Routine Immunohistochemistry Research Articles

Rapid Immunohistochemistry Based on Ultrasonic Thermal Steam Heating for Improvement of Intraoperative Diagnosis.

To evaluate the role of rapid immunohistochemistry (RIHC) based on ultrasonic thermal steam heating in improving diagnostic accuracy of intraoperative frozen section diagnosis and to recommend RIHC antibody panels for pathologic differential diagnosis. RIHC based on ultrasonic thermal steam heating was tested for intraoperative frozen diagnosis with difficulty in diagnosis, and all slides were reviewed and compared with the final diagnosis. Ninety-three cases of surgical specimens involving RIHC examination were studied. Discordance rates with paraffin immunohistochemistry were calculated. In 93 cases where RIHC was performed, 85 cases (91%) were proven to be helpful for the diagnosis. A total of 58 antibodies were used for RIHC 276 times, of which 19 antibodies were not effective 25 times. Fifteen RIHC antibody panels are recommended based on staining stability and utilization frequency. After improving the staining method, ultrasonic thermal steam heating RIHC is practical, convenient, and cost-effective, making it suitable for use in any pathology department with routine immunohistochemistry reagents. It plays an important auxiliary role in improving the accuracy of intraoperative rapid pathologic diagnosis.

Hypertriglyceridemia in Apoa5-/- mice results from reduced amounts of lipoprotein lipase in the capillary lumen.

Why apolipoprotein AV (APOA5) deficiency causes hypertriglyceridemia has remained unclear, but we have suspected that the underlying cause is reduced amounts of lipoprotein lipase (LPL) in capillaries. By routine immunohistochemistry, we observed reduced LPL staining of heart and brown adipose tissue (BAT) capillaries in Apoa5-/- mice. Also, after an intravenous injection of LPL-, CD31-, and GPIHBP1-specific mAbs, the binding of LPL Abs to heart and BAT capillaries (relative to CD31 or GPIHBP1 Abs) was reduced in Apoa5-/- mice. LPL levels in the postheparin plasma were also lower in Apoa5-/- mice. We suspected that a recent biochemical observation - that APOA5 binds to the ANGPTL3/8 complex and suppresses its capacity to inhibit LPL catalytic activity - could be related to the low intracapillary LPL levels in Apoa5-/- mice. We showed that an ANGPTL3/8-specific mAb (IBA490) and APOA5 normalized plasma triglyceride (TG) levels and intracapillary LPL levels in Apoa5-/- mice. We also showed that ANGPTL3/8 detached LPL from heparan sulfate proteoglycans and GPIHBP1 on the surface of cells and that the LPL detachment was blocked by IBA490 and APOA5. Our studies explain the hypertriglyceridemia in Apoa5-/- mice and further illuminate the molecular mechanisms that regulate plasma TG metabolism.

Abstract C092: Genetic profiling of high grade serous ovarian cancer among women of African ancestry

Abstract (A) AN INTRODUCTORY SENTENCE INDICATING THE PURPOSES OF THE STUDY: Ovarian cancer (OC) is a heterogeneous disease with high grade serous ovarian cancer (HGSOC) as the commonest, the most aggressive tumor, which is often diagnosed at advanced stages, and with poor overall survival. Unfortunately, like many other cancer diagnoses, women of West African ancestry (Black women) with ovarian cancer experience worse outcomes than White women. In the US, Black women have higher morbidity and mortality rates and higher un-staged or unclassified tumors compared with White women, resulting in undertreatment with subsequent compromise in progression-free survival. Genomic instability is one of the most common underlying features of ovarian cancer and defective DNA damage repair has been implicated. The goal is to uncover canonical genes associated with the aggressive behavior of HGSOC in a cohort of black women that could explain the biology of this tumor. (B) A BRIEF DESCRIPTION OF PERTINENT EXPERIMENTAL PROCEDURES: Formalin fixed paraffin embedded (FFPE) HGSOC tissue blocks from Nigeria in West Africa (WAB), Bahama in Caribbean (CBB), and Miami in USA (USB) were identified and selected after rigorous review and confirmation with immunohistochemical (IHC) markers when needed. Integration of digital histopathology and IHC markers (estrogen receptor, progesterone receptor, Ki67 and P53), whole exome sequence, RNA sequence of the FFPE samples was used to identify the proportion of genomic instability and defective DNA damage repair genes. We use spatial genomic library on tissue microarray (TMA) slide prepared on GeoMx digital spatial profiler (DSP) Nanostring platform to study the biology of the tumor and the tumor microenvironment (TME) and proteogenomics.(C) A SUMMARY OF THE NEW, UNPUBLISHED DATA: The unsupervised principal component analysis of the tumor showed a distinct population of WAB and USB while the CBB showed admixture population. BACH2, ARG1, NAFT2 and FRA10A1 are commonly differentially expressed genes among the cohort. There is low expression of routine IHC markers among the WAB compared to the other groups. Conclusion: There is variation on gene expression by nativity and we hope to explore this variation for therapeutic purpose among black women with ovarian cancer. Citation Format: Ayodele Omotoso, Uchenna Ezenkwa, Raleigh Butler, Melissa Castillo, Alex Sanchez-Covarrubias, Matthew Schlumbrecht, Andre Pinto, Bala Audu, Sophia George. Genetic profiling of high grade serous ovarian cancer among women of African ancestry [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C092.

Kaposi's sarcoma: Problems of differential diagnosis

Kaposi's sarcoma is a multifocal malignant disease of vascular origin with a primary lesion of the skin, as well as internal organs and lymph nodes. According to various authors, it is believed that Kaposi's sarcoma is associated with herpes simplex virus type 8 (HHV-8).
 According to the literature, the following types of Kaposi's sarcoma are distinguished: classical (European), immunosuppressive, endemic (African) and epidemic (this form of sarcoma is associated with the state of acquired immunodeficiency). Clinically, with Kaposi's sarcoma, rashes can vary depending on the form of the disease, among which there are spotty, papular and tumor forms. Also in the literature, some authors distinguish the 4th form of Kaposi's sarcoma ― bullous.
 Diagnostic search for Kaposi's sarcoma is a difficult problem due to the large number of diseases of vascular origin, as well as their clinical similarity. The diagnostic methods described in the literature include many studies, including a blood test for HHV-8, as well as routine histological examination and immunohistochemistry.
 The article presents a rare clinical case of Kaposi's sarcoma in a young girl with a long history of the disease.

Immunohistochemical expression of TROP‑2 (TACSTD2) on the urothelial carcinoma of the urinary bladder and other types of cancer.

In metastatic or locally advanced urothelial carcinoma (UC), therapeutic options have been limited to chemotherapy and immune checkpoint inhibitors. Novel targets and drugs such as antibody drug conjugates have been developed, and enfortumab vedotin targeting nectin-4 and sacituzumab govitecan (SG) targeting trophoblast cell surface antigen 2 (TROP-2), the protein product of the TACSTD2 gene, have been approved. The expression of TROP-2 was investigated within UC and other types of carcinomas, and within the tissue of different healthy organs to understand treatment responses and toxicities. The expression of TROP-2 in the tissues of 42 patients with UC, 13 patients with other types of cancer and in the normal tissues of 11 patients was retrospectively analyzed. Immunohistochemical staining of the TROP-2 protein was performed on a BenchMark ULTRA IHC/ISH System (Roche Tissue Diagnostics; Roche Diagnostics, Ltd.) according to accredited staining protocols in a routine immunohistochemistry accredited and certified facility of the laboratory of immunohistochemistry at the Institute of Pathology (Gerhard-Domagk Institute)- University Hospital Muenster (UKM)-Muenster-Germany]. Different expression levels of TROP-2 were observed, and the highest expression rate of TROP-2 was observed in UC, independent of the tumor stage. However, normal urothelial cells had similar expression levels. Except for ductal carcinoma in situ, the expression of TROP-2 was reduced in other types of cancer and in the healthy tissues from other organs, including pancreas, gall bladder, colon and prostate. Given the treatment response based on the expression level of TROP-2, SG would be effective in almost all cases of UC. However, it would also have an effect on the normal urothelium.

The significance of immune microenvironment in patients with endometriosis.

Endometriosis represents an estrogen-dependent disease of the female reproductive system and intra- and extraperitoneal regions, with chronic feature. Currently, immune cells, such as macrophages and lymphocytes, are considered to play a pivotal role in angiogenesis and invasion of endometriotic cells through matrix remodeling. Additionally, various studies have revealed the role of E-cadherin, β-catenin, along with steroid hormone receptors in endometriosis development. In this context, our study aimed to analyze the relationship between the cellular immune profile and E-cadherin, β-catenin, estrogen receptor alpha (ERα), and progesterone receptor (PR) immunoexpression in endometriosis tissues, along with an analysis of the possible association between serological parameters and immunohistochemical (IHC) markers. The study included 53 patients diagnosed with ovarian or cutaneous abdominal wall endometriosis, which have been investigated by routine histology, immunohistochemistry, and serum analysis. The IHC exam showed an increased density of cluster of differentiation (CD)4+ T-cells, CD8+ T-cells, and CD68+ macrophages, along with variable increased expressions of E-cadherin, β-catenin, ERα, and PR. Statistical analysis revealed an intense positive correlation between CD68 and PR expression (p<0.05), without any other statistically significant correlations between IHC markers or between IHC and serological markers. Our study supports that endometriosis is an immune-dependent disease characterized by an abnormal morphological profile of T-cells and macrophages in endometriotic implants. Our study provides additional data useful in the understanding the immune milieu of endometriosis in the context of its complex pathogenic molecular mechanism. Further research is needed to develop new immunological therapeutic approaches, like immune checkpoint inhibitors administration or T-cell-targeted immunotherapy in these patients.

Effects of Calcium, Available Phosphorus and Microbial Phytase on Ovarian FSHR and LHR Expression in Laying Hens

Folliculogenesis, steroidogenesis, ovulation, and vitellogenesis are regulated by the effect of follicle stimulating hormone (FSH) and luteinizing hormone (LH) in the hypothalamus-pituitary-ovary axis and these hormones act via follicle stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) in the ovary. Poultry ration and food additives are essential in the regulation of reproductive activity. Phytase is a supplement frequently added to laying hen diets to increase phosphorus (P) utilization. The aim of this study was to reveal the effects of a newly isolated microbial phytase together with different concentrations of calcium (Ca2+) and available phosphorus (AP) on ovarian FSHR and LHR expressions. For this purpose, 90 Lohmann LSL-White layers were first divided into three main diet groups (standard Ca2+ and AP, standard Ca2+ and low AP, low Ca2+ and AP) and then into three subgroups (no-phytase, commercial phytase, and microbial phytase). At the end of the experiment, all chickens were slaughtered and ovarian tissues were fixed in formalin. Routine avidin-biotin complex immunohistochemistry was performed using anti-FSHR and anti-LHR primary antibodies. Immunohistochemically, FSHR and LHR were expressed in granulosa/theca cells, oocytes, interstitial cells, and vitellus. While the expression intensity of the receptors increased in the microbial phytase-treated groups, the strongest expression was obtained in the granulosa/theca cells and oocytes in the standard Ca and low AP group. In conclusion, we suggest that the addition of newly isolated microbial phytase to diets of laying hens and feeding standard Ca and low AP may have positive effects on reproductive performance by increasing the FSHR and LHR expression in ovaries.

Histomorphometric analysis of angiogenesis in oral submucous fibrosis and oral squamous cell carcinoma associated with oral submucous fibrosis

Background: Oral submucous fibrosis (OSMF), now worldwide acknowledged as the disease of “Southeast Asia and Indian subcontinent”, has the utmost malignant transformation prevalence amongst oral precancerous disorders. Increased vascularity that is neoangiogenesis has been observed in the superficial connective tissue region of pre-cancerous lesions showing dysplasia. This could be an initial occurrence of the carcinogenesis process. The aim of this study will be to analyse neoangiogenesis by examining immunoexpression of CD105 in OSMF, and OSCC with OSMF cases. Methods: The project will comprise 30 normal oral mucosa samples (group I), 30 clinicopathologically diagnosed cases of OSMF (group II), and on the basis of association of OSMF, 30 surgically operated and histopathologically diagnosed cases of OSCC associated with OSMF (group III). Hematoxylin and eosin stains will be used for routine staining procedures and immunohistochemistry for the expression of CD105. Oral epithelial dysplasia (OED) cases of OSMF will be categorized into two groups, low-risk epithelial dysplasia (LRED) and high-risk epithelial dysplasia (HRED). The micro vessel density (MVD), total microvessel area (TVA), and mean microvessel area (MVA) are within and surrounding the tumor tissue sections immunostained with the CD105 antibody will be determined. ANOVA will be applied for evaluation of the mean scores of MVD, TVA, and MVA of groups II and III. The obtained score will be compared with different parameters of OSCC (TNM stage, lymph node metastasis, and histopathological grades). Conclusions: We postulate the progressively increased vascularity with the disease progression from LRED to HRED and further its transformation to invasive oral squamous cell carcinoma This increased vascularity will be evident by enhanced MVD, TVA and mean MVA, which is expressed by CD105 immunoexpression. This observation will emphasize the significance of neoangiogenesis in cases of OSMF with epithelial dysplasia and its further progression to OSCC.

Artificial intelligence for HPV status prediction based on disease-specific images in head and neck cancer: A systematic review and meta-analysis.

Accurate early detection of the human papillomavirus (HPV) status in head and neck cancer (HNC) is crucial to identify at-risk populations, stratify patients, personalized treatment options, and predict prognosis. Artificial intelligence (AI) is an emerging tool to dissect imaging features. This systematic review and meta-analysis aimed to evaluate the performance of AI to predict the HPV positivity through the HPV-associated diseased images in HNC patients. A systematic literature search was conducted in databases including Ovid-MEDLINE, Embase, and Web of Science Core Collection for studies continuously published from inception up to October 30, 2022. Search strategies included keywords such as "artificial intelligence," "head and neck cancer," "HPV," and "sensitivity & specificity." Duplicates, articles without HPV predictions, letters, scientific reports, conference abstracts, or reviews were excluded. Binary diagnostic data were then extracted to generate contingency tables and then used to calculate the pooled sensitivity (SE), specificity (SP), area under the curve (AUC), and their 95% confidence interval (CI). A random-effects model was used for meta-analysis, four subgroup analyses were further explored. Totally, 22 original studies were included in the systematic review, 15 of which were eligible to generate 33 contingency tables for meta-analysis. The pooled SE and SP for all studies were 79% (95% CI: 75-82%) and 74% (95% CI: 69-78%) respectively, with an AUC of 0.83 (95% CI: 0.79-0.86). When only selecting one contingency table with the highest accuracy from each study, our analysis revealed a pooled SE of 79% (95% CI: 75-83%), SP of 75% (95% CI: 69-79%), and an AUC of 0.84 (95% CI: 0.81-0.87). The respective heterogeneities were moderate (I2 for SE and SP were 51.70% and 51.01%) and only low (35.99% and 21.44%). This evidence-based study showed an acceptable and promising performance for AI algorithms to predict HPV status in HNC but was not comparable to the routine p16 immunohistochemistry. The exploitation and optimization of AI algorithms warrant further research. Compared with previous studies, future studies anticipate to make progress in the selection of databases, improvement of international reporting guidelines, and application of high-quality deep learning algorithms.

Evaluation of the routine use of E-cadherin immunohistochemistry in the typing of breast carcinomas: results of a randomized diagnostic study.

Invasive lobular carcinoma (ILC) has distinct morphology and association with loss of E-cadherin function. It has special clinical and imaging features, and its proper recognition is important. Following a recent proposal, we tested the value of the routine useofE-cadherin immunohistochemistry (IHC) in recognizing ILC. Five pathologists with experience in breast pathology from four Hungarian institutions histotyped 1001 breast cancers from diagnostic core biopsies or excision specimens randomly assigned to haematoxylin and eosin (HE) diagnosis first, followed by E-cadherin IHC; or to immediate HE and E-cadherin-based diagnosis. Of 524 cases with HE diagnosis, 73(14%) were deemed uncertain. E-cadherin made the initial histological type change in 14/524 cases (2.7%), including three with confident HE-based type allocation. Use of E-cadherin immunostaining was considered useful in 88/477 cases (18%) with immediate dual assessment, and typing uncertainty went down to 5% (25/477 cases), but was not zero. Collective assessment of 171 uncertain, difficult, nonclassical cases resulted in consensus diagnosis in most cases, but 15 cases were still doubtful as concerns their proper histological type. CDH1 gene sequencing was attempted and successful in 13; pathogenic genetic alterations were identified in seven cases. The routine use of E-cadherin IHC decreases the uncertainty in typing and improves the typing accuracy at the cost of potentially redundant additional immunostains. Furthermore, this procedure does not exclude uncertainty due to E-cadherin-positive ILCs, which are occasionally difficult to confidently label as ILC, especially when the growth pattern is not classic.

Does hyperthermic povidone-iodine lavage increase the apoptotic rate of residual cancer cell in patients with malignant pleural mesothelioma?-a prospective pilot study.

Malignant pleural mesothelioma (MPM) is an incurable, late presenting primary cancer, conferring a survival of 8-14 months. Different intrapleural treatments have been tested as part of a multimodality approach to treat a select group of patients with limited disease, increasing survival. Recently, povidone-iodine has been shown to induce apoptosis in microscopic tumour cells in vitro, with no reported complications. This is the first in vivo study assessing the apoptotic rate caused by intraoperative hyperthermic betadine lavage using routine immunohistochemistry combined with transmission electron microscopy (TEM). We included surgically fit patients aged >18, undergoing minimally invasive video-assisted thoracoscopic surgery (VATS) pleural biopsy between December 2016 and February 2018, for confirmed or presumed pleural malignancy. Parietal pleural biopsies were obtained at 7.5, 15 and 30 minutes after hyperthermic betadine lavage, and compared to pre-lavage biopsy samples, for apoptotic changes. Viable tumour samples underwent histological, immunohistochemical and ultrastructural analysis as well as TEM for features of apoptosis. N=6. Median age was 76 years. Median overall survival was 26.7 months. There was no statistical impact on survival of side of disease (left vs. right). There was no significant difference in expressions of markers of apoptotic index pre and post betadine treatment upon immunohistochemical analysis. There was no discernible effect on morphological features of apoptosis seen with betadine treatment, on TEM analysis. No side effects were identified post betadine lavage. Although hyperthermic betadine lavage is a safe antiseptic solution with no toxicity when performed intraoperatively, it confers no effect on apoptotic rate or necrosis. It is therefore unlikely that hyperthermic betadine lavage will have an impact on reducing the microscopic residual disease after pleurectomy decortication and enhancing survival.

Differentiating Thyroid Follicular Adenoma from Follicular Carcinoma via G-Protein Coupled Receptor-Associated Sorting Protein 1 (GASP-1).

Follicular neoplasms are classified as benign or malignant depending on the presence or absence of capsular and/or vascular invasion. Due to incomplete capsular penetration or equivocal vascular invasion, the evaluation of these features can be challenging using histologic examination. In the current study, we analyzed the involvement of G-protein coupled receptor-associated sorting protein 1 (GASP-1) in the development and progression of thyroid neoplasms. Affinity-purified anti-GASP-1 polyclonal antibodies were used for routine immunohistochemistry (IHC) analysis. Thyroid tissue microarrays containing normal thyroid tissue, follicular adenoma, follicular carcinoma, papillary thyroid carcinoma, and anaplastic carcinoma were analyzed. We found that the level of GASP-1 expression can differentiate follicular adenoma from follicular carcinoma. When numerous cases were scored for GASP-1 expression by a board-certified pathologist, we found that GASP-1 expression is 7-fold higher in thyroid malignant neoplasms compared to normal thyroid tissue, and about 4-fold higher in follicular carcinoma compared to follicular adenoma. In follicular adenoma tissues, we observed the presence of many mini-glands that are enriched in GASP-1 and some mini-glands contain as few as three cells. GASP-1 IHC also possesses several advantages over the conventional H&E and can be used to identify early thyroid cancer and monitor cancer progression.

Molecular classification of muscle-invasive bladder cancer based on a simplified immunohistochemical panel using GATA3, CK5/6 and p16.

The choice of therapy for muscle-invasive bladder cancer (MIBC) could be influenced by the tumor's molecular subtype. Currently, well-defined consensus subtypes are based on tumor microarray mRNA data. Clearly defined and easy-to-use surrogate molecular subtypes, based on immunohistochemistry (IHC) performed on whole slides, are needed to make subtyping cost-effective and useful in routine work and future research. To aid in the development of a simple immunohistochemical classifier, a retrospective single-center series of 92 cases of localized bladder cancer was identified. Routine IHC for GATA3, cytokeratins 5 and 6 (CK5/6), and p16 was performed on whole tissue blocks containing muscle-invasive disease. Electronic medical records were retrieved and searched for clinical variables, treatment, and survival data. The mean age was 69.6 years, and 73% were males. Conservative treatment was used in 55% of cases, while cystectomy with chemotherapy was used in 45%. GATA3 and CK5/6 expression divided cases into broad luminal and basal subtypes, respectively, while p16 expression was used to subclassify luminal cases into luminal papillary and luminal unstable types according to the consensus molecular classification. When subtyped in this way, GATA3 and CK5/6 negative cases showed worse overall survival. Molecular subtyping of MIBC on whole slides containing muscle-invasive tumor using only three commonly used, consensus-based antibodies, is a feasible and cost-effective method for detecting subtypes of invasive bladder cancer. Future work combining morphological analysis and IHC is needed to fully translate the consensus molecular classification into a comprehensive, cost-effective subtyping strategy.

Gastric amphicrine carcinoma: A histopathological diagnostic conundrum

Amphicrine carcinoma is rarely reported in literature. It is characterised by dual positivity of neuroendocrine markers and mucin stain in the same cells depicting divergent differentiation. Recently published WorldHealth Organisation (WHO) classification of neuroendocrine neoplasm of 2022 differentiates it from adenocarcinoma and mixed neuroendocrine-nonneuroendocrine neoplasms (MiNEN) it shows varied histomorphology and on rare occasions neuroendocrine features may not be evident in poorly differentiated carcinoma. Hence, diagnosis based solely on histomorphology can be misleading. This is a case of amphicrine carcinoma in an elderly female which showed poorly differentiated morphology along with signet ring cells. Same cells demonstrated immunopositivity for synaptophysin, chromogranin A and were also positive for mucin stains (PAS, PAS-D and Alcian blue). This case report, thus, emphasises the importance of neuroendocrine markers as a part of routine immunohistochemistry in poorly differentiated gastric carcinomas to aid diagnosis of amphicrine carcinoma.

Histotripsy ablation for the treatment of feline injection site sarcomas: a first-in-cat in vivo feasibility study

Purpose Feline soft tissue sarcoma (STS) and injection site sarcoma (fISS) are rapidly growing tumors with low metastatic potential, but locally aggressive behavior. Histotripsy is a non-invasive focused ultrasound therapy using controlled acoustic cavitation to mechanically disintegrate tissue. In this study, we investigated the in vivo safety and feasibility of histotripsy to treat fISS using a custom 1 MHz transducer. Materials and Methods Three cats with naturally-occurring STS were treated with histotripsy before surgical removal of the tumor 3 to 6 days later. Gross and histological analyses were used to characterize the ablation efficacy of the treatment, and routine immunohistochemistry and batched cytokine analysis were used to investigate the acute immunological effects of histotripsy. Results Results showed that histotripsy ablation was achievable and well-tolerated in all three cats. Precise cavitation bubble clouds were generated in all patients, and hematoxylin & eosin stained tissues revealed ablative damage in targeted regions. Immunohistochemical results identified an increase in IBA-1 positive cells in treated tissues, and no significant changes in cytokine concentrations were identified post-treatment. Conclusions Overall, the results of this study demonstrate the safety and feasibility of histotripsy to target and ablate superficial feline STS and fISS tumors and guide the clinical development of histotripsy devices for this application.

Hist2RNA: An Efficient Deep Learning Architecture to Predict Gene Expression from Breast Cancer Histopathology Images.

Gene expression can be used to subtype breast cancer with improved prediction of risk of recurrence and treatment responsiveness over that obtained using routine immunohistochemistry (IHC). However, in the clinic, molecular profiling is primarily used for ER+ breast cancer, which is costly, tissue destructive, requires specialised platforms, and takes several weeks to obtain a result. Deep learning algorithms can effectively extract morphological patterns in digital histopathology images to predict molecular phenotypes quickly and cost-effectively. We propose a new, computationally efficient approach called hist2RNA inspired by bulk RNA sequencing techniques to predict the expression of 138 genes (incorporated from 6 commercially available molecular profiling tests), including luminal PAM50 subtype, from hematoxylin and eosin (H&E)-stained whole slide images (WSIs). The training phase involves the aggregation of extracted features for each patient from a pretrained model to predict gene expression at the patient level using annotated H&E images from The Cancer Genome Atlas (TCGA, n = 335). We demonstrate successful gene prediction on a held-out test set (n = 160, corr = 0.82 across patients, corr = 0.29 across genes) and perform exploratory analysis on an external tissue microarray (TMA) dataset (n = 498) with known IHC and survival information. Our model is able to predict gene expression and luminal PAM50 subtype (Luminal A versus Luminal B) on the TMA dataset with prognostic significance for overall survival in univariate analysis (c-index = 0.56, hazard ratio = 2.16 (95% CI 1.12-3.06), p < 5 × 10-3), and independent significance in multivariate analysis incorporating standard clinicopathological variables (c-index = 0.65, hazard ratio = 1.87 (95% CI 1.30-2.68), p < 5 × 10-3). The proposed strategy achieves superior performance while requiring less training time, resulting in less energy consumption and computational cost compared to patch-based models. Additionally, hist2RNA predicts gene expression that has potential to determine luminal molecular subtypes which correlates with overall survival, without the need for expensive molecular testing.

Color Deconvolution as a Simple and Rapid Tool in Quantitative Biomedical Research

The quality, efficiency, and speed of a quantitative analysis are critical factors in cytopathology. In this scenario, reliable and reproducible tools are needed to improve results in a shorter period,[1] mainly routine immunohistochemistry (IHC) slides and biomedical research.

Multiorgan Involvement of Dormant Uveal Melanoma Micrometastases in Postmortem Tissue From Patients Without Coexisting Macrometastases.

Almost half of all patients diagnosed with uveal melanoma will die of metastatic disease. This has been attributed to early seeding of micrometastases. We investigate the presence, density, organ involvement, and characteristics of micrometastases of uveal melanoma in tissue obtained at autopsy of patients with and without coexisting macrometastases. Patients diagnosed with primary uveal melanoma at a national referral center between 1960 and 2020 (n = 4,282) were cross-referenced with autopsy registers at nearby hospitals. Eleven patients were included. Formalin-fixed, paraffin-embedded tissue samples obtained during autopsy were examined with routine histology, immunohistochemistry, and immunomagnetic separation. Micrometastases were detected in 5 of 5 patients with and in 5 of 6 patients without coexisting macrometastases. Micrometastases were identified in several sites, including lungs, kidneys, myocardium, and bone marrow. Their highest density per mm2 of tissue was seen in the liver. Of 11 examined patients, 2 had at least 1 BAP-1-positive metastasis. All micrometastases had immune cell infiltrates and no or very low proliferative activity. We demonstrate multiorgan involvement of apparently dormant micrometastases in patients with uveal melanoma. This suggests that micrometastases are present in nearly all patients diagnosed with primary uveal melanoma, regardless of coexisting macrometastases.

MP14-15 MACROPHAGES AND REGULATORY T-CELLS AS PROGNOSTIC FACTORS IN PATIENTS WITH UROTHELIAL MUSCLE-INVASIVE BLADDER CANCER

You have accessJournal of UrologyCME1 Apr 2023MP14-15 MACROPHAGES AND REGULATORY T-CELLS AS PROGNOSTIC FACTORS IN PATIENTS WITH UROTHELIAL MUSCLE-INVASIVE BLADDER CANCER Florestan Koll, Severine Banek, Luis Kluth, Jens Köllermann, Andreas Weigert, Felix Chun, Peter Wild, and Henning Reis Florestan KollFlorestan Koll More articles by this author , Severine BanekSeverine Banek More articles by this author , Luis KluthLuis Kluth More articles by this author , Jens KöllermannJens Köllermann More articles by this author , Andreas WeigertAndreas Weigert More articles by this author , Felix ChunFelix Chun More articles by this author , Peter WildPeter Wild More articles by this author , and Henning ReisHenning Reis More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003234.15AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The prognosis of muscle-invasive urothelial bladder cancer (MIBC) is poor and response rates to systemic therapy are insufficient. Infiltration of tumors by immune cells has been described to be associated with response rates to immune- and chemotherapy and patient’s outcome. However, tumor-associated macrophages (TAMs) are abundant in many tumors and can promote cancer progression. In this study we evaluate different subsets of immune cells in the tumor microenvironment (TME) as predictors of prognosis in MIBC and survival rates with adjuvant chemotherapy. METHODS: We used multiplex immunohistochemistry (IHC) to quantify and characterize immune, tumor and stroma cells: double negative T-cells (CD3+CD4-CD8-); T-helper cells (CD4+); cytotoxic T-cells (CD8+); PD-1 positive T-cells; macrophages (CD163+); regulatory T-cells (Tregs; FoxP3+)); fibroblasts (Vimentin+); immune cells (CD45+); tumor cells (panCK+); PD-L1 positive tumor cells; proliferating immune cells (CD45+Ki67+); proliferating tumor cells (panCK+Ki67+). Analysis was performed in 101 MIBC patients receiving radical cystectomy. Uni- and multivariate Cox regression were used to identify cell types that predict prognosis. Patients were divided into three clusters for their macrophage and Treg infiltration. RESULTS: High Treg infiltration was associated with better overall survival (OS) (HR 0.1, 95% CI 0.01-0.7; p=0.03) and high macrophage infiltration was associated with decreased OS (HR 10.9, 95% CI 2.8-40.5; p=0.0004) in the multivariate Cox-regression model adjusting for tumor stage, lymph node status and application of adjuvant chemotherapy. The cluster with high Treg concentration was also enriched for other immune cells and cells showed high PD1 and PD-L1 expression. Patients in the cluster with high macrophage concentration had the worst OS also when adjuvant chemotherapy was given. We validated the findings using standardized IHC for CD163 as a macrophage marker using a digital analysis software in 141 patients. CONCLUSIONS: In MIBC high macrophage concentration is an independent predictor of poor prognosis. High levels of Tregs are associated with other immune cells in the TME which might lead to better survival rates. Using routine IHC for TAMs (CD163) should be prospectively validated to confirm findings and to evaluate the predictive value for response to systemic therapies. Source of Funding: Deutsche Krebshilfe (German Cancer Aid). Wilhelm-Sander Foundation. Hessen State Ministry for Higher Education, Research and the Arts © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e188 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Florestan Koll More articles by this author Severine Banek More articles by this author Luis Kluth More articles by this author Jens Köllermann More articles by this author Andreas Weigert More articles by this author Felix Chun More articles by this author Peter Wild More articles by this author Henning Reis More articles by this author Expand All Advertisement PDF downloadLoading ...

Immunohistochemical Profiling of HER-2/neu, Steroid Hormone Receptors and KI-67 Biomarkers in A Cohort of Nigerian Women with Breast Cancer.

Progressive improvement in the accuracy of profiling of hormone receptors in breast cancer provides the basis for targeted endocrine therapy, a major pillar of multimodal breast cancer treatment. However, the disparity in findings from comparatively smaller sample-sized studies in West Africa has led to somewhat conflicting conclusions and recommendations. This study investigates the immunohistochemical (IHC) profile of breast cancer specimens for estrogen receptor (ER), progesterone receptor (PR), human epidermal receptor-2 (HER2)/neu, and Ki-67 in a tertiary hospital in Ibadan, Nigeria over 12 years. We reviewed 998 IHC reports, documented clinicopathologic parameters, computed patterns of biomarkers, and stratified them based on the American Society of Clinical Oncology/College of American Pathologists recommendations. Descriptive analysis including frequency, mean, and median were generated from the data extracted. Out of the 998 cases, 975 (97.7%) were females and 23 (2.3%) were males. The mean age was 48.84 ± 11.99 years. Open biopsies were the most common types of specimens (320, 41.6%): lumpectomy and incisional biopsy of ulcerated, fungating or unresectable tumours. In those cases, 246 (32.0%) were samples of breast-conserving or ablative surgical extirpation (mastectomy/wide local excision/quadrantectomy), and 203 (26.4%) were obtained by core needle biopsies. Invasive ductal carcinoma was the most common histopathological type (673, 94.5%). The majority of graded tumours were intermediate grade (444, 53.5%). Four hundred and sixty-nine (48.4%) were ER positive, 414 (42.8%) were PR positive, and 180 (19.4%) were HER2/neu positive. Three hundred and thirty-four (34.0%) were triple-negative. Eighty-nine cases had Ki-67 staining done, and of these 61 (68.5%) had positive nuclear staining. Steroid hormone receptors and HER-2/neu proportions in our cohort are likely to be more representative than the widely varied figures hitherto reported in the sub-region. We advocate routine IHC analysis of breast cancer samples as a guide to personalized endocrine therapy.