Clinicopathological Significance Research Articles

Expression of Forkhead Box M1 and Anticancer Effects of FOXM1 Inhibition in Epithelioid Sarcoma

Epithelioid sarcoma (ES) is a rare aggressive sarcoma that, unlike most soft-tissue sarcomas, shows a tendency toward local recurrence and lymph node metastasis. Novel antitumor agents are needed for ES patients. Forkhead box transcription factor 1 (FOXM1) is a member of the Forkhead transcription factor family and is associated with multiple oncogenic functions; FOXM1 is known to be overexpressed and correlated with pathogenesis in various malignancies. In this study, we immunohistochemically analyzed FOXM1 expression levels and their clinical, clinicopathologic, and prognostic significance in 38 ES specimens. In addition, to investigate potential correlations between FOXM1 downregulation and oncologic characteristics, we treated ES cell lines with thiostrepton, a naturally occurring antibiotic that inhibits both small interfering RNA (siRNA) and FOXM1. In the analyses using ES samples, all 38 specimens were diagnosed as positive for FOXM1 by immunohistochemistry. We separated specimens into high (n = 19) and low (n = 19) FOXM1–protein expression groups by staining index score, and into large (n = 12), small (n = 25), and unknown (n = 1) tumor-size groups using a cutoff of 5 cm maximum diameter. Although there were significantly more samples with high FOXM1 expression in the large tumor group (P = .013), there were no significant differences with respect to age (P = 1.00), sex (P = .51), primary site of origin (P = .74), histologic subtypes (P = 1.00), depth (P = .74), or survival rate (P = .288) between the high and low FOXM1–protein expression groups. In the in vitro experiments using ES cell lines, FOXM1 siRNA and thiostrepton successfully downregulated FOXM1 mRNA and protein expression. Furthermore, downregulation of FOXM1 inhibited cell proliferation, drug resistance against chemotherapeutic agents, migration, and invasion and caused cell cycle arrest in the ES cell lines. Finally, cDNA microarray analysis data showed that FOXM1 regulated cIAP2, which is one of the apoptosis inhibitors activated by the TNFα-mediated NF-κB pathway. In conclusion, the FOXM1 gene may be a promising therapeutic target for ES.

Exploring the role of trifarotene against RAR-α: an investigation of expression pattern and clinicopathological significance of RAR-α in breast cancer.

The members retinoic acid receptors (RARs) (α, β, and γ) and retinoid X receptors (RXRs) (α, β, and γ) belong to the retinoid receptor family. They regulate the biological action of classical retinoids through nuclear retinoid receptors, a transcription factor that is regulated by ligands. Through the binding of particular retinoic acid-responsive elements (RAREs) located in target gene promoters, RARs and members of the RXRs form heterodimers. By binding to its nuclear receptors and triggering the transcription of the target genes downstream, retinoic acid (RA) mediates the expression of certain genes. Retinoids so mainly control gene expression to carry out their biological actions. RARs are essential for many biological processes, such as development, immunity, reproduction, organogenesis, and homeostasis. Apart from their physiological functions, RARs are also linked to pathologies and tumors due to mutations, protein fusions, changes in expression levels, or abnormal post-translational changes that lead to aberrant functions and homeostasis breakdown. The oncogenic development of animal tissues or cultured cells is linked to altered expression of retinoid receptors. The RAR-α is over-expressed in several malignancies. Increased invasion and migration in several cancer forms, including HNSC carcinoma, pediatric low-grade gliomas, lung adenocarcinoma, and breast cancer, have been linked to its upregulated expression. Numerous approved therapeutic regimens targeting RAR-α have been developed, improving patient survival rates. This study's main objective was to identify novel RAR-α-targeting drugs and evaluate the expression patterns of RAR-α in breast cancer patients. In-silico investigation using a variety of bioinformatics tools like UALCAN, TISCH, TIMER 2.0, ENRICHR, and others were employed to examine the expression of RAR-α. Further we evaluated in-silico inhibition of RAR-α with trifarotene and also tested the cytotoxicity of trifarotene in breast cancer cells. Our research indicates that RAR-α is upregulated in several malignancies including Breast Cancer. It regulates granulocyte differentiation and has an association with the retinoic acid receptor signaling pathway and cellular response to estrogen stimulus. Furthermore, trifarotene was found as a potential synthetic compound that targets RAR-α through in silico and in-vitro study. Overall, this research indicates that elevated expression of RAR-α enhances the onset of breast cancer. Using trifarotene medication to target RAR-α will significantly boost the response of breast cancer individuals to treatment and delay the development of resistance to drugs.

The emerging role and clinicopathological significance of MFSD12 in cancer and lysosomal storage diseases.

MFSD12 protein has recently risen as a key factor in malignancy and plays a potential role in a variety of complex oncogenic signaling cascades. Current studies suggest that MFSD12 has a positive complex role in the growth and progression of tumors such as melanoma, breast cancer, and lung cancer. At the same time, as a transporter of cysteine, MFSD12 is also involved in the development of lysosomal storage diseases. Therefore, MFSD12 may be an effective target to inhibit tumor development, block metastasis, and expand the therapeutic effect. This article reviews the molecular mechanisms of MFSD12 in a variety of cancers and lysosomal storage diseases.

Cytokeratin 15 is a novel and independent predictor of poor outcome in luminal B HER2-negative breast carcinomas

Cytokeratin 15 (CK15) has been described as a stem cell marker in human organs and its expression is seen in breast tissue. CK15 expression is associated with aggressive features in endometrial and oesophageal cancers, but data on the breast are lacking. This study aims to investigate the clinicopathological associations and prognostic significance of CK15 in breast carcinomas. A multi-institute cohort of breast carcinomas were retrieved. Clinicopathological and outcome data were obtained and compared with immunohistochemical expression CK15 and a panel of biomarkers. In total, 1,476 cases were included, with an expression rate of 3.5%, preferentially expressed in luminal subtypes (p=0.024), with luminal B carcinomas being the highest (4.7%), as opposed to basal-like (1%) and HER2-overexpressed carcinomas (0%). Except for nodal stage (p=0.013) and nodal metastasis (p=0.048), oestrogen (p=0.035) and progesterone receptor (p=0.001) positivity, there were no associations with other clinicopathological parameters. A trend was observed with shorter breast cancer specific survival (BCSS) in CK15-positive luminal B carcinomas (p=0.062). On further subgroup multivariate analysis of luminal B HER2-negative carcinomas, CK15 expression exhibited robust correlation with shorter BCSS (HR=9.004, p=0.001) and disease-free survival (HR=7.085, p<0.001). Restricted to luminal breast carcinomas, specifically luminal B HER2-negative, CK15 is demonstrated to be a robust independent predictor of higher risk of recurrence and shorter survival, with potential as a clinical prognostic marker and an exclusive stem cell marker for this subgroup of carcinomas.

The Clinicopathological Significance of Tumor Cell Subtyping in Appendiceal Neuroendocrine Tumors: A Series of 135 Tumors

Appendiceal neuroendocrine tumors (NETs) are common and often are identified as incidental lesions at the time of appendectomy. The guidelines for management are based on tumor size, degree of invasion, and the Ki67 proliferation index. Most small bowel NETs are composed of serotonin-producing EC-cells, but there are multiple other neuroendocrine cell types. In the rectum, there are L-cell tumors that express peptide YY (PYY), glucagon-like peptides (GLPs), and pancreatic polypeptide (PP); they are thought to have a better prognosis than serotonin-producing tumors. We investigated whether the appendix has distinct neuroendocrine tumor types based on cell type and whether that distinction has clinical significance. We collected 135 appendiceal NETs from the pathology archives of UHN Toronto and UHCMC (Cleveland). We analyzed the expression of biomarkers including CDX2, SATB2, PSAP, serotonin, glucagon (that detects GLPs), PYY, and pancreatic polypeptide (PP) and correlated the results with clinicopathologic parameters. Immunohistochemistry identified three types of appendiceal NETs. There were 75 (56%) classified as EC-cell tumors and 37 (27%) classified as L-cell tumors; the remaining 23 (17%) expressed serotonin and one of the L-cell biomarkers and were classified as mixed. EC-cell tumors were significantly larger with more extensive invasion involving the muscularis propria, subserosa, and mesoappendix compared with L-cell tumors. Mixed tumors were intermediate in all of these parameters. Both EC-cell and mixed tumors had lymphatic and/or vascular invasion while L-cell tumors had none. Unlike EC-cell NETs, L-cell tumors were not associated with lymph node metastasis. Tumor type correlated with pT stage and the only patient with distant metastatic disease in this series had an EC-cell tumor. Our study confirms that appendiceal NETs are not a homogeneous tumor population. There are at least three types of appendiceal NET, including EC-cell, L-cell, and mixed tumors. This information is important for surveillance of patients, as monitoring urinary 5HIAA levels is only appropriate for patients with serotonin-producing tumors, whereas measurement of GLPs and/or PP is more appropriate for patients with L-cell tumors. Our data also show that tumor type is of significance with EC-cell tumors exhibiting the most aggressive behavior.

METTL3 as a potential predictive biomarker for immunotherapy response in metastatic head and neck cancer.

e18019 Background: Head and neck squamous cell carcinoma (HNSCC) that has metastasized presents a significant challenge for cancer treatment, requiring innovative approaches to predict immunotherapy response. Methyltransferase-like 3 (METTL3), a key component of the m6A RNA methylation machinery, has been shown to have various roles in cancer, including its potential impact on immunotherapy responses. In this study, we aim to explore the expression and related function of METTL3 in metastatic HNSCC. Methods: We investigated the expression level and its clinicopathological significance of METTL3 using The Cancer Genome Atlas Head-Neck Squamous Cell Carcinoma (TCGA-HNSC) datasets. Further validation of the METTL3 mRNA and protein expression were carried out with real-time PCR and Western blot on tissues from 112 cases of HNSCC patients. In addition, the functional role of METTL3 was investigated in vitro studies. The treatment response prediction values of METTL3 in metastatic HNSCC were calculated using ROC-plotter. Results: Our investigation revealed a significant overexpression of METTL3 in HNSCC tissue samples than in normal tissues ( p &lt; 0.001). The high expression of METTL3 was significantly correlated with nodal metastasis, advanced cancer stages, and higher tumor grades in HNSCC samples, indicating that METTL3 plays an important role in driving HNSCC progression and aggressiveness. The functional study revealed intricate connections between METTL3 and immune regulatory genes expression, highlighting its potential to influence the tumor immune microenvironment. Silencing or pharmacological inhibition of METTL3 by METTL3 inhibitor STM2457 reduced tumorigenic activity through decreasing PD-L1 expression. ROC-plotter calculations showed that the high METTL3 expression predicted powerful immunotherapy (AUC=0.588, p=6.3e-06) in metastatic HNSCC patients. Conclusions: In conclusion, METTL3 appears to be a promising predictive biomarker for the response to immunotherapy in patients with metastatic HNSCC. This research provides valuable insights into the multifaceted role of METTL3 and promising therapeutic response predictor in metastatic HNSCC.

Clinicopathological significance of epidermal growth factor receptor expression in papillary thyroid carcinoma: a meta-analysis.

This study aimed to determine the relationship between the expression of epidermal growth factor receptor (EGFR) and pathological indicators in papillary thyroid carcinoma (PTC). PubMed, Embase, Web of Science, MEDLINE, and Cochrane Library databases were searched for relevant clinical trials. The odds ratio (OR) and 95% confidence interval (CI) showed the effect magnitude of the expression of EGFR, age, gender, tumor size, lymph node metastasis (LNM), extrathyroid extension, and tumor, lymph node, metastasis) stage. Stata 12.0 was used for statistical analysis of data. A total of 845 cases of PTC were included through the retrieval of 8 studies performed abroad. EGFR significantly correlated with extrathyroid extension (OR=3.25; 95% CI: 1.25-8.43; Z=2.42; P=0.015), LNM (OR=8.40; 95% CI: 5.44-12.97; Z=9.61; P=0.000), TNM stage (OR=2.30, 95% CI: 1.51-3.51; Z=3.87; P=0.000), and tumor size (OR=1.68; 95% CI: 1.06-2.68; Z=2.19; P=0.03). EGFR had no correlation with age (OR=1.13; 95% CI: 0.83-1.53; Z=0.77; P=0.44), gender (OR=0.93; 95% CI: 0.66-1.33; Z=0.38; P=0.70). Sensitivity analysis demonstrated that the studies by Cui Tang and Alfred King Yin Lam in LNM impacted the pooled OR. After removing these two studies, relatively stable results between the expression of EGFR and LNM were obtained. The results in the expression of EGFR is frequent and cancer-specific event in PTC. Besides, the expression of EGFR was involved in the progression and metastasis of PTC.

Expression of CD109 in oral squamous cell carcinoma and its clinical significance.

The purpose of this study was to investigate the expression of CD109 and its clinicopathological significance in oral squamous cell carcinoma. Data from TIMER2.0 and UALCAN were analyzed to assess CD109 mRNA levels in OSCC. The immunohistochemical method was used to investigate the expressions of CD109 in 20 normal oral mucosa and 75 OSCC and analyzed the relationship between the expression of CD109 and the clinical variables. The mRNA levels of CD109 in OSCC tissues were significantly higher than in adjacent normal tissues (p<0.05). Immunohistochemical analysis revealed that CD109 protein expression was increased in OSCC tissues compared to normal tissues, and this difference was statistically significant (P<0.05). The positive rate of CD109 expression was 94% (16/117) in the group with lymph node metastasis, while it was 55% (32/58) in the group without metastasis (P<0.05). Similarly, the positive rate of CD109 expression was 91% (22/23) in the low differentiation group and 59% (26/52) in the high differentiation group (P<0.05). CD109 expression is markedly higher in OSCC, contributes to the pathological grading of OSCC and predicts lymph node metastasis.

SOAT1 in gallbladder cancer: Clinicopathological significance and avasimibe therapeutics.

The aim of this investigation was to evaluate the differential expression of the sterol O-acyltransferase 1 (SOAT1) protein in gallbladder cancer tissues and cells, investigate the impact of Avastin on the proliferation, migration, invasion capabilities of gallbladder cancer cells, and its potential to induce cell apoptosis. Immunohistochemical analysis of samples from 145 gallbladder cancer patients was conducted, along with analysis of SOAT1 protein, mRNA expression levels, and cholesterol content in gallbladder cancer cell lines SGC-996, NOZ, and gallbladder cancer (GBC)-SD using Western blot and q-PCR techniques. Furthermore, the effects of Avastin on the proliferation, migration, and invasion capabilities of these gallbladder cancer cell lines were studied, and its ability to induce cell apoptosis was evaluated using flow cytometry, Western blot, and immunohistochemical methods. Additionally, gene expression and pathway analysis were performed, and the synergistic therapeutic effects of Avastin combined with gemcitabine were tested in a gallbladder cancer xenograft model. The study found that SOAT1 expression was significantly upregulated in GBC tissues and positively correlated with lymph node metastasis and TNM staging. In vitro experiments demonstrated that Avastin significantly inhibited the proliferation, migration, and invasion capabilities of SGC-996 and GBC-SD cell lines and induced apoptosis. RNA sequencing analysis revealed multiple differentially expressed genes in cells treated with Avastin, primarily enriched in biological pathways such as signaling transduction, malignant tumors, and the immune system. In vivo, experiments confirmed that Avastin could effectively suppress tumor growth in a gallbladder cancer xenograft model and enhanced the treatment efficacy when used in combination with gemcitabine. Overall, these findings provide new insights and strategies for targeted therapy in gallbladder cancer.

Tumor budding is an optimal indictor of occult cervical metastasis in clinical early-stage buccal mucosa squamous cell carcinoma.

Buccal mucosa squamous cell carcinoma (BMSCC) is an aggressive disease. This study investigated the clinicopathological significance of tumor budding (TB), depth of invasion (DOI), and mode of invasion (MOI) on occult cervical metastasis (CM) of BMSCC. Seventy-one cT1-2N0 BMSCC patients were included in this retrospective study. TB, DOI, MOI, and other clinicopathological features were reviewed. Risk factors for occult CM, locoregional recurrence-free survival (LRRFS), and overall survival (OS) were analyzed using logistic regression and Cox's proportional hazard models, respectively. Multivariate analysis with the logistic regression model revealed that MOI, DOI, and TB were significantly associated with occult CM in early-stage BMSCC after adjusting for variates. However, multivariate analysis with the Cox's proportional hazard model found only TB to be a prognostic factor for LRRFS (hazard ratio 15.03, 95% confidence interval [CI] 1.94-116.66; p = 0.01; trend test p = 0.03). No significant association was found between MOI, DOI, or TB and OS. The optimal predictor of occult CM and prognosis of early-stage BMSCC is TB, which may assist clinicians in identifying patients at high risk of cervical metastasis.

Stage-Specific Alteration and Prognostic Relationship of Serum Fumarate Hydratase Autoantibodies in Gastric Cancer.

The relationship between energy production and cancer is attracting attention. This study aimed to investigate the clinicopathological significance of fumarate hydratase (FH), a tricarboxylic acid cycle enzyme, in gastric cancer using autoantibodies as biomarkers. The study analyzed 116 patients who underwent gastric cancer surgery and 96 healthy controls. Preoperative serum FH autoantibody (s-FH-Ab) titers were analyzed using an immunosorbent assay with an amplified luminescent proximity homogeneous assay. Receiver operating characteristic analysis was used to determine the cutoff s-FH-Ab titer. Clinicopathological factors and prognosis were compared between the high and low s-FH-Ab groups. The s-FH-Ab levels were significantly higher in the gastric cancer group than in the control group (p = 0.01). Levels were elevated even in patients with stage I gastric cancer compared with healthy controls (p = 0.02). A low s-FH-Ab level was significantly associated with distant metastasis (p = 0.01), peritoneal dissemination (p < 0.05), and poor overall survival (p < 0.01). Multivariate analysis revealed that low s-FH-Ab levels were an independent risk factor for poor prognosis (p < 0.01). Therefore, s-FH-Ab levels may be a useful biomarker for early diagnosis and the prediction of prognosis in patients with gastric cancer.

The prognostic implications and tumor-promoting functions of CHSY3 in gastric cancer.

Chondroitin sulfate synthase 3 (CHSY3) is an important enzyme that regulates glycosylation, but its role in tumors has not been determined. Here, we showed that high CHSY3 expression promotes proliferation in gastric cancer (GC) cells and is associated with poor prognosis in GC patients. We analyzed the immunohistochemistry data of 150 gastric cancer patients to determine the clinicopathological and survival significance of CHSY3. Immunofluorescence was used to detect the colocalization of CHSY3 with infiltrating immune cells. Additionally, CHSY3 was predominantly found in tumor tissues and showed higher abundance compared to matched adjacent tissues. High CHSY3 expression was associated with more advanced tumor stage, higher recurrence risk and worse survival. Immunohistochemistry and bioinformatic analysis revealed that CHSY3 expression was significantly positively correlated with tumor-associated macrophage (TAM) infiltration. Moreover, after knocking down CHSY3, the proliferation of cells was decreased, and the migration ability was reduced, as shown by scratch, monoclonal and transwell assays. In conclusion, this study revealed that CHSY3 has a tumor-promoting effect on GC, suggesting a novel therapeutic strategy against this disease.

Clinicoradiological Parameters and Biochemical and Molecular Alterations Predicting Remission and Recurrence After Surgical Treatment of Corticotroph Adenomas—Cushing Disease

ObjectivesEndonasal endoscopic transsphenoidal surgery (TSS) and resection of pituitary adenomas is considered to be the gold standard treatment for Cushing’s Disease (CD). Even with various recent advances in management, disease persistence and recurrences are common in these patients. The remission rate in the global population after surgery has been reported to vary widely from 64 to 93%. This study aims to determine various clinical, biochemical, radiological and histological factors that correlate with persistence and recurrence in patients with CD. This study also aims to understand the clinicopathological significance of EGFR-MAPK, NF-kB and SHH pathway activation and to study the protein expression levels of activation markers of these pathways viz. c-Fos , c-Jun, GLI-1 , pMEK, NR4A1 and p44 in functioning corticotroph pituitary adenomas. MethodsFrom January 2009 to September 2022, the clinical data of 167 patients who underwent surgical treatment (n=174 surgeries) for Cushing’s disease was ambispectively analysed with a median follow up of 8.1 years (1-13.29 years). The preoperative clinical, biochemical, radiological features, operative findings, postoperative clinical and biochemical data, histopathological and molecular profile, were retrieved from electronic records. The patients were followed up to assess the remission status. ResultsAmong the 174 surgeries performed, 140 were primary surgeries, 22 were revision surgeries, 24 surgeries were for paediatric patients and 12 surgeries were performed in patients with Nelson syndrome. In the primary surgery cohort, 74.3% were females and the average age was 28.73 years (SD 10.15). 75% of the patients experienced remission after primary surgery compared to 47.4% in revision surgery. The remission rate in paediatric patients was 55.5%. Post-operative day 1 plasma cortisol (p<0.001; AUC 0.8894 [0.8087-0.9701]) and ACTH (p<0.001; AUC 0.9 [0.7386-1]) levels were seen to be strong independent predictors of remission in the primary surgery cohort. Remission rates after Endoscopic TSS were more than that after microscopic TSS, in patients undergoing primary surgery (81.08% vs 57.14%; p=0.008). Presence of adenoma on histopathological examination (HPE) was also a strong predictor of disease remission (p=0.020). On categorising based on surgical approach and HPE, microscopically operated patients without histopathological evidence of adenoma had significantly higher odds of non-remission (OR 38.1 95% CI 4.2-348.3) compared to endoscopically operated patients with adenoma on HPE. Lower immune reactivity score (IRS) of NR4A1 was found to correlate with higher remission rates (p=0.074). However, none of the molecular markers studied viz. c-Fos , c-Jun, GLI-1, pMEK and p44 showed a significant correlation with the preoperative cortisol values. ConclusionRemission rate after primary surgery is higher as compared to that after revision surgery, and that in paediatric patients is lower as compared to adults. Post-operative day 1 plasma cortisol and ACTH levels are strong independent predictors of remission in the primary surgery cohort. Endoscopic approach with histopathological evidence of adenoma is associated with a higher remission rate and thus endoscopy should be the approach of choice in these patients with the goal of identification of an adenoma on histopathological analysis.

Keratin 6A Is Expressed at the Invasive Front and Enhances the Progression of Colorectal Cancer

Keratins (KRTs) are intermediate filament proteins in epithelial cells, and they are important for cytoskeletal organization. KRT6A, classified as a type II KRT, is normally expressed in stratified squamous epithelium and squamous cell carcinomas. Little is known about the expression and role of KRT6A in adenocarcinomas. We investigated the clinicopathologic and molecular biological significance of KRT6A in colorectal adenocarcinoma. Immunostaining of colorectal adenocarcinoma cases treated at our institution demonstrated that KRT6A showed significantly stronger expression at the invasive front than that at the tumor center (P < .0001). The high KRT6A–expression cases (n = 47) tended to have a high budding grade associated with significantly worse prognoses. A multivariate analysis revealed that the KRT6A expression status was an independent prognostic factor for overall survival (P = .0004), disease-specific survival (P = .0097), and progression-free survival (P = .0033). The correlation between KRT6A and patient prognoses was also validated in an external cohort from a published data set. To determine the function of KRT6A in vitro, KRT6A was overexpressed in 3 colon cancer cell lines: DLD-1, SW620, and HCT 116. KRT6A overexpression increased migration and invasion in DLD-1 but did not in SW620 and HCT116. In 3-dimensional sphere-forming culture, KRT6A expression enhanced the irregular protrusion around the spheroid in DLD-1. Our findings in this study indicated that KRT6A expression is a valuable prognostic marker of colorectal cancer and KRT6A may be involved the molecular mechanism in the progression of invasive areas of colorectal cancer.

SKA3 Expression as a Prognostic Factor for Patients with Pancreatic Adenocarcinoma.

The spindle and kinetochore-associated complex subunit 3 (SKA3) is a protein essential for proper chromosome segregation during mitosis and thus responsible for maintaining genome stability. Although its involvement in the pathogenesis of various cancer types has been reported, the potential clinicopathological significance of SKA3 in pancreatic ductal adenocarcinoma (PDAC) has not been fully elucidated. Therefore, this study aimed to assess clinicopathological associations and prognostic value of SKA3 in PDAC. For this purpose, in-house immunohistochemical analysis on tissue macroarrays (TMAs), as well as a bioinformatic examination using publicly available RNA-Seq dataset, were performed. It was demonstrated that SKA3 expression at both mRNA and protein levels was significantly elevated in PDAC compared to control tissues. Upregulated mRNA expression constituted an independent unfavorable prognostic factor for the overall survival of PDAC patients, whereas altered SKA3 protein levels were associated with significantly better clinical outcomes. The last observation was particularly clear in the early-stage tumors. These findings render SKA3 a promising prognostic biomarker for patients with pancreatic ductal adenocarcinoma. However, further studies are needed to confirm this conclusion.

Lactylproteome analysis indicates histone H4K12 lactylation as a novel biomarker in triple-negative breast cancer.

The established link between posttranslational modifications of histone and non-histone lysine (K) residues in cell metabolism, and their role in cancer progression, is well-documented. However, the lactylation expression signature in triple-negative breast cancer (TNBC) remains underexplored. We conducted a comprehensive lactylproteome profiling of eight pairs of TNBC samples and their matched adjacent tissues. This was achieved through 4-Dimensional label-free quantitative proteomics combined with lactylation analysis (4D-LFQP-LA). The expression of identified lactylated proteins in TNBC was detected using immunoblotting and immunohistochemistry (IHC) with specific primary antibodies, and their clinicopathological and prognostic significance was evaluated. Our analysis identified 58 lactylation sites on 48 proteins, delineating the protein lactylation alteration signature in TNBC. Bioinformatic and functional analyses indicated that these lactylated proteins play crucial roles in regulating key biological processes in TNBC. Notably, lactylation of lysine at position 12 (H4K12lac) in the histone H4 domain was found to be upregulated in TNBC. Further investigations showed a high prevalence of H4K12lac upregulation in TNBC, with positive rates of 93.19% (137/147) and 92.93% (92/99) in TNBC tissue chip and validation cohorts, respectively. H4K12lac expression correlated positively with Ki-67 and inversely with overall survival (OS) in TNBC (HR [hazard ratio] =2.813, 95%CI [credibility interval]: 1.242-6.371, P=0.0164), suggesting its potential as an independent prognostic marker (HR=3.477, 95%CI: 1.324-9.130, P=0.011). Lactylation is a significant post-translational modification in TNBC proteins. H4K12lac emerges as a promising biomarker for TNBC, offering insights into the lactylation profiles of TNBC proteins and linking histone modifications to clinical implications in TNBC.

Clinicopathological Significance of Cyclin-Dependent Kinase 2 (CDK2) in Ductal Carcinoma In Situ and Early-Stage Invasive Breast Cancers.

Cyclin-dependent kinase 2 (CDK2) is a key cell cycle regulator, with essential roles during G1/S transition. The clinicopathological significance of CDK2 in ductal carcinomas in situ (DCIS) and early-stage invasive breast cancers (BCs) remains largely unknown. Here, we evaluated CDK2's protein expression in 479 BC samples and 216 DCIS specimens. Analysis of CDK2 transcripts was completed in the METABRIC cohort (n = 1980) and TCGA cohort (n = 1090), respectively. A high nuclear CDK2 protein expression was significantly associated with aggressive phenotypes, including a high tumour grade, lymph vascular invasion, a poor Nottingham prognostic index (all p-values < 0.0001), and shorter survival (p = 0.006), especially in luminal BC (p = 0.009). In p53-mutant BC, high nuclear CDK2 remained linked with worse survival (p = 0.01). In DCIS, high nuclear/low cytoplasmic co-expression showed significant association with a high tumour grade (p = 0.043), triple-negative and HER2-enriched molecular subtypes (p = 0.01), Comedo necrosis (p = 0.024), negative ER status (p = 0.004), negative PR status (p < 0.0001), and a high proliferation index (p < 0.0001). Tumours with high CDK2 transcripts were more likely to have higher expressions of genes involved in the cell cycle, homologous recombination, and p53 signaling. We provide compelling evidence that high CDK2 is a feature of aggressive breast cancers. The clinical evaluation of CDK2 inhibitors in early-stage BC patients will have a clinical impact.

Abstract PO1-24-03: The clinicopathological and prognostic significance of HER2-low breast cancer: A comparative analysis between HER2-low and HER2-zero subtypes

Abstract Background: HER2-low breast cancer (BC) is a newly defined subset of HER2-negative BC that has a HER2 immunohistochemical (IHC)-1+ or 2+/ISH negative phenotype. Recent clinical trials have demonstrated the clinical benefits of novel HER2 directing ADCs in treating this group. It is uncertain whether HER2-low BC represents a distinct biological/clinical subgroup or if the HER2-low categorization has any prognostic significance. We retrospectively investigated the clinicopathological and prognostic significance of HER2-low BC and compared it with HER2-0 BC. Methods: Primary BC patients who underwent surgery from January 2000 to March 2023 were enrolled in this retrospective study. A total of 10,215 invasive BC cases with Stage I-III were analyzed. The HER2 status was divided into 3 groups: HER2-0, HER2-low, and HER2-positive (3+ or 2+ with positive ISH). The clinicopathological factors investigated were age, nodal status, tumor size, nuclear grade (NG), ER/PgR (cutoff points; 1% and 10%), and the Ki-67 index value (cutoff points: 15% and 30%). The pCR rate in patients who underwent neoadjuvant chemotherapy (NAC) was examined. The disease-free (DFS) and overall survival (OS) were calculated using the Kaplan-Meier method and tested with the log-rank procedure. Uni- and multivariate analyses for recurrent factors were performed using the Cox proportional hazard model. Results: 1. The HER2 status was classified as follows; HER2-0 in 1,227 cases (12.0%), HER2-low in 7,209 cases (70.6%), and HER2-positive in 1,779 cases (17.4%). In the recurrent cases, HER2-low was observed in 648 cases (66.1%), and in cases treated with NAC, HER2-low was observed in 544 cases (52.7%). 2. The HER2 status significantly correlated with age, tumor size, lymph nodal status, ER, PgR, NG, and the Ki-67 index value. A comparison between HER2-low and HER2-0 revealed that patients with HER2-low tumors were significantly more likely to be under 50 years of age and had more positive nodes. Moreover, HER2-low was significantly associated with positive ER/PgR, lower NG, and lower Ki-67 index values. 3. There was no significant difference in terms of DFS between the three HER2 groups. However, a difference was observed between the three groups in terms of OS. HER2-0 had the lowest OS rate. Moreover, there was a significant difference in OS after recurrence between HER2-0 and HER2-low. 4. When the DFS for the ER status was examined, a significant difference between the HER2 status in cases with ER &amp;lt; 1.0% and ER&amp;gt;10% was observed. Moreover, the DFS in the HER2-low cases was equivalent to the DFS in the HER2-0 cases. However, the DFS in HER2-low was lower in the ER 1-10% group. Although all the HER2 groups showed a significant difference in OS, HER2-low in the ER 1-10% group had the lowest OS, and HER2-0 in the ER&amp;gt;10% group had the lowest OS and was significantly different from HER2-low in the same group. 5. A multivariate analysis revealed that there was no significant difference between HER2-low and HER2-0 as a prognostic factor after recurrence. 6. The pCR rate for NAC was lowest in the HER2-low group, and the pCR rate in the HER2-low group was significantly lower than the HER2-0 group. 7. The DFS after NAC was significantly better in all the pCR cases, regardless of the HER2 status. However, the DFS was significantly lower in the HER2-low non-pCR cases. Conclusion: HER2-low accounted for 70% of the cases. There were more cases with positive ER/PgR, lower NG, and lower Ki-67 index values in the HER2-low group. The HER2-low group had a significantly better OS than the HER2-0 group. However, the response to NAC was low in the HER2-low group and this group had the poorest prognosis among all the non-pCR cases. These findings indicate that HER2-low has a different biology and prognosis compared to HER2-0 and it may be a new subtype. Citation Format: Reiki Nishimura, Yoshitaka Fujiki, Tetsuhiko Taira, Toshiko Miyaki, Shuichi Kanemitsu, Daisuke Yotsumoto, Megumi Teraoka, Junko Kawano, Naomi Gondo, Reiko Mitsueda, Shinichi Baba, Yasuyo Ohi, Yoshiaki Rai, Yoshiaki Sagara, Yasuaki Sagara. The clinicopathological and prognostic significance of HER2-low breast cancer: A comparative analysis between HER2-low and HER2-zero subtypes [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-24-03.

Abstract PO4-26-04: Immunolocalization of cytoplasmic ER in ER-negative breast carcinoma as a potent favorable prognostic predictor

Abstract Background: It is known that estrogen receptor (ER) has extranuclear signaling functions in addition to classical genomic pathway, and estrogenic actions have been reported in ER-negative breast carcinoma cells. However, significance of cytoplasmic-ER immunoreactivity has not been reported in ER-negative breast carcinoma tissues. Methods: We immunolocalized cytoplasmic ER in 155 ER-negative breast carcinoma tissues and evaluated its clinicopathological significance including the prognosis. As a comparative cohort set, we also used 142 ER-positive breast carcinomas. Results: Cytoplasmic-ER immunoreactivity was detected in the carcinoma cells, but not in the non-neoplastic mammary epithelium. Cytoplasmic-ER immunoreactivity was positive in the 35 out of 155 (23%) ER-negative breast carcinoma cases, whereas it was detected only in 2 out of 142 (1.4%) ER-positive cases. Cytoplasmic ER status was positively associated with cytoplasmic-PR status, but inversely associated with Ki67 labeling index or distant free-relapse survival rate. Moreover, cytoplasmic-ER status turned out to be an independent good prognostic factor for both distant relapse-free survival and breast cancer specific survival. Conclusion: These findings suggested that cytoplasmic ER plays important roles in the ER-negative breast carcinoma, and cytoplasmic ER is a potent good prognostic factor. (accepted in Acta Histochemica et Cytochemica, 2023/Jun/27) Citation Format: Akiko Ebata, Takashi Suzuki, Narumi Harada-Shoji, Yohei Hamanaka, Minoru Miyashita, Erina Iwabuchi, Kiyoshi Takagi, Yasuhiro Miki, Hiroshi Tada, Takanori Ishida. Immunolocalization of cytoplasmic ER in ER-negative breast carcinoma as a potent favorable prognostic predictor [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-26-04.

Radiologic tumor border can further stratify prognosis in patients with pancreatic neuroendocrine tumor

Background and objectivesPancreatic neuroendocrine tumor (PanNET), although rare in incidence, is increasing in recent years. Several clinicopathologic and molecular factors have been suggested for patient stratification due to the extensive heterogeneity of PanNETs. We aimed to discover the prognostic role of assessing the tumor border of PanNETs with pre-operative computed tomography (CT) images and correlate them with other clinicopathologic factors. MethodsThe radiologic, macroscopic, and microscopic tumor border of 183 surgically resected PanNET cases was evaluated using preoperative CT images (well defined vs. poorly defined), gross images (expansile vs. infiltrative), and hematoxylin and eosin-stained slides (pushing vs. infiltrative). The clinicopathologic and prognostic significance of the tumor border status was compared with other clinicopathologic factors. ResultsA poorly defined radiologic tumor border was observed in 65 PanNET cases (35.5 %), and were more frequent in male patients (P = 0.031), and tumor with larger size, infiltrative macroscopic growth pattern, infiltrative microscopic tumor border, higher tumor grade, higher pT category, lymph node metastasis, lymphovascular and perineural invasions (all, P < 0.001). Patients with PanNET with a poorly defined radiologic tumor border had significantly worse overall survival (OS) and recurrence-free survival (RFS; both, P < 0.001). Multivariable analysis revealed that PanNET with a poorly defined radiologic border is an independent poor prognostic factor for both OS (P = 0.049) and RFS (P = 0.027). ConclusionPre-operative CT-based tumor border evaluation can provide additional information regarding survival and recurrence in patients with PanNET.