Expression of Cathepsin D in early-stage breast cancer and its prognostic and predictive value.

PURPOSE: Cathepsin D is a proteolytic enzyme that is normally localized in the lysosomes and is involved in the malignant progression of breast cancer. There are conflicting results regarding Cathepsin D significance as prognostic and predictor marker in breast cancer. This study aimed to evaluate the expression and prognostic significance of Cathepsin D in early-stage breast cancer. METHODS: Expression of Cathepsin D was assessed by immunohistochemical staining of tissue microarrays, in a large well-characterized series of early-stage operable breast cancer (n = 954) from Nottingham Primary Breast Carcinoma Series between the period of 1988 and 1998 who underwent primary surgery. Correlation of Cathepsin D expression with clinicopathological parameters and prognosis was evaluated. RESULTS: Cathepsin D expression was positive in 71.2% (679/954) of breast cancer tumours. Positive expression of Cathepsin D was significantly associated with high histological grade (p = 0.007), pleomorphism (p = 0.002), poor Nottingham Prognostic Index (NPI) score (p < 0.002), recurrence (p = 0.005) and distant metastasis (p < 0.0001). Kaplan-Meier analysis showed that Cathepsin D expression was significantly associated with shorter breast cancer-specific survival (p = 0.001), higher risk of recurrence (p = 0.001) and distant metastasis (p < 0.0001). ER-positive tumours expressing Cathepsin D and treated with tamoxifen demonstrated a significantly higher risk of distant metastasis. CONCLUSION: Cathepsin D expression significantly predicts poor prognosis in breast cancer and is associated with variables of poor prognosis and shorter outcome. The strong association of Cathepsin D with aggressive tumour characteristics and poor outcomes warrants further research of its potential as a therapeutic target The results also suggest a possible interaction between Cathepsin D and tamoxifen therapy in ER-positive breast cancer which needs further investigation to elucidate the underlying mechanisms.

KLK7 expression in human tumors: a tissue microarray study on 13,447 tumors.

BACKGROUND: Kallikrein-related peptidase 7 (KLK7) is a chymotrypsin-like serine protease which is essential for the desquamation of corneocytes and thus plays a pivotal role in maintaining skin homeostasis. In cancer, KLK7 overexpression was suggested to represent a route for metastasis through cleavage of cell junction and extracellular matrix proteins of cancer cells. METHODS: To comprehensively determine KLK7 protein expression in normal and neoplastic tissues, a tissue microarray containing 13,447 samples from 147 different tumor types and subtypes as well as 608 samples of 76 different normal tissue types was analyzed by immunohistochemistry. RESULTS: KLK7 positivity was found in 64 of 147 tumor categories, including 17 tumor categories with at least one strongly positive case. The highest rate of KLK7 positivity was found in squamous cell carcinomas from various sites of origin (positive in 18.1%-63.8%), ovarian and endometrium cancers (4.8%-56.2%), salivary gland tumors (4.8%-13.7%), bilio-pancreatic adenocarcinomas (20.0%-40.4%), and adenocarcinomas of the upper gastrointestinal tract (3.3%-12.5%). KLK7 positivity was linked to nodal metastasis (p = 0.0005), blood vessel infiltration (p = 0.0037), and lymph vessel infiltration (p < 0.0001) in colorectal adenocarcinoma, nodal metastasis in hepatocellular carcinoma (p = 0.0382), advanced pathological tumor stage in papillary thyroid cancer (p = 0.0132), and low grade of malignancy in a cohort of 719 squamous cell carcinomas from 11 different sites of origin (p < 0.0001). CONCLUSIONS: These data provide a comprehensive overview on KLK7 expression in normal and neoplastic human tissues. The prognostic relevance of KLK7 expression and the possible role of KLK7 as a drug target need to be further investigated.

Higher expression of SALL4-A isoform is correlated with worse outcomes and progression of the disease in subtype of testicular germ cell tumours.

BACKGROUND: The transcription factor SALL4 is associated with embryonic pluripotency and has proposed as a novel immunohistochemistry (IHC) marker for diagnosing germ cell tumours. SALL4 comprises three isoforms, and SALL4-A being the full-length isoform. Studying its isoforms could revolutionize testicular cancer prognosis and subtype differentiation. METHODS: The expression and clinical significance of isoform 'A' of SALL4 was evaluated in 124 testicular germ cell tumours (TGCTs) subtypes, adjacent 67 normal tissues and 22 benign tumours, using immunohistochemistry on tissue microarrays (TMA). RESULTS: A statistically significant higher expression of nuclear and cytoplasmic SALL4-A was detected in TGCTs histological subtypes and benign tumours compared to the normal tissues. Seminoma and yolk sac tumours had the highest nuclear and cytoplasmic expression of SALL4-A. A significant correlation was detected between the higher nuclear expression of SALL4-A and increased pT stages (P = 0.026) in seminomas. Whereas in embryonal carcinomas, cytoplasmic expression of SALL4-A was associated with the tumour recurrence (P = 0.04) and invasion of the epididymis (P = 0.011). CONCLUSIONS: SALL4-A isoform expression in the cytoplasm and nucleus of TGCTs may be associated with histological differentiation. In the seminoma subtype of TGCTs, higher expression of SALL4-A may be used as a predictive indicator of poorer outcomes and prognosis.

Hourglass, a rapid analysis framework for heterogeneous bioimaging data, identifies sex disparity in IL-6/STAT3-associated immune phenotypes in pancreatic cancer.

Integration and mining of bioimaging data remains a challenge and lags behind the rapidly expanding digital pathology field. We introduce Hourglass, an open-access analytical framework that streamlines biology-driven visualization, interrogation, and statistical assessment of multiparametric datasets. Cognizant of tissue and clinical heterogeneity, Hourglass systematically organizes observations across spatial and global levels and within patient subgroups. Applied to an extensive bioimaging dataset, Hourglass promptly consolidated a breadth of known interleukin-6 (IL-6) functions via its downstream effector STAT3 and uncovered a so-far unknown sexual dimorphism in the IL-6/STAT3-linked intratumoral T-cell response in human pancreatic cancer. As an R package and cross-platform application, Hourglass facilitates knowledge extraction from multi-layered bioimaging datasets for users with or without computational proficiency and provides unique and widely accessible analytical means to harness insights hidden within heterogeneous tissues at the sample and patient level. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Expression of integrin αvß3 in medullary thyroid carcinoma.

Aim: Tumor markers often remain elevated after intended curative resection of medullary thyroid carcinoma (MTC). The aim of this study was to determine the expression of αvß3, a promising theranostics target, in MTC and its metastases. Materials & methods: Avß3 expression was analyzed in 104 patients using a tissue microarray and correlated with clinicopathological variables and survival. Results: Cytoplasmic αvß3 positivity was seen in 70 patients and was associated with lymph node metastases at time of initial surgery. Membranous positivity was considered positive in 30 patients and was associated with sporadic MTC. Conclusion: Avß3 was expressed in the cytoplasm of 67% of MTC patients. Membranous expression, which is presumably most relevant for the theranostic use of αvß3, was seen in 29%.

[Box: see text].

Interferon γ Expressing Mucosal Cells in Pediatric Chronic Inflammatory Bowel Disease.

The pathogenesis of Crohn's disease (CD) and ulcerative colitis (UC) is multifactorial and includes aberrations in the composition of gastrointestinal mucosal inflammatory cells. Accurate identification of CD and UC is important as treatment and prognosis differs; however, CD and UC may be difficult to differentiate. Interferon γ (IFNγ) expression appears to be increased in ileal mucosa from CD patients, implying that IFNγ could be a diagnostically useful marker to differentiate CD from UC. This study uses automated assessment of IFNγ immunohistochemical expression in archival GI mucosal biopsies from stomach, duodenum, terminal ileum, and colon in a pediatric population to address this possibility. IFNγ positive mucosal cells are increased in the colon in both CD and UC compared to normal colon and in the ileum of CD compared to normal and UC. The abundance of IFNγ positive cells is not correlated with the presence of active inflammation, indicating that active inflammation is not responsible for the variance in abundance of IFNγ positive cells between cohorts and sites. Overlap between CD, UC, and normal suggests that IFNγ immunohistochemistry may only be clinically useful in select situations such as undetermined inflammatory bowel disease and additional study in these areas is warranted.

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.

BACKGROUND: A 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. METHODS: To 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. RESULTS: Tumor 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). CONCLUSION: It 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.

Full-Automatic High-Efficiency Mueller Matrix Microscopy Imaging for Tissue Microarray Inspection.

This paper proposes a full-automatic high-efficiency Mueller matrix microscopic imaging (MMMI) system based on the tissue microarray (TMA) for cancer inspection for the first time. By performing a polar decomposition on the sample's Mueller matrix (MM) obtained by a transmissive MMMI system we established, the linear phase retardance equivalent waveplate fast-axis azimuth and the linear phase retardance are obtained for distinguishing the cancerous tissues from the normal ones based on the differences in their polarization characteristics, where three analyses methods including statistical analysis, the gray-level co-occurrence matrix analysis (GLCM) and the Tamura image processing method (TIPM) are used. Previous MMMI medical diagnostics typically utilized discrete slices for inspection under a high-magnification objective (20×-50×) with a small field of view, while we use the TMA under a low-magnification objective (5×) with a large field of view. Experimental results indicate that MMMI based on TMA can effectively analyze the pathological variations in biological tissues, inspect cancerous cervical tissues, and thus contribute to the diagnosis of postoperative cancer biopsies. Such an inspection method, using a large number of samples within a TMA, is beneficial for obtaining consistent findings and good reproducibility.

Upregulated Nuclear Expression of Soluble Epoxide Hydrolase Predicts Poor Outcome in Breast Cancer Patients: Importance of the Digital Pathology Approach.

Breast cancer (BC) is the most common cancer in women, with incidence rates increasing globally in recent years. Therefore, it is important to find new molecules with prognostic and therapeutic value to improve therapeutic response and quality of life. The polyunsaturated fatty acids (PUFAs) metabolic pathway participates in various physiological processes, as well as in the development of malignancies. Although aberrancies in the PUFAs metabolic pathway have been implicated in carcinogenesis, the functional and clinical relevance of this pathway has not been well explored in BC. To evaluate the clinical significance of soluble epoxide hydrolase (EPHX2) expression in Mexican patients with BC using tissue microarrays (TMAs) and digital pathology (DP). Immunohistochemical analyses were performed on 11 TMAs with 267 BC samples to quantify this enzyme. Using DP, EPHX2 protein expression was evaluated solely in tumor areas. The association of EPHX2 with overall survival (OS) was detected through bioinformatic analysis in public databases and confirmed in our cohort via Cox regression analysis. Clear nuclear expression of EPHX2 was identified. Receiver operating characteristics (ROC) curves revealed the optimal cutoff point at 2.847062 × 10-3 pixels, with sensitivity of 69.2% and specificity of 67%. Stratification based on this cutoff value showed elevated EPHX2 expression in multiple clinicopathological features, including older age and nuclear grade, human epidermal growth factor receptor 2 (HER2) and triple negative breast cancer (TNBC) subtypes, and recurrence. Kaplan-Meier curves demonstrated how higher nuclear expression of EPHX2 predicts shorter OS. Consistently, multivariate analysis confirmed EPHX2 as an independent predictor of OS, with a hazard ratio (HR) of 3.483 and a 95% confidence interval of 1.804-6.724 (p < 0.001). Our study demonstrates for the first time that nuclear overexpression of EPHX2 is a predictor of poor prognosis in BC patients. The DP approach was instrumental in identifying this significant association. Our study provides valuable insights into the potential clinical utility of EPHX2 as a prognostic biomarker and therapeutic target in BC.

Oral epithelial dysplasia detection and grading in oral leukoplakia using deep learning.

BACKGROUND: The grading of oral epithelial dysplasia is often time-consuming for oral pathologists and the results are poorly reproducible between observers. In this study, we aimed to establish an objective, accurate and useful detection and grading system for oral epithelial dysplasia in the whole-slides of oral leukoplakia. METHODS: Four convolutional neural networks were compared using the image patches from 56 whole-slide of oral leukoplakia labeled by pathologists as the gold standard. Sequentially, feature detection models were trained, validated and tested with 1,000 image patches using the optimal network. Lastly, a comprehensive system named E-MOD-plus was established by combining feature detection models and a multiclass logistic model. RESULTS: EfficientNet-B0 was selected as the optimal network to build feature detection models. In the internal dataset of whole-slide images, the prediction accuracy of E-MOD-plus was 81.3% (95% confidence interval: 71.4-90.5%) and the area under the receiver operating characteristic curve was 0.793 (95% confidence interval: 0.650 to 0.925); in the external dataset of 229 tissue microarray images, the prediction accuracy was 86.5% (95% confidence interval: 82.4-90.0%) and the area under the receiver operating characteristic curve was 0.669 (95% confidence interval: 0.496 to 0.843). CONCLUSIONS: E-MOD-plus was objective and accurate in the detection of pathological features as well as the grading of oral epithelial dysplasia, and had potential to assist pathologists in clinical practice.

Vimentin expression correlates with immune checkpoint inhibitor efficacy in non-small cell lung cancer.

BACKGROUND: Although vimentin is often expressed in non-small cell lung cancer (NSCLC), the association between vimentin expression and immune-checkpoint inhibitor (ICI) efficacy remains unclear. METHODS: This retrospective multicenter study enrolled patients with NSCLC who received ICI treatment between December 2015 and July 2020. The authors constructed tissue microarrays and performed immunohistochemical staining with vimentin. They analyzed the relationship between vimentin expression rate and objective response rate (ORR), progression-free survival (PFS), and overall survival (OS). RESULTS: Immunohistochemically evaluable specimens on microarray blocks were available for 397 patients, of whom 343 (86%) were negative (<10%), 30 (8%) were positive (10%-49%), and 24 (6%) were highly positive (≥50%) for vimentin expression. Both rates of programmed death-ligand 1 (PD-L1) tumor proportion score ≥1% and ≥50% were significantly higher in the vimentin-positive group (≥10%) than the vimentin-negative group (<10%) (96% vs. 78%, p = .004; 64% vs. 42%, p = .006, respectively). In patients treated with ICI monotherapy, ORR, PFS, and OS were significantly better in the vimentin-positive group (10%-49%) than in the vimentin-negative group (<10%) (54% vs. 25%, p = .003, median = 7.9 vs. 3.2 months, p = .011; median = 27.0 vs. 13.6 months, p = .015, respectively), whereas there was no significant difference in PFS and OS between the vimentin highly positive group (≥50%) and the vimentin-negative group (<10%) (median = 3.4 vs. 3.2 months, p = .57; median = 7.2 vs. 13.6 months, p = .086, respectively). CONCLUSIONS: Vimentin expression correlated with PD-L1 expression and ICI efficacy. PLAIN LANGUAGE SUMMARY: We constructed tissue microarrays and performed immunohistochemical staining with vimentin on 397 patients with advanced non-small cell lung cancer who were treated with immune-checkpoint inhibitor (ICI). The vimentin-positive group who were treated with ICI monotherapy showed significantly better objective response rate, progression-free survival, and overall survival than the vimentin negative group. The measurement of vimentin expression will aid in determining appropriate immunotherapy strategies.

High-throughput MALDI-MSI metabolite analysis of plant tissue microarrays.

A novel metabolomics analysis technique, termed matrix-assisted laser desorption/ionization mass spectrometry imaging-based plant tissue microarray (MALDI-MSI-PTMA), was successfully developed for high-throughput metabolite detection and imaging from plant tissues. This technique completely overcomes the disadvantage that metabolites cannot be accessible on an intact plant tissue due to the limitations of the special structures of plant cells (e.g. epicuticular wax, cuticle and cell wall) through homogenization of plant tissues, preparation of PTMA moulds and matrix spraying of PTMA sections. Our study shows several properties of MALDI-MSI-PTMA, including no need of sample separation and enrichment, high-throughput metabolite detection and imaging (>1000 samples per day), high-stability mass spectrometry data acquisition and imaging reconstruction and high reproducibility of data. This novel technique was successfully used to quickly evaluate the effects of two plant growth regulator treatments (i.e. 6-benzylaminopurine and N-phenyl-N'-1,2,3-thiadiazol-5-ylurea) on endogenous metabolite expression in plant tissue culture specimens of Dracocephalum rupestre Hance (D. rupestre). Intra-day and inter-day evaluations indicated that the metabolite data detected on PTMA sections had good reproducibility and stability. A total of 312 metabolite ion signals in leaves tissues of D. rupestre were detected, of which 228 metabolite ion signals were identified, they were composed of 122 primary metabolites, 90 secondary metabolites and 16 identified metabolites of unknown classification. The results demonstrated the advantages of MALDI-MSI-PTMA technique for enhancing the overall detection ability of metabolites in plant tissues, indicating that MALDI-MSI-PTMA has the potential to become a powerful routine practice for high-throughput metabolite study in plant science.

Clinical, pathological, and comprehensive molecular analysis of the uterine clear cell carcinoma: a retrospective national study from TMRG and GINECO network.

BACKGROUND: Uterine clear cell carcinomas (CCC) represent less than 5% of uterine cancers. Their biological characteristics and clinical management remain uncertain. A multicenter study to explore both clinical and molecular features of these rare tumors was conducted. METHODS: This multicenter retrospective national study was performed within the French TMRG (Rare Gynecologic Malignant Tumors) network. Clinical data and, when available, FFPE blocks were collected. Clinical features, treatments, and outcome (progression-free survival (PFS) and overall survival (OS)) were analyzed and correlated to the protein (tissue micro-array), RNA (Nanostring nCounter® technology), and DNA (array-Comparative Genomic hybridization and target-next generation sequencing) levels using the tumor samples available. RESULTS: Sixty-eight patients with uterine CCC were enrolled, 61 from endometrial localization and 5 with cervix localization. Median age at diagnosis was 68.9 years old (range 19-89.7). Most tumors were diagnosed at an early stage (78% FIGO stage I-II). Hysterectomy (performed in 90%) and lymph node dissection (80%) were the most frequent surgical treatment. More than 70% of patients received external beam radiotherapy and 57% received brachytherapy. Nearly half (46%) of the patients received chemotherapy. After a median follow-up of 24.7 months, median PFS was 64.8 months (95 CI [5.3-124.4]) and median OS was 79.7 (IC95 [31.0-128.4]). Low hormone receptor expression (13% estrogen-receptor positive), frequent PI3K pathway alterations (58% PTEN loss, 50% PIK3CA mutations), and P53 abnormalities (41%) were observed. Mismatch repair deficiency was identified in 20%. P16 expression was associated with shorter PFS (HR = 5.88, 95 CI [1.56-25], p = 0.009). Transcriptomic analyzes revealed a specific transcriptomic profile notably with a high expression of immune response-associated genes in uterine CCC displaying a very good overall prognosis. CONCLUSIONS: Uterine CCC reported to be potentially MSI high, hormone receptors negative, and sometimes TP53 mutated. However, some patients with immune response-associated features and better prognosis may be candidate to treatment de-escalation and immunotherapy.

Racial differences in transcriptomics and reactive oxygen species burden in myometrium and leiomyoma.

STUDY QUESTION: Are there differences in Mediator Complex Subunit 12 mutations (MED12) mutation, transcriptomics, and protein expression in uterine myometrium and leiomyomas of Black and White women? SUMMARY ANSWER: RNA sequencing, tissue microarray, and immunohistochemistry data revealed that Black and White women have significant differences in their myometrium and leiomyoma profiles. WHAT IS KNOWN ALREADY: Black women develop uterine leiomyoma earlier than White women, and are more likely to be anemic, have multiple tumors, undergo hysterectomy at an earlier age, have a higher uterine weight, and report very severe pelvic pain. STUDY DESIGN, SIZE, DURATION: Uterine tissues were collected from premenopausal women undergoing hysterectomy or myomectomy at Northwestern University Prentice Women's Hospital (Chicago, IL) from 2010 to 2021. Tissues were collected from a total of 309 women, including from 136 Black women, 135 White women, and 38 women from other racial groups. A total of 529 uterine leiomyomas (290 from Black women, 184 from White women, and 55 from women of other racial groups) were subjected to molecular analysis. Leiomyoma and matched myometrium from a total of 118 cases including 60 Black women and 58 White women, were used for tissue microarrays, along with 34 samples of myometrium without leiomyoma from White women. PARTICIPANTS/MATERIALS, SETTING, METHODS: Tissues from the above patient cohorts were analyzed by tissue microarray, immunohistochemistry, RNA sequencing, and mutation analysis. MAIN RESULTS AND THE ROLE OF CHANCE: The results indicated that leiomyoma from Black women have a higher rate of MED12 mutations (79.0%) than those from White women (68.5%) (*P ≤ 0.05). RNA-sequencing analysis in myometrium revealed differentially expressed genes (270 upregulated, 374 downregulated) dependent on race, wherein reactive oxygen species, hypoxia, and oxidative phosphorylation pathways were positively correlated with samples derived from Black patients. The levels of proteins associated with oxidative DNA damage and repair, 8-hydroxyguanosine (8-OHdG), 8-oxoguanine glycosylase (OGG1), heme oxygenase-1 (HO-1), and kelch-like ECH-associated protein 1 (KEAP1), were higher in leiomyoma and matched myometrium, particularly those from Black patients, compared to the control myometrium (with leiomyoma) (***P ≤ 0.001). LARGE SCALE DATA: The datasets are available in the NCBI (The BioProject number: PRJNA859428). LIMITATIONS, REASONS FOR CAUTION: Myometrium without leiomyoma derived from White patients was used as a control in the tissue microarray analysis, as myometrium without leiomyoma from Black patients was not accessible in large numbers. The RNA sequencing was performed on myometrium tissue with leiomyoma present from 10 White and 10 Black women. However, one sample from a Black woman yielded low-quality RNA-sequencing data and was excluded from further analysis. WIDER IMPLICATIONS OF THE FINDINGS: Women with symptomatic leiomyomas have a considerable loss in their quality of life. This study provides information on underlying genetic and molecular defects that may be necessary for future therapeutics targeted at leiomyomas. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by grants from NCI (R01CA254367) and NICHD (P01HD057877). The authors declare no conflict of interest.

GATA3 Expression in Human Tumors: A Tissue Microarray Study on 16,557 Tumors.

INTRODUCTION: GATA3 is a transcription factor involved in epithelial cell differentiation. GATA3 immunostaining is used as a diagnostic marker for breast and urothelial cancer but can also occur in other neoplasms. METHODS: To evaluate GATA3 in normal and tumor tissues, a tissue microarray containing 16,557 samples from 131 different tumor types and subtypes and 608 samples of 76 different normal tissue types was analyzed by immunohistochemistry. RESULTS: GATA3 positivity was found in 69 different tumor types including 23 types (18%) with at least one strongly positive tumor. Highest positivity rates occurred in noninvasive papillary urothelial carcinoma (92-99%), lobular carcinoma (98%), carcinoma of no special type of the breast (92%), basal cell carcinoma of the skin (97%), invasive urothelial carcinoma (73%), T-cell lymphoma (23%), adenocarcinoma of the salivary gland (16%), squamous cell carcinoma of the skin (16%), and colorectal neuroendocrine carcinoma (12%). In breast cancer, low GATA3 staining was linked to high pT stage (p = 0.03), high BRE grade (p < 0.0001), HER2 overexpression (p = 0.0085), estrogen and progesterone receptor negativity (p < 0.0001 each), and reduced survival (p = 0.03). CONCLUSION: Our data demonstrate that GATA3 positivity can occur in various tumor entities. Low levels of GATA3 reflect cancer progression and poor patient prognosis in breast cancer.

PRAME expression in cutaneous melanoma does not correlate with disease-specific survival.

BACKGROUND: Immunohistochemistry-based protein biomarkers can provide useful prognostic information in cutaneous melanoma. The independent prognostic value of Ki-67 has been studied with variable results. PReferentially expressed Antigen in MElanoma (PRAME) immunohistochemistry is a useful new ancillary tool for distinguishing cutaneous nevi from melanoma; however, its prognostic value has not been well studied. We evaluated PRAME as a prognostic marker in cutaneous melanoma, compared to Ki-67. METHODS: We analyzed the immunohistochemical expression of PRAME and Ki-67 in 165 melanocytic lesions, including 92 primary melanomas, 19 metastatic melanomas, and 54 melanocytic nevi using tissue microarrays. PRAME immunostaining was scored based on the percentage of positive nuclei: 0 <1%, 1+ 1%-25%, 2+ 26%-50%, 3+ 51%-75%, and 4+ >75%. The percentage of Ki-67-positive tumor nuclei was used to calculate the proliferation index. RESULTS: PRAME and Ki-67 both showed significantly increased expression in melanomas compared to nevi (p < 0.0001 and p < 0.001, respectively). There was no significant difference in PRAME expression in primary versus metastatic melanomas. By contrast, the Ki-67 proliferation index was higher in metastatic melanoma than in primary melanoma (p = 0.013). Increased Ki-67 index correlated with ulceration (p < 0.001), increased Breslow depth (p = 0.001), and higher mitotic rate (p < 0.0001), whereas increased PRAME expression correlated with higher mitotic rate (p = 0.047) and Ki-67 index (p = 0.007). Increased Ki-67 index correlated with worse disease-specific survival in patients with primary melanoma (p < 0.001), but PRAME expression did not show prognostic significance in disease-specific survival (p = 0.63). In a multivariable analysis of patients with primary melanoma, tumor Breslow depth, ulceration, mitotic rate, and Ki-67 index were each independent predictors of disease-specific survival (p = 0.006, 0.02, 0.001, and 0.04, respectively); however, PRAME expression was not predictive of disease-specific survival (p = 0.64). CONCLUSION: Ki-67 is an independent prognostic marker; although increased PRAME expression correlates with the Ki-67 proliferation index and mitotic rate, PRAME is not an independent prognostic marker for cutaneous melanoma. PRAME and Ki-67 are useful ancillary tools for distinguishing benign from malignant melanocytic lesions.

Pattern of MUC6 expression across 119 different tumor types: A tissue microarray study on 15 412 tumors.

Mucin 6 (MUC6) is a secreted gel-forming mucin covering the surfaces of gastrointestinal and other tissues. Published work demonstrates that MUC6 can also be expressed in several cancer types and can aid in the distinction of different tumor entities. To systematically analyze MUC6 expression in normal and cancerous tissues, a tissue microarray containing 15 412 samples from 119 different tumor types and subtypes as well as 608 samples of 76 different normal tissue types was analyzed by immunohistochemistry. At least a weak MUC6 positivity was seen in 50 of 119 (42%) tumor entities. Thirty-three tumor entities included tumors with strong positivity. MUC6 immunostaining was most frequent in mucinous carcinomas of the breast (44%), adenocarcinomas of the stomach (30%-40%) and esophagus (35%), and neuroendocrine carcinomas of the colon. Strong MUC6 staining was linked to advanced pT stage (p = 0.0464), defective mismatch repair status and right-sided tumor location (p < 0.0001 each) in colorectal cancer, as well as to high tumor grade (p = 0.0291), nodal metastasis (p = 0.0485), erb-b2 receptor tyrosine kinase 2 positivity (p < 0.0001) and negative estrogen receptor (p = 0.0332)/progesterone receptor (p = 0.0257) status in breast carcinomas of no special type. The broad range of tumor types with MUC6 expression limits the utility of MUC6 immunohistochemistry for the distinction of different tumor types.

Methods for preparing tissue microarray slides using xenografts with different levels of HER2 expression to standardize HER2 detection.

Gene amplification and protein overexpression of human epidermal growth factor receptor type 2 (HER2) are specific targets for HER2-targeting drugs in breast, gastric, salivary gland, and colorectal cancers. The histopathological determination of HER2 status is crucial for treatment, highlighting the importance of improving HER2 detection accuracy in clinical practice. We prepared tissue microarray (TMA) slides for use as control slides for the standardization of gastric HER2 testing. Four human gastric cancer cell lines with HER2 scores of 3+, 2+, 1+, and 0 were xenografted in NOG mice. The TMA slides were constructed using samples from three different areas in these tumors. Staining properties were determined using six clinical kits for HER2. In TMA, HER2-positive tumors with HER2 scores of 3+ and 2+ showed good staining with all diagnostic kits, and the tissue images were similar to those of clinical samples. Xenograft tumor slides could potentially be used as external controls to standardize staining conditions for a variety of kits and may improve the accuracy of HER2 detection in clinical practice.

Expression pattern and prognostic value of key regulators for N7-methylguanosine RNA modification in prostate cancer.

Alterations in the regulators of RNA methylation modifications, such as N7-methylguanosine (m7G), have been implicated in a variety of diseases. Therefore, the analysis and identification of disease-related m7G modification regulators will accelerate advances in understanding disease pathogenesis. However, the implications of alterations in the regulators of m7G modifications remain poorly understood in prostate adenocarcinoma. In the present study, we analyze the expression patterns of 29 m7G RNA modification regulators in prostate adenocarcinoma using The Cancer Genome Atlas (TCGA) and perform consistent clustering analysis of differentially expressed genes (DEGs). We find that 18 m7G-related genes are differentially expressed in tumor and normal tissues. In different cluster subgroups, DEGs are mainly enriched in tumorigenesis and tumor development. Furthermore, immune analyses demonstrate that patients in cluster 1 have significantly higher scores for stromal and immune cells, such as B cells, T cells, and macrophages. Then, a TCGA-related risk model is developed and successfully validated using a Gene Expression Omnibus external dataset. Two genes ( EIF4A1 and NCBP2) are determined to be prognostically significant. Most importantly, we construct tissue microarrays from 26 tumor specimens and 20 normal specimens, and further confirm that EIF4A1 and NCBP2 are associated with tumor progression and Gleason score. Therefore, we conclude that the m7G RNA methylation regulators may be involved in the poor prognosis of patients with prostate adenocarcinoma. The results of this study may provide support for exploring the underlying molecular mechanisms of m7G regulators, especially EIF4A1 and NCBP2.

Comprehensive clinicopathological significance and putative transcriptional mechanisms of Forkhead box M1 factor in hepatocellular carcinoma.

BACKGROUND: The Forkhead box M1 factor (FOXM1) is a crucial activator for cancer cell proliferation. While FOXM1 has been shown to promote hepatocellular carcinoma (HCC) progression, its transcriptional mechanisms remain incompletely understood. METHODS: We performed an in-house tissue microarray on 313 HCC and 37 non-HCC tissue samples, followed by immunohistochemical staining. Gene chips and high throughput sequencing data were used to assess FOXM1 expression and prognosis. To identify candidate targets of FOXM1, we comprehensively reanalyzed 41 chromatin immunoprecipitation followed by sequencing (ChIP-seq) data sets. We predicted FOXM1 transcriptional targets in HCC by intersecting candidate FOXM1 targets with HCC overexpressed genes and FOXM1 correlation genes. Enrichment analysis was employed to address the potential mechanisms of FOXM1 underlying HCC. Finally, single-cell RNA sequencing analysis was performed to confirm the transcriptional activity of FOXM1 on its predicted targets. RESULTS: This study, based on 4235 HCC tissue samples and 3461 non-HCC tissue samples, confirmed the upregulation of FOXM1 in HCC at mRNA and protein levels (standardized mean difference = 1.70 [1.42, 1.98]), making it the largest multi-centered study to do so. Among HCC patients, FOXM1 was increased in Asian and advanced subgroups, and high expression of FOXM1 had a strong ability to differentiate HCC tissue from non-HCC tissue (area under the curve = 0.94, sensitivity = 88.72%, specificity = 87.24%). FOXM1 was also shown to be an independent exposure risk factor for HCC, with a pooled hazard ratio of 2.00 [1.77, 2.26]. The predicted transcriptional targets of FOXM1 in HCC were predominantly enriched in nuclear division, chromosomal region, and catalytic activity acting on DNA. A gene cluster encoding nine transcriptional factors was predicted to be positively regulated by FOXM1, promoting the cell cycle signaling pathway in HCC. Finally, the transcriptional activity of FOXM1 and its targets was supported by single-cell analysis of HCC cells. CONCLUSIONS: This study not only confirmed the upregulation of FOXM1 in HCC but also identified it as an independent risk factor. Moreover, our findings enriched our understanding of the complex transcriptional mechanisms underlying HCC pathogenesis, with FOXM1 potentially promoting HCC progression by activating other transcription factors within the cell cycle pathway.