relative, the recently defined STK11 adnexal tumor are rare neoplasms thought to arise from mesonephric remnants. These tumors typically arise in the broad ligament, fallopian tube, and ovarian hilum and although most are associated with a good prognosis, up to 50% of STK11 adnexal tumors demonstrate aggressive clinical behavior. The chief differential diagnoses include endometrioid adenocarcinoma and sex cord stromal tumors. However, the morphologic and immunohistochemical features of these tumors exhibit considerable overlap with peritoneal mesothelioma. To fully characterize their immunophenotypic signature, we examined a total of 21 cases (18 Wolffian and 3 STK11 adnexal tumors) with standard markers used in the diagnosis of mesothelioma. Morphologic and immunohistochemical (IHC) features were reviewed and additional IHC performed for cases with available material. Patient age ranged from 25 to 73 (mean: 51) years. Sites included adnexa/broad ligament (6, 28%), paratubal (5, 24%), ovary/paraovarian (5, 24%), tubal (intraluminal) (2, 9.5%), pelvis (2, 9.5%), and liver (1, 5%). The mean tumor size was 9.3cm (range: 0.2 to 22cm). The histomorphology in most cases (14/21, 66%) consisted of tubular to solid sheets of neoplastic cells lined by columnar to cuboidal cells containing uniform round to oval nuclei. Compressed tubules with slit-like lumens and sieve-like pattern were also seen in at least 7 (33%) cases. Three cases demonstrated interanastomosing cords and trabeculae of epithelioid cells with cribriform and microacinar patterns growing within prominent myxoid stroma as described in STK11 adnexal tumors. In the cases with available IHC for 3 mesothelial markers (calretinin, WT1, D2-40), 55.5% (5 of 9) showed reactivity with all 3 markers. In cases with at least 2 available mesothelial markers, 69% (11/16) were positive for 2 markers (mostly calretinin and WT1). Claudin-4, MOC31, and BER-EP4 were negative in most cases tested (78% [7/9], 71.4% [5/7], and 100% [6/6], respectively). Given the resemblance to mesothelioma, there was initial strong consideration and/or actual misdiagnosis of mesothelioma in 3 cases (14%). In summary, the morphologic and immunohistochemical features of Wolffian tumor and its recently defined relative, STK11 adnexal tumor, can lead to misdiagnosis of mesothelioma, particularly when encountered in the disseminated or metastatic setting. Wolffian tumor and STK11 adnexal tumor should be considered in the differential diagnosis of all pelvic and peritoneal mesotheliomas.

Immunohistochemistry of p53 surrogates TP53 mutation as an accurate predictor for early-relapse of surgically resected stage I-III lung adenocarcinoma

IntroductionTP53 is a strong tumor suppressor gene; its deactivation contributes to carcinogenesis and influences clinical outcomes. However, the prognostic influence of p53 deactivation on early relapse in patients with surgically resected non–small cell lung cancer remains unclear. Materials and methodsA cohort of 170 patients with primary stage I through III lung adenocarcinoma (LADC) and lung squamous cell carcinoma who underwent complete resection at Tokyo Medical and Dental University was screened for TP53 mutations using panel testing, and association studies between TP53 mutations and clinical data, including histology and postoperative recurrence, were performed. The association between TP53 mutations and postoperative recurrence was validated using data from 604 patients with MSK-IMPACT from The Cancer Genome Atlas. Additional immunohistochemistry for p53 was performed on some subsets of the Tokyo Medical and Dental University population. ResultsMutations in TP53 were recurrently observed (35.9%; 61 out of 170) in the Tokyo Medical and Dental University cohort. In the histology-stratified analysis, patients with LADC histology showed TP53 mutations that were associated with poor relapse-free survival (log-rank test; P = .020), whereas patients with lung squamous cell carcinoma histology showed TP53 mutations that were not (P = .99). The poor prognosis of TP53 mutation-positive LADCs was validated in The Cancer Genome Atlas-LADC cohort (log-rank test; P = .0065). Additional immunohistochemistry for p53 in patients with LADC histology in the Tokyo Medical and Dental University cohort showed a significant correlation between TP53 mutations and abnormal IHC pattern of p53 (Cramer's correlation coefficient V = 0.67). ConclusionsTP53 mutation is a potential marker for worse prognosis in surgically resected LADC; immunohistochemistry for p53 could be a surrogate method to identify patients with LADC with a worse prognosis.

A blind validation of a deep-learning solution providing HER2, ER, and PR results from H&amp;E-stained breast cancer specimens.

562 Background: Newly diagnosed breast cancer specimens are routinely tested for oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER2) status. This requires additional immunohistochemistry (IHC) +/- in situ hybridisation (ISH) assessment. This can increase laboratory/pathologist workloads and turnaround times. A computer-based approach could help to alleviate these issues. PANProfiler Breast is a deep-learning solution designed to predict ER/PR status and detect HER2 negative cases from whole slide images (WSIs) of haematoxylin and eosin (H&amp;E)-stained breast cancer tissue. Here we present a blind validation of PANProfiler Breast. Methods: Three cohorts of H&amp;E-stained archival breast cancers (400 cases, 692 WSIs) from St James’s University Hospital, Leeds, UK, were assigned for calibration (200 cases, 344 WSIs) and blind validation (200 cases, 348 WSIs). Slides were scanned at 40x magnification on an Aperio GT450 (Leica, Illinois, USA) scanner. PANProfiler returned “Positive”, “Negative” or “Indeterminate” for ER and PR, and “Negative” or “Indeterminate” for HER2. Results were compared to the ER/PR/HER2 status given in the corresponding pathology report. For calibration and blind validation, the WSI(s) corresponding to the IHC/ISH testing block was used for analysis. One case with missing HER2 status was excluded from HER2 analyses. Confusion matrices enabled analysis of concordance. While a conventional Positive/Negative confusion matrix was used for ER/PR, “Negative” results were used as the reference Positive event for HER2. Results: Case concordance for ER and PR status across the three cohorts ranged from 89.74-92.86% and 85.71-90.91% respectively. Additionally, sensitivity for ER ranged from 89.19-100%, with specificity ranging between 62.50-100%. For PR, sensitivity was between 87.50% and 93.48% and specificity between 75.00% and 83.33%. False negative rates for both ER and PR were between 0-10.81% and 6.52-12.50% respectively. Regarding HER2, sensitivity was relatively low, ranging between 30.77% and 37.04%. However, over 90% concordance was seen across all cohorts for cases PANProfiler predicted as HER2 “Negative”. Specificity and positive predictive value were also over 90% throughout the three cohorts. Additionally, false positive rates were 0.00%, 4.35% and 9.52% for HER2 across these same three cohorts. Test replacement rates varied amongst the cohorts with 70-84% for ER, 55-84% for PR and 22-27% for HER2. Conclusions: This blind validation of PANProfiler Breast shows impressive levels of performance for predicting ER and PR status in breast cancer WSIs. For HER2, the technology demonstrated a striking level of confidence for cases predicted as HER2 “Negative”. Further development and analysis are warranted to increase the number of HER2 negative BC WSIs that PANProfiler is able to identify.

Chronic consequences of ischemic stroke: Profiling brain injury and inflammation in a mouse model with reperfusion.

Stroke is a pervasive and debilitating global health concern, necessitating innovative therapeutic strategies, especially during recovery. While existing literature often focuses on acute interventions, our study addresses the uniqueness of brain tissue during wound healing, emphasizing the chronic phase following the commonly used middle cerebral artery (MCA) occlusion model. Using clinically relevant endpoints in male and female mice such as magnetic resonance imaging (MRI) and plasma neurofilament light (NFL) measurement, along with immunohistochemistry, we describe injury evolution. Our findings document significant alterations in edema, tissue remodeling, and gadolinium leakage through MRI. Plasma NFL concentration remained elevated at 30 days poststroke. Microglia responses are confined to the region adjacent to the injury, rather than continued widespread activation, and boron-dipyrromethene (BODIPY) staining demonstrated the persistent presence of foam cells within the infarct. Additional immunohistochemistry highlighted sustained B and T lymphocyte presence in the poststroke brain. These observations underscore potentially pivotal roles played by chronic inflammation brought on by the lipid-rich brain environment, and chronic blood-brain barrier dysfunction, in the development of secondary neurodegeneration. This study sheds light on the enduring consequences of ischemic stroke in the most used rodent stroke model and provides valuable insights for future research, clinical strategies, and therapeutic development.

Abstract PS03-06: Clinical implementation of artificial-intelligence-assisted detection of breast cancer metastases in sentinel lymph nodes: saving costs and time (the CONFIDENT-B trial)

Abstract Background Metastases in sentinel lymph nodes (SN) of breast cancer (BC) patients are strongly associated with a worse survival and consequently guide treatment. If metastases are absent upon pathologist’ assessment of the regular hematoxylin and eosin (HE)-slide, additional immunohistochemistry (IHC)-stains are performed to ensure that no metastases are missed. However, these stains come at high additional costs, which may exceed specimen reimbursement. Fortunately, digital pathology is becoming more common, thereby creating an avenue of opportunities for artificial intelligence (AI) assistance. Although the number of studies on (promising) AI-algorithms increases exponentially, studies on actual clinical implementation are lacking. In this single-center prospective trial, we investigated to which extent an artificial intelligence (AI)-assisted clinical workflow for the detection of SN-metastases reduces IHC-use, while maintaining current diagnostic safety standards. Methods We enrolled 190 SN-specimens of 182 patients with invasive or in situ BC from September 2022 to May 2023. SN-specimens were allocated bi-weekly to either the control-arm (n=90) or the intervention-arm (n=100). In the control-arm, SN-specimens were digitally assessed according to the current clinical workflow, while pathologists in the intervention-arm assessed the SN-specimen with the output the ‘Metastasis-Detection-App’ (Visiopharm©) available. In both groups, IHC was performed in all morphologically negative cases. Main outcome was the relative risk (RR) of IHC-use per detected case of SN-metastases. Case-mix adjustment was performed by log-binomial regression. Results Overall, 59 SN-specimens contained metastases (31.1%). AI-assistance resulted in a significantly lower risk of IHC-use per detected case of SN-metastases (adjusted RR: 0.680, 95% CI: 0.347-0.878). Besides preventing IHC-use, thereby reducing costs, AI-assisted pathologists also spent significantly less time on their assessment of the SN-specimen (3m:41s vs. 6m:04s, p = 0.028). Furthermore, the sensitivity of AI-assisted pathologists was up to 30% higher. The AI-assisted pathologists missed two cases of micro-metastases in the intervention arm, one of which was in retrospect highlighted by the algorithm, while in the other case the tumor cells were located in a heavily cauterized area of the HE-slide and therefore only visible on the (serial) IHC-slides. In the control arm, the algorithm in retrospect picked up all micro- and macro-metastases and nearly half of the isolated tumor cells (ITC). In addition, all participating pathologists stated that AI was easy to use, that they felt confident using AI, and that besides saving them time, AI made their work more enjoyable. Cost reductions on IHC by AI-assistance depend on laboratory policy (i.e. when and on how many levels IHC is performed), but, at a cost of €25,- per IHC-stain, range from €1.500 to €3.500 per 100 SN’s in a scenario where IHC is performed in all morphologically negative cases. In a scenario where IHC is only performed in patients in whom finding ITC has clinical consequences (i.e. patients who received neoadjuvant treatment), cost savings on IHC range from €7.500-€12.500 per 100 SNs, depending on laboratory policy. Conclusion AI-implementation for the detection of SN-metastases in BC-patients leads to a significant reduction of IHC-use and subsequent costs, while saving pathologists time and making their work more enjoyable. Importantly, AI-implementation during this trial was safe and patients were not at risk of an inferior diagnosis. By doing this trial alone, an estimated €3,000 on IHC-use was saved. Such tangible cost savings are crucial to build a viable business case for AI implementation in diagnostic pathology. Citation Format: Carmen van Dooijeweert, Rachel Flach, Natalie ter Hoeve, Celien Vreuls, Roel Goldschmeding, Jan-Erik Freund, Paul Pham, Tri Nguyen, Elsken Van der Wall, Geert Frederix, Nikolas Stathonikos, Paul Van Diest. Clinical implementation of artificial-intelligence-assisted detection of breast cancer metastases in sentinel lymph nodes: saving costs and time (the CONFIDENT-B trial) [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 PS03-06.

Abstract PO2-20-08: Metastatic Lung Cancer Masquerading as Metaplastic Breast Cancer

Abstract Background Metaplastic breast carcinoma (MBC) is an unusual malignancy that presents a diagnostic challenge due to the presence of varying cytomorphologies that can be seen in benign and malignant tumors. We posit that metastatic disease to the breast, itself an uncommon entity, should be considered in the differential diagnosis of MBC. We report a unique case of metastatic non-small cell lung cancer (NSCLC) with an actionable driver mutation that presented as a symptomatic breast mass and was initially considered to represent MBC. Report A 74-year-old woman with a 22-pack-year smoking history presented to the breast clinic complaining of a palpable left breast mass that had grown rapidly over the previous month. Ultrasound-guided biopsy of the breast mass revealed high-grade poorly differentiated carcinoma. On immunohistochemical (IHC) evaluation, tumor cells were strongly positive for epithelial markers CAM5.2 and keratin AE1/AE3, weakly positive for breast cancer (BC) marker TRPS1, and negative for BC marker GATA3, squamous cell carcinoma markers p63 and CK5, and melanoma marker SOX10. Metaplastic triple-negative BC was diagnosed. However, concern remained for metastasis from a non-breast primary tumor due to the patient’s constitutional symptoms such as fatigue, night sweats, and 60-pound unintentional weight loss over the previous year, as well as interval development of a suspicious nodular rash on her right upper extremity. An FDG-PET-CT was obtained and demonstrated a hypermetabolic left lung consolidation (maximum SUV 33.3) measuring 7.2 cm, pulmonary nodules, and extensive thoracic lymphadenopathy. Due to rising suspicion for a primary lung tumor, additional IHC staining on the original breast biopsy was requested. Tumor cells were positive for TTF-1, consistent with metastatic lung adenocarcinoma. Tissue next-generation sequencing (NGS) identified a MET exon 14 skipping mutation, making the patient eligible for treatment with capmatinib, a first-line kinase inhibitor targeted therapy for metastatic NSCLC. At the time of this report, the patient has completed a course of palliative stereotactic body radiation therapy to the left lung and started capmatinib 400 mg BID, which she is tolerating well. Her first restaging scans after treatment initiation will be obtained one week after the time of this report. Conclusion In the absence of heightened suspicion for a non-breast primary tumor, our patient had initially received a diagnosis of triple-negative MBC. This diagnosis would have exposed her to increased treatment-related toxicities from cytotoxic chemotherapy and poor prognosis from MBC without survival benefit from targeted therapy for NSCLC. We advocate for the use of supplemental body imaging, expert and comprehensive IHC evaluation, and multidisciplinary discussion in cases of MBC and undifferentiated breast malignancy to facilitate accurate diagnosis and treatment. Citation Format: Tanmayi Pai, Marte Wasserman, Jason Lewis, Miglena Komforti, James Jakub, Augustine Lee, Yanyan Lou, Pooja Advani, Rohit Rao. Metastatic Lung Cancer Masquerading as Metaplastic 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 PO2-20-08.

Abstract PO4-25-01: Selective elimination of CD169+ macrophages in lymph nodes invaded by breast cancers

Abstract Lymph node metastasis is a prognostically significant factor in breast cancer, though the colonization mechanism of cancer cells in this immune cell-rich organ remains elusive. This study aimed to elucidate the impact of breast cancer on the lymph node immune cell landscape. Multiscale transcriptomic analyses were performed on both metastatic and non-metastatic lymph node samples from breast cancer patients with lymph node metastasis. Twenty laser-micro-dissected sections were obtained from 17 lymph nodes across six breast cancer patients at stages II–III, with each patient contributing both types of lymph nodes for direct comparison. Comparing the transcriptomes of paired lymph nodes with and without metastasis from the same patients revealed selective downregulation of CD169+ macrophage-related genes in metastatic lymph nodes. The spatial transcriptome indicated a potential depletion of CD169+ macrophages, initiators of anticancer immunity, from their residence (sinuses) in metastatic lymph nodes, while other principal immune cell types were unaltered. Mass spectrometry imaging revealed that the numbers of CD169+ macrophages were smaller in the metastatic lymph nodes than in the non-metastatic lymph nodes, suggesting that cancer cells uprooted CD169+ macrophages from the lymph nodes. Conversely, the count of B, T, Treg, and CD11c+ cells remained comparable in both lymph node types, albeit an enrichment of Treg cells around metastasized cancer tissues was observed. Additional immunohistochemistry analysis of 315 non-metastatic lymph nodes and 159 metastatic lymph nodes from 58 patients with breast cancer showed that a reduced CD169+ macrophage population was prevalent in various breast cancer subtypes. A subset of metastatic lymph nodes (37 out of 159) displayed a complete absence of detectable CD169+ macrophages. The data also depicted a gradual decline in CD169+ macrophages correlating with the pN classification, while no correlation was identified with pathological tumor size classification (pT) or metastasized cancer volume. The data suggest precedence of CD169+ macrophage elimination over other reported immune cell abnormalities, such as cell number and metabolic irregularities. CD169+ macrophages are a unique type of resident macrophages in the lymphoid organs that present cancer-derived antigens to CD8+ cells. The antigen-presenting role of CD169+ macrophages to T cells, a pivotal step in adaptive immunity, signifies the catastrophic implications of their suppression. This study underscores CD169+ macrophage suppression as a pronounced pathological phenotype in lymph nodes with breast cancer metastasis, thereby establishing it as a critical future therapeutic target. Citation Format: Yurina Maeshima, Tatsuki R. Kataoka, Alexis Vandenbon, Masahiro Hirata, Yasuhide Takeuchi, Yutaka Suzuki, Yukiko Fukui, Yumiko Ibi, Hironori Haga, Satoshi Morita, Masakazu Toi, Shinpei Kawaoka, Kosuke Kawaguchi. Selective elimination of CD169+ macrophages in lymph nodes invaded by breast cancers [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 PO4-25-01.

Longitudinal imaging of vitreal hyperreflective foci in mice with acute optic nerve damage using visible-light optical coherence tomography.

Hyperreflective foci (HRFs) appear in optical coherence tomography (OCT) images of the retina and vitreous of patients with various ocular diseases. HRFs are hypothesized to be immune cells that appear in response to ischemia or tissue damage. To accurately identify HRFs and establish their clinical significance, it is necessary to replicate the detection of similar patterns in vivo in a small animal model. We combined visible-light OCT with temporal speckle averaging (TSA) to visualize and track vitreal HRFs (VHRFs) densities for three days after an optic nerve crush (ONC) injury. Resulting vis-OCT images revealed that VHRF density significantly increased approximately 10-fold at 12 h after ONC and returned to baseline three days after ONC. Additional immunohistochemistry results confirmed these VHRFs as inflammatory cells induced from optic nerve damage.

Recent advances and future challenges in the diagnosis of neuroendocrine neoplasms.

Neuroendocrine neoplasms (NEN) are a heterogeneous group of malignancies with increasing incidence, whose diagnosis is usually delayed, negatively impacting on patients' prognosis. The latest advances in pathological classifications, biomarker identification and imaging techniques may provide early detection, leading to personalized treatment strategies. In this narrative review the recent developments in diagnosis of NEN are discussed including progresses in pathological classifications, biomarker and imaging. Furthermore, the challenges that lie ahead are investigated. By discussing the limitations of current approaches and addressing potential roadblocks, we hope to guide future research directions in this field. This article is proposed as a valuable resource for clinicians and researchers involved in the management of NEN. Update of pathological classifications and the availability of standardized templates in pathology and radiology represent a substantially improvement in diagnosis and communication among clinicians. Additional immunohistochemistry markers may now enrich pathological classifications, as well as miRNA profiling. New and multi-analytical circulating biomarkers, as liquid biopsy and NETest, are being proposed for diagnosis but their validation and availability should be improved. Radiological imaging strives for precise, non-invasive and less harmful technique to improve safety and quality of life in NEN patient. Nuclear medicine may benefit of somatostatin receptors' antagonists and membrane receptor analogues. Diagnosis in NEN still represents a challenge due to their complex biology and variable presentation. Further advancements are necessary to obtain early and minimally invasive diagnosis to improve patients' outcomes.

Abstract 6527: Fucosyltransferase 5 promotes immune evasion in ovarian cancer by suppressing double negative T cells

Abstract Emerging evidence suggests that aberrant glycosylation in cancer impact on various stages in the disease progression including immunomodulation, but the underlying mechanisms remain poorly understood. Our previous data demonstrated that fucosyltransferase 5 (FUT5), a glycosyltransferase responsible for the terminal step in synthesizing Lewis antigens, may contribute to peritoneal metastasis of ovarian cancer. In this study, using clinical samples of high grade serous ovarian cancer (HGSOC), the most common subtype, we found that high expression of FUT5 is associated with a worse outcome. By employing multiplex immunohistochemistry (IHC) to examine T cell subsets, we found a significant decrease in CD3+CD4−CD8− (double negative, DN) T cells in FUT5high (n=10) compared to FUT5low samples (n=10), while there is no difference in the abundance of CD8+ cytotoxic T cells, CD4+ helper T cells or CD4+Foxp3+ regulatory T cells. Tumor-infiltrating DN T cells has been shown to mediate potent anti-tumor response. In addition to their cytotoxic capabilities, DN T cells largely exhibit modulatory functions on other T cell subsets. To further investigate, we performed additional IHC staining and observed a significant increase in proliferating (Ki67+) CD8+ tissue resident memory cells, which are known to be important for cancer immunity. In addition, we observed a decrease in metastases in the FUT5 knockdown group, accompanied by an increase in DN T cells in ascites and omental metastatic tumors in humanised mice, as revealed by flow cytometry, confirming a direct immunomodulatory role of FUT5. Together, these findings reveal a novel role of FUT5 in promoting immune evasion by suppressing anti-tumor DN T cells, which could provide novel insights for immunotherapy in HGSOC, which has a low response rate to immune checkpoint inhibitors (This research is supported by RGC SRFS2223-7S05, HMRF 08192286 and LSCCB under the Health@InnoHK Program launched by ITC, HKSAR). Citation Format: S. W. Fung, Sally K. Y. To, A. A. Hassan, Phillip P.C. Ip, Alice S. T. Wong. Fucosyltransferase 5 promotes immune evasion in ovarian cancer by suppressing double negative T cells [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 6527.

Abstract 7391: Beyond traditional staining: A virtual-IHC digital assay for HER2 score prediction from routine H&amp;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&amp;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&amp;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&amp;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&amp;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&amp;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&amp;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&amp;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&amp;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 106: Selective elimination of CD169+ macrophages in lymph nodes invaded by breast cancers

Abstract Background: Lymph node metastasis is known as one of the strongest prognostic factors in breast cancer. The invasion mechanism of metastasized cancer cells in such immune cell-rich organs remains clear. Here, we aimed to elucidate the impact of metastasized cancer cells on the lymph node immune cell landscape. Methods: Multiscale transcriptomic analyses were performed on both metastatic and nonmetastatic lymph node samples from breast cancer patients with lymph node metastasis. As cancer cells interact with lymphocytes in lymph nodes in direct, proximal, and distant manners, we tried to capture the direct and proximal impact on immune cells in invaded lymph nodes by comparing paired metastatic and non-metastatic lymph node samples. Laser-micro-dissected sections were obtained from 17 lymph nodes across six breast cancer patients at stages II‒III, with each patient contributing both types of lymph nodes for direct comparison. We performed bulk transcriptome, spatial transcriptome, and imaging mass cytometry to analyze the obtained lymph nodes. Furthermore, we conducted immunohistochemistry analyses against a larger patient cohort (574 lymph nodes from 58 patients). Results: Comparing the transcriptomes of paired lymph nodes with and without metastasis from the same patients revealed selective downregulation of CD169+ macrophage-related genes in metastatic lymph nodes. The spatial transcriptome indicated a potential depletion of CD169+macrophages, initiators of anticancer immunity, from their residence in metastatic lymph nodes, while other principal immune cell types were unaltered. Mass cytometry imaging revealed that the numbers of CD169+ macrophages were smaller in the metastatic lymph nodes than in the nonmetastatic lymph nodes. Conversely, the number of B cells, CD4+ T cells, CD8+T cells, Treg cells, and CD11c+ cells remained comparable in both lymph node types. Additional immunohistochemistry analysis of 315 nonmetastatic lymph nodes and 159 metastatic lymph nodes from 58 patients with breast cancer showed that a reduced CD169+ macrophage population was prevalent in all breast cancer subtypes. CD169+ macrophages gradually decreased in correlation with pathological lymph node metastasis status, while no correlation was identified with pathological tumor size classification or metastasized cancer volume in invaded lymph nodes. Discussion: CD169+ macrophages are a unique type of resident macrophages in the lymphoid organs that present cancer-derived antigens to CD8+ T cells. The antigen-presenting role of CD169+ macrophages to T cells is a pivotal step in adaptive immunity. The elimination of CD169+ macrophage implies the disruption of the first step of the initial immune response. This study uncovered CD169+ macrophage suppression as a pronounced pathological phenotype in lymph nodes with breast cancer metastasis, thereby establishing it as a critical future therapeutic target. Citation Format: Yurina Maeshima, Tatsuki R. Kataoka, Alexis Vandenbon, Masahiro Hirata, Yasuhide Takeuchi, Yutaka Suzuki, Yukiko Fukui, Yumiko Ibi, Hironori Haga, Satoshi Morita, Masakazu Toi, Shinpei Kawaoka, Kosuke Kawaguchi. Selective elimination of CD169+ macrophages in lymph nodes invaded by breast cancers [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 106.

Abstract 1155: Spatial colocalization and molecular crosstalk of myofibroblastic CAFs and tumor cells shape lymph node metastasis in oral squamous cell carcinoma

Abstract Lymph node metastasis (LNM) is a critical prognostic factor for patients with oral squamous cell carcinoma (OSCC). While previous research has implicated partial epithelial-to-mesenchymal transition (p-EMT) of tumor cells and cancer-associated fibroblasts (CAFs), specifically myofibroblastic CAFs (myCAFs), in LNM, the underlying molecular mechanisms remain poorly understood. Furthermore, the lack of diagnostic and prognostic biomarkers related to LNM has impeded the improvement in clinical outcomes for patients with OSCC. Here, we conducted a comprehensive molecular analysis integrating original and publicly available OSCC data of the bulk genome and transcriptome analyses, single-cell transcriptome profiling, and spatial transcriptome analyses. We found that myCAFs were both quantitatively and functionally activated in LNM-positive samples and spatially colocalized with OSCC cells within the invasive tumor front (ITF), providing a niche that may facilitate LNM. We validated spatial colocalization of myCAFs and OSCC cells using 50 additional Immunohistochemistry (IHC) samples. Mechanistically, myCAFs provided increased extracellular matrix (ECM) signals, such as COLLAGEN, Fibronectin1(FN1), and LAMININ to OSCC cells. Consequently, OSCC cells upregulate ECM receptors such as αβ integrins (ITGA-B), syndecans (SDC), and CD44, leading to upregulating stemness-related genes in OSCC cells. Further analysis using CRISPRGeneEffect data from DepMap found that ITGB1, ITGA3, and CD44 are among the most essential genes for the survival and growth of OSCC cells. By analyzing the spatial transcriptomic data of matched primary tumor sites (HUH001-P1 and HUH001-P2) and metastatic site (HUH001-met) from the same patient, we identified the origins of metastatic cells in the primary tumor site. We further extracted a 23-gene signature from the metastatic origin in ITF, where OSCC and myCAFs colocalize. This spatially-resolved 23-gene signature predicted LNM status and poor overall survival (OS) in clinical OSCC patients. The prognostic significance of the 23-gene signature we extracted from the spatial analysis was also validated in separate clinical cohorts from TCGA study and two other microarray datasets. Our findings provide novel insight into the molecular crosstalk between myCAFs and OSCC cells that facilitates LNM and identifies clinically valuable biomarkers for better prognostication in patients with OSCC. Citation Format: Ken Furudate, Shuya Kasai, Tadashi Yoshizawa, Yuya Sasaki, Kohei Fujikura, Shintaro Goto, Ryohei Ito, Tomoyuki Tanaka, Hiroshi Kijima, Kosei Kubota, Ken Itoh, Wataru Kobayashi, Koichi Takahashi. Spatial colocalization and molecular crosstalk of myofibroblastic CAFs and tumor cells shape lymph node metastasis in oral squamous cell carcinoma [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 1155.

Primary pancreatic lymphoma: Histopathological pattern of 8 cases.

To report the histopathological pattern of primary pancreatic lymphoma (PPL) in 2 tertiary hospitals. The pathology slides and reports of all the cases diagnosed in pathology departments in 2 referral hospitals were reviewed. An additional immunohistochemistry study was done to reclassify lymphomas according to the current system. Eight patients with PPL have been identified. The ages ranged from 36 to 71 years. Clinical presentation includes abdominal pain, weight loss, jaundice, abdominal mass, nausea, and vomiting. Pathological evaluation revealed 5 diffuse large B-cell lymphomas, one high-grade B-cell lymphoma, one MALT lymphoma, and one follicular lymphoma. Primary pancreatic lymphoma is a very rare tumor without specific clinical, laboratory tests, or radiological findings. Abdominal pain is the most common clinical presentation. Diffuse large b-cell lymphoma is the most common pathological subtype. Primary pancreatic lymphoma should be taken into consideration when evaluating pancreatic mass to avoid unnecessary surgical resection.

Clinical application of the HM-1000 image processing for HER2 fluorescence in situ hybridization signal quantification in breast cancer

BackgroundAccurate quantification of human epidermal growth factor receptor 2 (HER2) gene amplification is important for predicting treatment response and prognosis in patients with breast cancer. Fluorescence in situ hybridization (FISH) is the gold standard for the diagnosis of HER2 status, particularly in cases with equivocal status on immunohistochemistry (IHC) staining, but has some limitations of non-classical amplifications and such cases are diagnosed basing on additional IHC and FISH. This study investigated the clinical utility of a novel super-resolution fluorescence microscopy technique for the better FISH signal visualization and HER2 FISH classification.MethodsFourteen breast cancer tissue samples were retrospectively collected between September 2018 and February 2022, and FISH HER2 signal quantification was evaluated by determining the HER2/chromosome 17 centromere (CEP17) ratio and the number of HER2 signals per nucleus in super- versus conventional-resolution images.ResultsSuper-resolution images maintained the same overall HER2 diagnosis from routine, but HER2 FISH amplification changed negative to monosomy in two cases. Two Letrozole non-response relapses coincided to monosomy samples. The median number of HER2 signals per nucleus was 7.5 in super-resolution images and 4.0 in conventional-resolution images in HER2-positive samples and 2.8 and 2.1 signals per nucleus, respectively, in HER2-negative samples.ConclusionsSuper-resolution images improved signal visualization, including a significant difference in the number of countable HER2 and CEP17 signals in a single nucleus compared with conventional-resolution images. Increased accuracy of signal quantification by super-resolution microscopy may provide clinicians with more detailed information regarding HER2 FISH status that allows to better FISH classification such as HER2-low samples.

Clinicopathological Features of Primary Renal Mesenchymal Neoplasms in Adults: A Cross-sectional Study

Introduction: Renal mesenchymal tumours are a subset of primary renal tumours arising from the mesenchymal tissue in the kidney. They are a heterogeneous group of mostly benign tumours that exhibit varied behaviours and molecular characteristics. Aim: To analyse the wide spectrum of histological subtypes, their unique clinical presentation, and pathological features of primary renal mesenchymal neoplasms. Materials and Methods: This was a retrospective crosssectional observational clinicopathological study conducted in Department of General Pathology, Christian Medical College, tertiary care hospital, Vellore, Tamil Nadu, India, looking at data of adult Primary Renal Mesenchymal Tumours (PRMT) for a 15- year period between January 2006 and March 2021. Clinical details such as age, presenting symptoms, and tumour size were obtained from the hospital information system. Histopathology and immunohistochemical slides were reviewed for all the cases. Additional Immunohistochemistry (IHC) and molecular studies were performed for the undifferentiated sarcomas. The tumours were categorised into three groups as recommended by the World Health Organisation (WHO) 2020 classification of soft-tissue tumours based on biological behaviour: benign, intermediate, and malignant. Continuous variables are expressed as mean and Standard Deviation (SD). Comparison of categorical variables between groups was performed using the Chi-squared test. Continuous variables between groups were compared using the Student’s t-test for significance. A p-value of less than 0.05 was considered statistically significant. Results: Of the 2164 nephrectomies performed for neoplastic conditions, 97 (4.5%) were diagnosed as renal mesenchymal tumours. There were 59 (60.8%) benign, 10 (10.3%) with intermediate biologic behaviour and 28 (28.9%) malignant tumours. The mean ages at presentation were 40.5 years for benign, 43.4 years for intermediate, and 41 years for malignant tumours, respectively. Haematuria was seen in 11 of 38 (29%) intermediate grade and malignant tumours and in only 6 of 59 (10%) benign tumours, a difference that was statistically significant (p-value=0.017). Malignant tumours 24 of 28 (86%) were more likely to be larger (&gt;7 cm) when compared to benign tumours 28 of 59 (47%) at the time of presentation (p-value=0.027). Classical angiomyolipomas constituted 53/59 (90%) of the benign tumours. Of the 10 intermediate grade tumours, epithelioid angiomyolipomas and solitary fibrous tumours were the most common, accounting for 50% and 30% of the intermediate group, respectively. Undifferentiated small round cell sarcoma was the most common malignant neoplasm, making up 12/28 (43%) of the malignant tumours. Conclusion: The present study found that renal mesenchymal tumours constitute a small but unique group of renal tumours. They are predominantly benign, but up to a quarter are malignant. Malignant tumours tend to be larger and more often present with haematuria. The present study highlights the importance of IHC in the diagnosis of intermediate and malignant mesenchymal tumours and the requirement of exhaustive molecular studies individually tailored to the immuno-profile of malignant tumours.

Androgen Receptor Influenced Recurrence Score Correlation in Hormone Positive and HER2 Negative Breast Cancer Indian Patients: A Comparative Approach

Breast cancer (BC) recurrence is a major concern for both patients and healthcare providers. Accurately predicting the risk of BC recurrence can help guide treatment decisions and improve patient outcomes for a disease-free survival. There are several approaches and models that have been developed to predict BC recurrence risk. These include derived clinical assays such as genetic profiling (Oncotye Dx, MammaPrint, CanAssist and others), and algorithm derived open access tools such as Magee equations (ME), CTS5 Calculator and Predict Breast cancer. All the clinical assays are well accepted, but affordability and feasibility remain the challenge due to a noteworthy price tag of USD 3000. As per The American Society of Clinical Oncology (ASCO) updates, open access tools are possible substitutes but the availability of limited information on their applicability is a concern. These tools take into consideration the histopathologic parameters and immunohistochemistry (IHC) biomarkers data for estrogen receptor/progesterone (ER/PR), human epidermal growth factor receptor 2 (HER2), and Ki67. The current study focuses on the application of these tools in a subset of 55 Indian BC patients considering the influence of the androgen receptor (AR) IHC expression profile. AR is a potent target and a close interacting neighbor protein to ER and available literature also suggests their crosstalk expression in BC clinical models. Our comparative recurrence scores (RSs) predictive data showed a statistically significant AR expression correlation with average ME scores. No significance was noted across different prediction tools. The findings are suggestive that ME predictive scores are more relevant and informative compared to other online tools and with an additional AR IHC expression analysis the recurrence prediction might prove to be beneficial and feasible to many deprived BC patients.

Palato-Antral Involvement of a Primary Extracranial Sinonasal Meningioma

Primary extracranial sinonasal meningiomas are one of the rarest tumours involving the head and neck region. Very few reports exist with additional involvement of the palato-antral region. This paper describes a case of primary extracranial sinonasal meningioma with palatal involvement and the management of the same. The patient was taken up for surgical excision via intra-oral and endoscopic approaches. Postoperative healing was uneventful with no recurrence noted over 2 years. A palatal obturator was used for rehabilitation. The diagnosis of this pathology needs additional immunohistochemistry testing for confirmation and treatment entails complete surgical excision which assures no recurrence or delayed presentation of residual disease in follow-up.&#x0D; Keywords: primary extra cranial meningioma; sinonasal; maxilla ; palate.