Staining Of Tumor Cells Research Articles

Evidence for Unified Assessment Criteria of HER2 IHC in Colorectal Carcinoma

HER2 expression is an important biomarker for the management of RAS wild-type metastatic colorectal carcinoma (CRC). Immunohistochemistry (IHC) with reflex in situ hybridization (ISH) is accepted as a standard method of assessment, yet there are currently two sets of criteria used to interpret results: the HERACLES criteria, and the My Pathway criteria. The HERACLES criteria require ISH confirmation when IHC staining is 3+ in 10-49% of cells, while the My Pathway criteria mirror those for gastric HER2 assessment and do not recommend ISH confirmation in the previously referenced scenario. We aimed to assess the prevalence of HER2 3+ heterogeneity and its association with ERBB2 copy number amplification (CNA) to evaluate the necessity of ISH testing when IHC staining is 3+ in <50% of cells.Next-generation sequencing (NGS) of DNA (592-gene panel or whole exome sequencing; WES) was performed for 13 208 colorectal carcinoma (CRC) tumors submitted to Caris Life Sciences (Phoenix, AZ). HER2 (4B5) expression was tested by IHC. A subset of tumors was tested for ERBB2 amplification via chromogenic ISH and/or via NGS (copy number amplification [CNA]). X2/Fisher-Exact tests were applied where appropriate, with p-values adjusted for multiple comparisons (p< 0.05).Of 13 208 CRCs with HER2 IHC, 87.4% (11 541/13 208) were negative for HER2 expression (intensity ≤3+ and < 10% tumor-cell staining) and 11.2% (1 473/13 208) demonstrated at least low HER2 expression (1-2+ and ≥10%). Only 1.5% (194/13 208) of all tested tumors were either positive or heterogeneously positive for HER2 overexpression (3+, ≥10%). Of these, 14% (28/194) had heterogenous HER2 overexpression (3+ staining of 10-49% of cells). Among 22 HER2-positive/heterogenous cases with successful ISH testing, 100% (22/22) demonstrated amplification via ISH.Because the classification of tumors as HER2-positive/heterogenous by IHC correlated very closely with ISH positivity, our results suggest that ISH is likely unnecessary for CRCs with 3+ HER2 overexpression in 10-49% of neoplastic cells.

Immunohistochemical Expression of Cyclin D1 in Invasive Breast Carcinoma

Objective: This study was conducted to determine the immunohistochemical expression of cyclin D1 in invasive breast carcinoma and its association with already established prognostic parameters like estrogen receptor (ER), progesterone receptor (PR), HER2/Neu, and Ki67 status. Study Design &amp; Setting: Cross sectional Observational. Department of Pathology, Armed Forces Institute of Pathology, Rawalpindi Methodology: The study included 350 cases of invasive breast cancer diagnosed between January 2023 and December 2023. Data collected included patient age, histological subtype, molecular subtypes, tumor size, and the presence of estrogen (ER) and progesterone (PR) receptors, as well as HER2/Neu and Ki67 status. Patients who had undergone chemotherapy, received radiation to the breasts, or experienced relapse were excluded from the study. Immunohistochemistry was conducted using a Cyclin D1 antibody to assess Cyclin D1 expression in tissue samples. The expression levels were categorized as negative, weak, moderate, strong staining in tumor cells. Data analysis was performed using SPSS 29.0, and statistical comparisons were made between Cyclin D1 staining and ER, PR, HER2/Neu, and Ki67 status. Results: Cyclin D1 moderate to strong staining was seen in 173/352 (49.14%) cases of invasive BC. Cyclin D1 expression was slightly statistically significantly associated with ER (x2 = 7.78, P value &lt;0.051) and Ki67 positivity (÷2 = 7.27, P value &lt;0.064). Conclusion: Cyclin D1 has the potential to serve as a prognostic marker. Incorporating it into the routine IHC workup for breast cancer could enhance patient management, especially with the development of new targeted therapies that inhibit the Cyclin D-CDK4/6 axis.

8401 Genetic Characterization of a Case of Primary Aldosteronism in an 11-Year-Old Girl carrying a Germline VUS of CACNA1H and a Somatic Mutation of CTNNB1

Abstract Disclosure: S. Parisien-La Salle: None. G. Van Vliet: None. G. Corbeil: None. M. Labrecque: None. M. Tétreault: None. I. Bourdeau: None. Introduction: Primary aldosteronism is a frequent cause of secondary hypertension in adults but is rare in children. We report the case and genetic studies of an 11 yo girl presenting with primary aldosteronism at puberty. Case presentation: An 11-year-old female patient was referred to the pediatric endocrinology clinic for hypertension (166/118). Her past medical history and family history were unremarkable. She reported polyuria, polydipsia and occasional headaches and abdominal pain. Her Tanner stage indicated that puberty was initiated (P2M2). An abdominal CT revealed a 9x10x11cm right adrenal mass with calcifications and a necrotic zone. On [1]8F-FDG PET/CT, the mass had an uptake of 2.9 SUVmax. Laboratory results revealed hypokalemia (K: 2.6 mmol/L [N: 3.5-4.5]) and elevated aldosterone (1712.8 pmol/L [N: 110-1330]) with suppressed renin (0.2 ng/ml/h [N: &amp;lt;5.3]). Her urinary aldosterone level was elevated (114 nmol/day [N: &amp;lt;67]). The rest of the biochemical adrenal panel was normal. Patient underwent a right adrenalectomy by laparotomy. The pathology report showed an adrenal tumor without capsular or vascular invasion. The only malignancy criterion was an area of necrosis and the ki-67% proliferation index was &amp;lt; 1%. Genetic investigations: After informed consent, the patient underwent germline analysis for the chimeric gene CYP11B2/CYP11B1 (Ruhr University Bochum, Germany) and a multigene panel including KCNJ5, CACNA1D, CACNA1H and CLCN2 genes (Fulgent, CA), which were both negative except for a variant of unknown significance (VUS) that was identified in CACNA1H (c.3868G&amp;gt;A, p.Val1290Ile) with a minor allele frequency of 3.291E-5 (GnomAD). Tumor DNA was extracted after adrenalectomy from fresh frozen tissues. Somatic genetic analysis of GNAS, CTNNB1, KCNJ5, ATP1A1, ATP2B3 and CACNA1D was performed using direct Sanger Sequencing (Genome Quebec Innovation Centre) and revealed a heterozygous missence pathogenic variant in the CTNNB1 gene (c.121 A&amp;gt;G p.Thr41Ala). This mutation was absent in leukocyte DNA of the patient. Immunohistochemistry of the adrenal tumor disclosed strong cytoplasmic β-catenin staining in most tumor cells (monoclonal anti-β-catenin antibody; BD Transduction Laboratories). RNA-sequencing of patient tumor RNA was performed (Research Center CHU Québec) and compared to human adrenal total RNA (Clontech, Mountain View, CA). Pathway enrichment analysis showed activation of the Wnt signaling pathway. No variant was identified in GNAQ or GNA11 genes. However, GNRHR was upregulated compared to the control sample with a log2 fold change of 3.96 (p=0.000074). Conclusion: We present a rare pediatric case of an aldosterone producing adenoma harbouring a somatic β-catenin pathogenic variant with upregulation of GNRHR. The implication of the germline CACNA1H VUS still needs to be clarified. Presentation: 6/3/2024

Aberrant CD45 Immunoreactivity in Neuroendocrine Neoplasms: A Diagnostic Pitfall-Report of 10 Specimens and Clinical Recommendations.

Leukocyte common antigen (LCA), or CD45, is classically thought of as a leukocyte-exclusive protein, and as such, CD45 immunohistochemistry (IHC) is often used as a key differentiator between non-Hodgkin lymphomas (NHLs) and morphologically similar neuroendocrine neoplasms (NENs). Herein, we report our experience regarding aberrant CD45 immunoreactivity in a series of NENs. A natural language search was used to retrieve desired archival patient files. All prior NENs which had a positive neuroendocrine diagnosis or IHC results (synaptophysin, chromogranin, CD56, and/or neuron-specific enolase), as well as CD45 staining performed, were reviewed for possible CD45 positivity (n = 686). Among these 686 NENs, 10 were aberrantly positive for CD45 staining. CD45 showed nuclear, cytoplasmic, and/or membranous staining in tumor cells. The significance of such staining is unclear. Albeit for a minority of patients, pathologists should be aware that NENs may aberrantly stain with CD45 and thereby pose a diagnostic pitfall. Therefore, broadening routine IHC panels is recommended to differentiate NENs more clearly from NHLs.

Glycoconjugate coumarins exploiting metabolism-enhanced fluorescence and preferential uptake: New optical tools for tumor cell staining

The possibility to visually discriminate cells based on their metabolism and capability to uptake exogenous molecules is an important topic with exciting fallback on translational and precision medicine. To this end, probes that combine several complementary features are necessary. The ideal probe is selectively uptaken and activated in tumor cells compared with control ones and is not fluorescent in the extracellular medium. Fluorogenic compounds that combine enzyme-activated pH sensitivity and good cell uptake can be an ideal solution, provided that the sensed enzymes are dysregulated in tumor cells. Here, we present synthesis and in vitro evaluation of a new class of glyco-coumarin based probes that merge all these features. These probes show uptake ratio in tumor vs. control cells up to 3:1, with a cell to background ratio upon administration of the probe up to 5:1. These features make this new family of fluorogenic targeted probes a promising tool in life science.

Concordance of HER2 Expression in Paired Primary and Metastatic Sites of Endometrial Serous Carcinoma and the Effect of Intratumoral Heterogeneity

Primary endometrial serous carcinoma, known for its aggressive nature and poor prognosis, shares similarities with breast and gastric cancers in terms of potential HER2 overexpression as a therapeutic target. Assessing HER expression is complicated by tumor heterogeneity and discrepancies between primary and metastatic sites. In this study, we retrospectively analyzed HER amplification and expression in 16 pairs of primary endometrial serous carcinoma resections and corresponding metastases. HER2 status was determined using immunohistochemistry (IHC), with criteria based on the percentage and intensity of tumor cell staining. Confirmatory techniques, such as dual in situ hybridization (DISH) and fluorescence in situ hybridization (FISH), were also employed. This study reports on the concordance rates and the presence and pattern of HER2 heterogeneity. Our results showed an 87.5% concordance rate in HER2 amplification status between primary and metastatic sites, with 33% of cases scored as 2+ being amplified. Heterogeneity was observed in 100% of amplified cases and 95% of non-amplified cases on in situ testing, with variations in heterogeneity patterns between techniques. In conclusion, our findings emphasize the importance of testing both primary and metastatic sites or recurrences, with a concordance rate of 87.5%. In addition, a review of the literature and combining the results showed a concordance rate of up to 68%. The presence and pattern of heterogeneity, particularly in cases of mosaic or clustered heterogeneity in the primary tumor, may serve as reliable indicators of concordance, predicting a non-amplified HER2 status in corresponding metastases.

Investigation of High Frequency Irreversible Electroporation for Canine Spontaneous Primary Lung Tumor Ablation.

In this study, the feasibility of treating canine primary lung tumors with high-frequency irreversible electroporation (H-FIRE) was investigated as a novel lung cancer treatment option. H-FIRE is a minimally invasive tissue ablation modality that delivers bipolar pulsed electric fields to targeted cells, generating nanopores in cell membranes and rendering targeted cells nonviable. In the current study, canine patients (n = 5) with primary lung tumors underwent H-FIRE treatment with an applied voltage of 2250 V using a 2-5-2 µs H-FIRE waveform to achieve partial tumor ablation prior to the surgical resection of the primary tumor. Surgically resected tumor samples were evaluated histologically for tumor ablation, and with immunohistochemical (IHC) staining to identify cell death (activated caspase-3) and macrophages (IBA-1, CD206, and iNOS). Changes in immunity and inflammatory gene signatures were also evaluated in tumor samples. H-FIRE ablation was evident by the microscopic observation of discrete foci of acute hemorrhage and necrosis, and in a subset of tumors (n = 2), we observed a greater intensity of cleaved caspase-3 staining in tumor cells within treated tumor regions compared to adjacent untreated tumor tissue. At the study evaluation timepoint of 2 h post H-FIRE, we observed differential gene expression changes in the genes IDO1, IL6, TNF, CD209, and FOXP3 in treated tumor regions relative to paired untreated tumor regions. Additionally, we preliminarily evaluated the technical feasibility of delivering H-FIRE percutaneously under CT guidance to canine lung tumor patients (n = 2). Overall, H-FIRE treatment was well tolerated with no adverse clinical events, and our results suggest H-FIRE potentially altered the tumor immune microenvironment.

Difficult Diagnosis of Interdigitating Dendritic Cell Sarcoma of the Retroperitoneum: A Case Report and A Brief Review of the Literature.

Interdigitating dendritic cell sarcoma (IDCS) is a rare and aggressive neoplasm classified within the M-group of malignant histiocytoses. Its diagnosis poses a significant challenge. This article aims to describe a rare clinical case of IDCS and to illustrate the differential diagnostic process undertaken by the authors in establishing this diagnosis. A 60-year-old woman was admitted for the resection of a retroperitoneal mass discovered via CT scan. Morphological examination revealed a 7.5×5.5×5.0 cm tumor, encapsulated by a thin fibrous capsule. The tumor was composed of 90-95% inflammatory infiltrate with lymphocyte-like cells showing mature nuclear morphology (CD3+ and CD20+ cells) mixed with histiocytes and plasma cells, and 5-10% large polymorphic spindle-shaped cells expressing expression of CD45, CD68, CD1a, CD21, CD35, CD31, and CD34. An extensive immunohistochemical panel was performed to exclude various other tumors. Based on the morphology and immunophenotype, a diagnosis of IDCS was established. Further literature analysis indicated the nonspecificity of symptoms in patients with this tumor localization and variability in CD45 and CD68 staining in tumor cells, with consistent lack of expression of CD21, CD23, CD35, CD1a, and specific T- and B-cell antigens. IDCS is a rare and poorly understood tumor with a poor prognosis. The nonspecificity of clinical symptoms and the need for extensive morphological differential diagnosis render this entity a diagnosis of exclusion, requiring significant diligence from the pathologist.

Expression and prognosis of DSG-2, CXADR, CD46 in head and neck squamous cell carcinoma

ObjectivesInvestigating the expression and prognostic significance of adenovirus receptors DSG-2, CXADR and CD46 in head and neck cancer. Methods104 patients with HNSCC (77 OPSCC, 27 LSCC) were retrospectively included in the study. Immunohistochemical staining was performed on all selected slides to detect the expression of DSG-2, CXADR, CD46 and the immunoreactive score (IRS) was determined from the number of positively stained tumor cells and their staining intensity. Furthermore, the respective HPV status was determined by immunohistochemical staining against p16 and HPV-PCR. Results81.7 % of the tumors showed DSG-2, 34.6 % of the tumors showed CXADR and 57.7 % of the tumors showed CD46 expression. A high DSG-2 IRS correlated significantly with an advanced tumor size (p= 0.003), increased grading (p=0.012) and positive HPV status (p=0.024) in OPSCC. A high CXADR IRS was significantly associated with a positive lymph node status (p= 0.041) in LSCC and an advanced AJCC stage (p= 0.012) and a positive HPV status (p= 0.009) in OPSCC. No significant correlation could be shown regarding CD46 expression and clinical tumor data. There was no effect of DSG-2, CXADR, and CD46 expression on 5-year overall and on 5-year disease-free survival. ConclusionNo prognostic significance of the expression of DSG-2, CXADR or CD46 in HNSCC was seen. DSG-2, CXADR and CD46 are expressed in HNSCC, so that optimization of oncotherapy with adenoviral vectors appears promising. Due to the significantly increased expression of DSG-2 and CXADR in advanced OPSCC tumors, there is potential for optimizing oncotherapy here in particular.

Deciphering the prognostic value of FGFR2b/2c isoform expression levels in advanced esophagogastric cancer through whole-transcriptome sequencing.

53 Background: Fibroblast growth factor receptor (FGFR) isoform switching from FGFR2b to FGFR2c has been implicated in epithelial-to-mesenchymal transition and enhanced invasive potential in various malignancies and potentially be involved in resistance mechanisms to FGFR2b targeted therapy (e.g., bemarituzumab). This study sought to investigate the potential of respective FGFR2 isoform expression evaluation utilizing whole-transcriptome sequencing (WTS) and its correlation with therapeutic efficacy and clinical outcomes in patients with advanced G/GEJ cancer. Methods: Patients with advanced G/GEJ cancer who received systemic therapy at the National Cancer Center Hospital East between May 2021 and September 2023, and were enrolled in the immunological profiling study (UMIN000019129) and MONSTAR-SCREEN-2 (UMIN000043899) were included. FGFR2 protein and RNA expression were assessed via immunohistochemistry (IHC) and WTS with Caris MI Profile, respectively. FGFR2b and FGFR2c isoforms were distinguished by the detection of their respective splice junctions using WTS data. The FGFR2b/(2b+2c) ratio was stratified into FGFR2b-dominat and -containing subtypes using the median value. The log-rank test was used to discern differences in overall survival (OS). Results: Among the 129 patients, 21 (16.3%) exhibited FGFR2-positivity by IHC (staining intensity 2+ or 3+≥1% membranous staining of tumor cells), and a significant correlation was observed between FGFR2 IHC status and RNA expression levels (TPM: transcript per million) in WTS (FGFR2-positive, median TPM=11.1; FGFR2-negative, median TPM=4.7; P=0.002). The distribution of FGFR2b/(2b+2c) ratio (median, 0.89) was: 10.9% (0≤x&lt;0.2), 5.4% (0.2≤x&lt;0.4), 7.0% (0.4≤x&lt;0.6), 12.4% (0.6≤x&lt;0.8), 43.4% (0.8≤x&lt;1.0), and 20.9% (x=1.0). Among 58 patients treated with 1st line therapy, no significant difference in treatment response was observed between the FGFR2b-dominat (67.9%) and FGFR2c-containing subtypes (53.3%) (P=0.259). Overall, the FGFR2c-containing subtype was associated with significantly shorter OS compared to the FGFR2b-dominat subtype (median OS, 10.1 vs. 17.9 months; hazard ratio, 1.72; 95% confidence interval, 1.03-2.86; log-rank P=0.037). Conclusions: Our findings underscore the substantial concordance between IHC and WTS analysis of FGFR2, the feasibility of distinguishing FGFR2 isoforms using WTS, and the prognostic implications of FGFR2c isoform in patients with advanced G/GEJ cancer. Further analysis is warranted in patients receiving FGFR2b targeted treatment.

The Expression of Cyclooxygenase-2 in Cervical Intraepithelial Neoplasia and Cervical Cancer.

Aim To examine the relationship between tumor differentiation, parametrial, and lymphovascular invasion, as well as the differential expression pattern of cyclooxygenase-2 (COX-2) in cervical intraepithelial neoplasia and various forms of cervical cancer. Methods Histologically diagnosed cases of in-situ and malignant lesions of the cervix were included in the study. Two sections were cut from paraffin blocks. One section was stained with Haematoxylin and Eosin (H&E) for morphologic diagnosis, and the other sections were subjected to COX-2 immunohistochemical staining. Cases of colon carcinoma were taken as positive controls. Cytoplasmic and membrane staining of tumor cells were considered as positive staining, and grading was done. Results Out of the 62 patients, 40 cases (64.5%) showed positive expression of COX-2 in squamous cell carcinoma when compared to in-situ cervical intraepithelial neoplasia and adenocarcinoma. The results were statistically significant, with a p-value of 0.003. Conclusion COX-2 expression is directly proportional to the level of grading of the tumor. The higher the grading, the higher the expression of COX-2. Selective COX-2 inhibitors increase the efficacy of chemotherapy or radiotherapy.

How Can "No Specific Molecular Profile" Heterogeneity be Reduced in Molecularly Classified Endometrial Cancer?: Prognostic Significance of L1 Cell Adhesion Molecule.

This study aims to investigate the role of L1 cell adhesion molecule (L1CAM) in the prognostic assessment of endometrial cancers that have been depicted as having no specific molecular profile (NSMP) in molecular classification. This is a retrospective review of 150 patients who received the diagnosis of endometrial cancer and underwent surgery at the study center between January 2008 and January 2022. When evaluating L1CAM immunohistochemical staining, scoring was done according to the percentage of positivity in tumor cells. Accordingly, score 0 = 0%, score 1=1% to 10%, score 2 = >10% to 50% and score 3 = >50%. If the staining in tumor cells was ≥10% (scores 2 and 3), it was considered positive. The patients with L1CAM positivity had significantly more frequent lymphovascular space invasion and lymph node metastasis than patients with L1CAM negativity (P = 0.013 and P = 0.007). L1CAM expression was strongly associated with mutant p53 (P = 0.003). Recurrence was significantly higher (P = 0.001) and overall survival and progression-free survival were significantly lower in patients with L1CAM positivity (P = 0.001 for both). Seventy-nine patients (52.7%) were put into NSMP group. About 84.8% of them (n = 67) were L1CAM negative and 15.2% of them (n = 12) were L1CAM-positive. Recurrence was significantly higher (P = 0.001) and overall survival and progression-free survival were significantly lower in patients with NSMP who were positive for L1CAM (P = 0.002 and P = 0.001, respectively). This study demonstrates that L1CAM expression status may add prognostic information to endometrial cancer, particularly in the NSMP subgroup. Considering the prognostic importance of L1CAM, its use as a marker may make significant contributions to reducing prognostic heterogeneity, especially in the NSMP subgroup.

Monitoring of three-dimensional live-cell cultures using a multimode, multiscale imaging system combining confocal fluorescence microscopy and optical coherence microscopy

Live-cell monitoring involves long-term observation and analysis of living cells in tissue cultures, which develop cells or tissues in an artificial environment and are essential for tissue engineering. Fluorescence microscopy (FM) is widely used as a monitoring tool owing to its powerful ability to visualize protein distribution within cells and tissues, thereby providing information on their functions in biological processes. To reduce the photobleaching effect, which is a major limiting factor in FM, FM is generally combined with other imaging modalities, such as phase contrast and differential interference contrast microscopy, which are nondestructive to fluorochromes, to selectively use fluorescence signals only in specific areas of interest within a sample. However, these methods are restricted to thin samples and cannot produce depth-resolved images. Here, we propose a method to determine three-dimensional (3D) positions in volumetric samples for application in high-resolution 3D fluorescence imaging using a multimode and multiscale imaging system that combines optical coherence microscopy (OCM) and line confocal FM (LC–FM). We also demonstrate the benefits of multimodal imaging in 3D cell culture monitoring. To evaluate the performance of the proposed system and method, we rapidly and accurately located the regions of interest in 3D tissue culture models, such as tumor spheroids and microvascular bed, and instantaneously acquired volumetric high-resolution fluorescence images. We also used an integrated system to monitor tumor spheroids mixed with extracellular matrix (ECM) over five days of the culture process, and the FM–OCM integrated technique successfully imaged the overall 3D structures, such as the distribution and boundaries of the spheroids and ECM as well as stained tumor cells. This complementary information is useful for understanding the relationship between cellular behaviors, such as the proliferation and migration of tumor cells, and microenvironments, such as the ECM.

Deregulation of Metalloproteinase Expression in Gray Horse Melanoma Ex Vivo and In Vitro.

The ability of human melanoma cells to switch from an epithelial to a mesenchymal phenotype contributes to the metastatic potential of disease. Metalloproteinases (MPs) are crucially involved in this process by promoting the detachment of tumor cells from the primary lesion and their migration to the vasculature. In gray horse melanoma, epithelial-mesenchymal transition (EMT) is poorly understood, prompting us to address MP expression in lesions versus intact skin by transcriptome analyses and the immunofluorescence staining (IF) of gray horse tumor tissue and primary melanoma cells. RNAseq revealed the deregulation of several MPs in gray horse melanoma and, notably, a 125-fold upregulation of matrix metalloproteinase 1 (MMP1) that was further confirmed by RT-qPCR from additional tumor material. The IF staining of melanoma tissue versus intact skin for MMP1 and tumor marker S100 revealed MMP1 expression in all lesions. The co-expression of S100 was observed at different extents, with some tumors scoring S100-negative. The IF staining of primary tumor cells explanted from the tumors for MMP1 showed that the metalloproteinase is uniformly expressed in the cytoplasm of 100% of tumor cells. Overall, the presented data point to MP expression being deregulated in gray horse melanoma, and suggest that MMP1 has an active role in gray horse melanoma by driving EMT-mediated tumor cell dissemination via the degradation of the extracellular matrix. Whilst S100 is considered a reliable tumor marker in human MM, gray horse melanomas do not seem to regularly express this protein.

Expression of PD-L1 (DAKO 28-8) in Urothelial Carcinoma of Urinary Bladder

Introduction: Around 90% of bladder cancers are urothelial carcinoma. Recent advancements in treatment have shifted towards targeted therapies, particularly immunotherapies targeting PD-L1 (Programmed Cell Death Ligand 1). PD-L1, a ligand for PD-1 receptors, helps cancer cells evade the immune system. Various factors, including genetic alterations, lead to its upregulation in cancer cells. Immunotherapies aim to block this pathway, making cancer cells more susceptible to the immune response. Objective: The study was designed to assess the frequency of PD-L1 expression by immunohistochemistry in urothelial carcinoma of urinary bladder and its association with histological grades. Methodology: This study was conducted at Department of Pathology, Pakistan Institute of Medical Sciences Islamabad. Fifty-six cases of urothelial carcinoma were included. Immunohistochemistry using PD-L1 Dako 28-8 clone was applied to prepared tissue sections. PD-L1 positivity was determined by brown membrane staining in tumor cells, scored using the Tumor Proportion Score (TPS). Results: The median patient age was 63±7 years, with the highest frequency (58.9%) of PD-L1 expression occurring in the age range of 56 to 65 years. However, there was no significant association between PD-L1 expression and age groups. Out of 56 cases, 64.3% had a TPS (Tumor Proportion Score) of ≥5%. Among the 76.7% high-grade cases, 72% had a TPS ≥5%, whereas 28% had a TPS &lt;5%. Among the 23.2% low-grade cases, 61.5% had a TPS &lt;5%, and 38.5% had a TPS ≥5%. Conclusion: Sixty-four percent urothelial carcinoma cases showed PD-L1 expression. Significant association (p value = 0.02) between PD-L1 expression and high-grade urothelial carcinoma was found using Pearson’s Chi-square test.

Galectin-3 expression by thyroid neoplasms in a center in southwest Nigeria

Background: Galectin-3 (GAL-3), which is known to be expressed in certain normal tissues and is implicated in tumor progression and metastasis, serves as a valuable marker for distinguishing malignant thyroid lesions from benign thyroid lesions. This study aimed to assess the clinical and histopathological features of thyroid neoplasms at OAUTHC Ile Ife utilizing GAL-3 immunohistochemistry and to compare these findings with those of studies conducted in other regions of the world. Materials and Methods: Histology slides from 56 patients with thyroid neoplasms spanning a 20-year period were subjected to immunohistochemical staining with monoclonal antibodies against GAL-3. Evaluation was based on the 2022 World Health Organization Classification of Thyroid Neoplasms, categorizing marker expression as follows: 1+ (10–25% stained cells), 2+ (26–50% stained cells), 3+ (51–75% stained cells), and 4+ (&gt;75% stained cells). Negative staining indicated no staining, while localized staining (focal) represented &lt;50% of cells stained (1+ or 2+), and diffuse staining indicated &gt;50% of cells stained (3+ or 4+). Results: A total of 56 patients with neoplastic thyroid disease were studied, including 15 males and 41 females. Among the 16 benign neoplastic lesions, four (25.0%) exhibited focal GAL-3 expression, while 12 (75.0%) showed no expression. Among the forty malignant neoplastic lesions, five (12.5%) tested negative for the GAL-3 immunomarker, while 35 (87.5%) demonstrated positive expression to varying degrees. Among the positive malignant lesions, two (5.0%) displayed focal GAL-3 expression, while 33 (82.5%) exhibited diffuse staining of tumor cells. The diffuse and strong expression of GAL-3 was significantly greater in malignant thyroid neoplasms than in benign neoplasms (p&lt;0.05). The sensitivity and specificity of GAL-3 were 88% and 75%, respectively. Conclusion: Our findings align with the previous research indicating that diffuse and strong immunohistochemical expression of GAL-3 is indicative of thyroid gland malignancy. GAL-3 serves as a valuable tool in diagnosing thyroid neoplasms, particularly in cases where histomorphologic characteristics are inconclusive or unclear.

PD-L1 expression in tumor and inflammatory cells is associated with favorable tumor features and favorable prognosis in muscle-invasive urothelial carcinoma of the bladder not treated by immune checkpoint inhibitors

BackgroundA high level of PD-L1 expression is the most relevant predictive parameter for response to immune checkpoint inhibitor (CPI) therapy in urinary bladder cancer. Existing data on the relationship between PD-L1 expression and the natural course of disease are controversial and sparse.MethodsTo expand our understanding of the relationship between PD-L1 expression and parameters of cancer aggressiveness, PD-L1 was analyzed on tissue microarrays containing 2710 urothelial bladder carcinomas including 512 patients with follow-up data who underwent radical cystectomy and follow-up therapies in the pre-immune checkpoint inhibitor therapy era.ResultsTumor cell positivity in ≥10% of cells were seen in 513 (20%) and an immune cell positivity occurred in 872 (34%) of 2566 interpretable cancers. PD-L1 positivity in tumor cells increased from pTaG2 low grade (0.9% positive) to pTaG3 high grade (4.1%; p = 0.0255) and was even higher in muscle-invasive (pT2–4) carcinomas (29.3%; p < 0.0001). However, within pT2–4 carcinomas, PD-L1 positivity was linked to low pT stage (p = 0.0028), pN0 (p < 0.0001), L0 status (p = 0.0005), and a better prognosis within 512 patients with cystectomy who never received CPIs (p = 0.0073 for tumor cells and p = 0.0086 for inflammatory cells). PD-L1 staining in inflammatory cells was significantly linked to PD-L1 staining in tumor cells (p < 0.0001) and both were linked to a positive p53 immunostaining (p < 0.0001).ConclusionIt cannot be fully excluded that the strong statistical link between PD-L1 status and favorable histological tumor features as well as better prognosis could influence the outcome of studies evaluating CPIs in muscle-invasive urothelial carcinoma.

Lectin-like Transcript-1 (LLT1) Expression in Oral Squamous Cell Carcinomas: Prognostic Significance and Relationship with the Tumor Immune Microenvironment.

Lectin-like transcript-1 (LLT1) expression is detected in different cancer types and is involved in immune evasion. The present study investigates the clinical relevance of tumoral and stromal LLT1 expression in oral squamous cell carcinoma (OSCC), and relationships with the immune infiltrate into the tumor immune microenvironment (TIME). Immunohistochemical analysis of LLT1 expression was performed in 124 OSCC specimens, together with PD-L1 expression and the infiltration of CD20+, CD4+, and CD8+ lymphocytes and CD68+ and CD163+-macrophages. Associations with clinicopathological variables, prognosis, and immune cell densities were further assessed. A total of 41 (33%) OSCC samples showed positive LLT1 staining in tumor cells and 55 (44%) positive LLT1 in tumor-infiltrating lymphocytes (TILs). Patients harboring tumor-intrinsic LLT1 expression exhibited poorer survival, suggesting an immunosuppressive role. Conversely, positive LLT1 expression in TILs was significantly associated with better disease-specific survival, and also an immune-active tumor microenvironment highly infiltrated by CD8+ T cells and M1/M2 macrophages. Furthermore, the combination of tumoral and stromal LLT1 was found to distinguish three prognostic categories (favorable, intermediate, and adverse; p = 0.029, Log-rank test). Together, these data demonstrate the prognostic relevance of tumoral and stromal LLT1 expression in OSCC, and its potential application to improve prognosis prediction and patient stratification.

Abstract CT230: Exploratory analyses of MGMT promoter methylation and PD-L1 expression from newly diagnosed glioblastoma patients in CheckMate 548 study suggest refinement in thresholds for predicting response to standard of care and nivolumab treatments

Abstract CheckMate 548 (NCT02667587, CM548) is a phase III, randomized trial evaluating first line nivolumab (NIVO) in combination with standard of care (SOC) [surgical resection followed by radiotherapy and temozolomide (TMZ)] versus placebo plus SOC in patients with newly diagnosed glioblastoma (GBM) positive for promoter methylation of O6-methylguanine DNA methyltransferase (MGMT). MGMT promoter methylation status and PD-L1 expression predict better outcomes in GBM patients treated with TMZ and in patients treated with NIVO in other indications, respectively. However, the impact of varying levels of MGMT promoter methylation and PD-L1 expression on patient outcome in GBM remains unclear. In CM548, MGMT promoter methylation levels were quantified through a central lab assay (LapCorp) and PD-L1 expression was determined by percentage of detectable membrane staining of tumor cells. MGMT methylation and PD-L1 data were prospectively generated at the time of enrolment. Our analyses showed that increasing levels of MGMT promoter methylation level were associated with longer overall survival in patients treated with SOC alone (n = 349). However, no survival benefit was observed with increasing levels of MGMT methylation in patients treated with NIVO in combination with SOC (n = 353). In addition, our data showed a non-linear relationship between PD-L1 expression and overall survival across both arms. In patients with intermediate PD-L1 expression (≥5%, &amp;lt;20%), longer survival was observed in the nivolumab + SOC arm compared to SOC alone. This benefit was not observed in patients with low (≥1%, &amp;lt;5%) and high (&amp;gt;20%) PD-L1 expression. Potential mechanisms driving these observed associations are currently explored using genomic data generated from patient samples. MGMT promoter methylation and PD-L1 expression are potential clinically relevant predictive biomarkers of response to treatments administered in the CM-548; however, the binary classification of positive/negative status of these markers does not fully capture their relationship with efficacy in CM548. Data generated from our exploratory analyses suggests that new, refined criteria are needed for better patient outcomes prediction. Citation Format: Yu-Han Hung, Aparna Chhibber, Michael Lim, Michael Weller, David A. Reardon, Antonio Omuro, Keith L. Ligon, Ana Lako, Ann Forslund. Exploratory analyses of MGMT promoter methylation and PD-L1 expression from newly diagnosed glioblastoma patients in CheckMate 548 study suggest refinement in thresholds for predicting response to standard of care and nivolumab treatments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT230.

Artificial intelligence algorithm accurately assesses oestrogen receptor immunohistochemistry in metastatic breast cancer cytology specimens: A pilot study.

The Visiopharm artificial intelligence (AI) algorithm for oestrogen receptor (ER) immunohistochemistry (IHC) in whole slide images (WSIs) has been successfully validated in surgical pathology. This study aimed to assess its efficacy in cytology specimens. The study cohort comprised 105 consecutive cytology specimens with metastatic breast carcinoma. ER IHC WSIs were seamlessly integrated into the Visiopharm platform from the Image Management System (IMS) during our routine digital workflow, and an AI algorithm was employed for analysis. ER AI scores were compared with pathologists' manual consensus scores. Optimization steps were implemented and evaluated to reduce discordance. The overall concordance between pathologists' scores and AI scores was excellent (99/105, 94.3%). Six cases exhibited discordant results, including two false-negative (FN) cases due to abundant histiocytes incorrectly counted as negatively stained tumour cells by AI, two FN cases owing to weak staining, and two false-positive (FP) cases where pigmented macrophages were erroneously counted as positively stained tumour cells by AI. The Pearson correlation coefficient of ER-positive percentages between pathologists' and AI scores was 0.8483. Optimization steps, such as lowering the cut-off threshold and additional training using higher input magnification, significantly improved accuracy. The automated ER AI algorithm demonstrated excellent concordance with pathologists' assessments and accurately differentiated ER-positive from ER-negative metastatic breast carcinoma cytology cases. However, precision in identifying tumour cells in cytology specimens requires further enhancement.