p53, Cytokeratin 19 Expression in Oral Squamous Cell Carcinoma and Correlation with Histopathologic Grading: An Immunohistochemical Study.

The purpose of this study was to examine the immunohistochemical expression of p53 and cytokeratin 19 (CK19) in normal oral mucosa (NOM) and oral squamous cell carcinoma (OSCC) and their association with histopathological differentiation grade. The secondary goal was to see if there was any correlation between p53 and CK19 expression in NOM and OSCC. A hospital-based retrospective analysis was conducted in which 40 NOM and 45 OSCC samples were acquired from archives and stained with mouse monoclonal antibodies p53 and CK19. For both the NOM and OSCC groups, the proportion of positively stained cells, staining intensity, and staining index were calculated. p53 immunoexpression revealed that 85% of positively stained cells in the NOM basal layer had a low staining index (mean ± SD 1.87 ± 0.34), whereas 66.7% of positively stained cells in the OSCC had a high staining index (mean ± SD 5.63 ± 3.02). When NOM and OSCC were compared, there was a statistically significant difference in staining intensity. However, despite a linear increase in the percentage of positive cells from well to poorly differentiated, the comparison between histopathological grades was non-significant. CK19 exhibited 18.5% positively stained cells in the NOM basal layer with a low staining index (mean ± SD 1.57 ± 0.53), whereas OSCC samples showed 4.44% immunopositivity with a high staining index. p53 is a marker of oral carcinogenesis independent of histological grade and CK19 expression. Further, CK19 is a marker of dysfunctional epithelial differentiation but lacks sensitivity and specificity; however, it demands further multicentric studies with a large sample size to draw definitive conclusions. Supplementary Information: The online version contains supplementary material available at 10.1007/s12070-023-04092-7.

Construction of biomimetic cell-sheet-engineered periosteum with a double cell sheet to repair calvarial defects of rats.

Background: The periosteum plays a crucial role in the development and injury healing process of bone. The purpose of this study was to construct a biomimetic periosteum with a double cell sheet for bone tissue regeneration. Methods: In vitro, the human amniotic mesenchymal stem cells (hAMSCs) sheet was first fabricated by adding 50 â€‹µg/ml ascorbic acid to the cell sheet induction medium. Characterization of the hAMSCs sheet was tested by general observation, microscopic observation, live/dead staining, scanning electron microscopy (SEM) and hematoxylin and eosin (HE) staining. Afterwards, the osteogenic cell sheet and vascular cell sheet were constructed and evaluated by general observation, alkaline phosphatase (ALP) staining, Alizarin Red S staining, SEM, live/dead staining and CD31 immunofluorescent staining for characterization. Then, we prepared the double cell sheet. In vivo, rat calvarial defect model was introduced to verify the regeneration of bone defects treated by different methods. Calvarial defects (diameter: 4 â€‹mm) were created of Sprague-Dawley rats. The rats were randomly divided into 4 groups: the control group, the osteogenic cell sheet group, the vascular cell sheet group and the double cell sheet group. Macroscopic, micro-CT and histological evaluations of the regenerated bone were performed to assess the treatment results at 8 weeks and 12 weeks after surgery. Results: In vitro, hAMSCs sheet was successfully prepared. The hAMSCs sheet consisted of a large number of live hAMSCs and abundant extracellular matrix (ECM) that secreted by hAMSCs, as evidenced by macroscopic/microscopic observation, live/dead staining, SEM and HE staining. Besides, the osteogenic cell sheet and the vascular cell sheet were successfully prepared, which were verified by general observation, ALP staining, Alizarin Red S staining, SEM and CD31 immunofluorescent staining. In vivo, the macroscopic observation and micro-CT results both demonstrated that the double cell sheet group had better effect on bone regeneration than other groups. In addition, histological assessments indicated that large amounts of new bone had formed in the calvarial defects and more mature collagen in the double cell sheet group. Conclusion: The double cell sheet could promote to repair calvarial defects of rats and accelerate bone regeneration. The translational potential of this article: We successfully constructed a biomimetic cell-sheet-engineered periosteum with a double cell sheet by a simple, low-cost and effective method. This biomimetic periosteum may be a promising therapeutic strategy for the treatment of bone defects, which may be used in clinic in the future.

Ultrasomics prediction for cytokeratin 19 expression in hepatocellular carcinoma: A multicenter study.

Objective: The purpose of this study was to investigate the preoperative prediction of Cytokeratin (CK) 19 expression in patients with hepatocellular carcinoma (HCC) by machine learning-based ultrasomics. Methods: We retrospectively analyzed 214 patients with pathologically confirmed HCC who received CK19 immunohistochemical staining. Through random stratified sampling (ratio, 8:2), patients from institutions I and II were divided into training dataset (n = 143) and test dataset (n = 36), and patients from institution III served as external validation dataset (n = 35). All gray-scale ultrasound images were preprocessed, and then the regions of interest were then manually segmented by two sonographers. A total of 1409 ultrasomics features were extracted from the original and derived images. Next, the intraclass correlation coefficient, variance threshold, mutual information, and embedded method were applied to feature dimension reduction. Finally, the clinical model, ultrasonics model, and combined model were constructed by eXtreme Gradient Boosting algorithm. Model performance was assessed by area under the receiver operating characteristic curve (AUC), sensitivity, specificity, and accuracy. Results: A total of 12 ultrasomics signatures were used to construct the ultrasomics models. In addition, 21 clinical features were used to construct the clinical model, including gender, age, Child-Pugh classification, hepatitis B surface antigen/hepatitis C virus antibody (positive/negative), cirrhosis (yes/no), splenomegaly (yes/no), tumor location, tumor maximum diameter, tumor number, alpha-fetoprotein, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, glutamyl-transpeptidase, albumin, total bilirubin, conjugated bilirubin, creatinine, prothrombin time, fibrinogen, and international normalized ratio. The AUC of the ultrasomics model was 0.789 (0.621 - 0.907) and 0.787 (0.616 - 0.907) in the test and validation datasets, respectively. However, the performance of the combined model covering clinical features and ultrasomics signatures improved significantly. Additionally, the AUC (95% CI), sensitivity, specificity, and accuracy were 0.867 (0.712 - 0.957), 0.750, 0.875, 0.861, and 0.862 (0.703 - 0.955), 0.833, 0.862, and 0.857 in the test dataset and external validation dataset, respectively. Conclusion: Ultrasomics signatures could be used to predict the expression of CK19 in HCC patients. The combination of clinical features and ultrasomics signatures showed excellent effects, which significantly improved prediction accuracy and robustness.

Diagnostic and prognostic impact of cytokeratin 19 expression analysis in human tumors: a tissue microarray study of 13,172 tumors.

To evaluate cytokeratin 19 (CK19) expression in normal and cancerous tissues, 15,977 samples from 122 tumor types and 608 samples of 76 normal tissue types were analyzed by immunohistochemistry (IHC). In normal tissues, CK19 expression occurred in epithelial cells of most glandular organs but was strictly limited to the basal cell layer of nonkeratinizing squamous epithelium and absent in the skin. CK19 expression in ≥90% of cases was seen in 34% of the tumor entities including the adenocarcinomas of the pancreas (99.4%), colorectum (99.8%), esophagus (98.7%), and stomach (97.7%), as well as breast cancer (90.0%-100%), high-grade serous (99.1%) or endometrioid (97.8%) ovarian cancer, and urothelial carcinoma (92.6%-100%). A low CK19 positivity rate (0.1-10%) was seen in 5 of 122 tumor entities including hepatocellular carcinoma and seminoma. A comparison of tumor versus normal tissue findings demonstrated that upregulation and downregulation of CK19 can occur in cancer and that both alterations can be linked to unfavorable phenotypes. CK19 downregulation was linked to high grade (p = 0.0017) and loss of estrogen receptor- and progesterone receptor-expression (p < 0.0001 each) in invasive breast carcinoma of no special type. CK19 upregulation was linked to nodal metastases in neuroendocrine tumors and papillary thyroid carcinomas (p < 0.05 each) and to poor grade in clear cell renal cell carcinoma (p < 0.05). CK19 upregulation was particularly common in squamous cell carcinomas. We concluded that CK19 IHC might separate primary liver cell carcinoma from liver metastases, seminoma from other testicular tumors, and helps in the detection of early neoplastic transformation in squamous epithelium.

MRI-Radiomics Prediction for Cytokeratin 19-Positive Hepatocellular Carcinoma: A Multicenter Study.

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and has poor prognosis. Cytokeratin (CK)19-positive (CK19+) HCC is especially aggressive; early identification of this subtype and timely intervention can potentially improve clinical outcomes. In the present study, we developed a preoperative gadoxetic acid-enhanced magnetic resonance imaging (MRI)-based radiomics model for noninvasive and accurate classification of CK19+ HCC. A multicenter and time-independent cohort of 257 patients were retrospectively enrolled (training cohort, n = 143; validation cohort A, n = 75; validation cohort B, n = 39). A total of 968 radiomics features were extracted from preoperative multisequence MR images. The maximum relevance minimum redundancy algorithm was applied for feature selection. Multiple logistic regression, support vector machine, random forest, and artificial neural network (ANN) algorithms were used to construct the radiomics model, and the area under the receiver operating characteristic (AUROC) curve was used to evaluate the diagnostic performance of corresponding classifiers. The incidence of CK19+ HCC was significantly higher in male patients. The ANN-derived combined classifier comprising 12 optimal radiomics features showed the best diagnostic performance, with AUROCs of 0.857, 0.726, and 0.790 in the training cohort and validation cohorts A and B, respectively. The combined model based on multisequence MRI radiomics features can be used for preoperative noninvasive and accurate classification of CK19+ HCC, so that personalized management strategies can be developed.

Molecular mechanism of CK19 involved in the regulation of postoperative recurrence of HBV-associated primary hepatocellular carcinoma in Guangxi.

BACKGROUND: Cytokeratin 19 (CK19/KRT19) is a marker of biliary epithelial cells and hepatic progenitor cells, which can be expressed in some hepatocellular carcinoma (HCC). However, its role in the occurrence, development, and recurrence of hepatitis B virus (HBV)-associated HCC remains to be clarified. This study is to analyze the relationship between the expression of CK19 protein and clinicopathological factors, as well as the effect of positive CK19 expression on the prognosis of HCC patients. METHODS: Small interfering RNA (siRNA) transfection was used to silence CK19 in MHCC-97H and Hep-3B. Real time polymerase chain reaction (qPCR), immunohistochemistry (IHC), and flow cytometry were used to detect the effects of CK19 silencing on cell function. High-throughput sequencing was used to explore the potential molecular mechanism of CK19 positive expression of HCC. RESULTS: In 24 patients with HCC, CK19 was only expressed in cancer tissues, regardless of primary or recurrent tumors, and the positive expression rate of recurrent tumors was higher than that of primary tumors. The HCC participants with positive primary CK19 expression had a shorter tumor-free survival time. Silencing of the CK19 gene in MHCC-97H and Hep-3B attenuated the migration and invasion ability of MHCC-97H, increased the G2 phase cell content of MHCC-97H and Hep-3B, and increased the proportion of apoptosis. High-throughput sequencing results suggested that changes in the function of the cell cycle regulating genes, drug, and carcinogenic metabolism might be the potential pathways of CK19 in regulating the biological behavior of HCC. CONCLUSIONS: Among HBV-related recurrent HCC, the positive rate of CK19 expression in recurrent HCC tumors was higher, and the tumor-free survival time of HCC patients with positive CK19 expression in primary HCC was shorter. After silencing of the CK19 gene, the migration and invasion ability of HCC cells were weakened, the content of G2-M cell cycle cells was increased, the invasion and migration of HCC cells were inhibited, and apoptosis was promoted. Changes in the function of the cell cycle regulating genes and the regulation of drug and carcinogenic metabolites-related pathways may be the pathways through which CK19 affects the biological behavior of HCC.

Does Neutrophil to Lymphocyte Ratio Have a Role in Identifying Cytokeratin 19-Expressing Hepatocellular Carcinoma?

BACKGROUND: Cytokeratin 19-positive (CK19(+)) hepatocellular carcinomas (HCC) are generally associated with poor prognosis after hepatectomy. It is typically detected from postoperative immunochemistry. We have analyzed several clinically available biomarkers, in particular, neutrophil to lymphocyte ratio (NLR) and aim to develop a panel of biomarkers in identifying CK19 expression in (HCC) preoperatively. METHODS: We retrospectively reviewed 36 HCC patients who underwent liver resections during January 2017 to March 2018 in Chang Gung Memorial Hospital. Patients were grouped based on the status of CK19 expression and their baseline characteristics, perioperative and oncologic outcomes were compared. Novel biomarkers including NLR, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA) and uric acid were analyzed and correlated with CK19 expression. RESULTS: NLR is highly associated with CK19 expression. NLR alone gave an AUROC of 0.728 (p-value = 0.043), higher than AFP, CEA or tumor size alone. NLR when combined with AFP, CEA and uric acid, gave an AUROC as high as 0.933 (p-value = 0.004). CONCLUSION: The current study demonstrated the predictive capability of NLR in combination with AFP, CEA and uric acid for CK19 expression in HCC patients preoperatively. Further prospective, large-scale studies are warranted to validate our findings.

Gene expression of cytokeratin 19 and its molecular detection in human breast cancer cell lines.

Cytokeratins have been identified as useful tools in oncology diagnostics. In this study, cytokeratin19 (CK19) expression was studied in three human breast cancer cell lines, SKBR3, BT549, and BT474 using RT-PCR. CK19 was expressed in tumor cell of different origin, showing higher expression in invasive breast cancer with ER(+) (BT474) than invasive breast cancer with ER(-) (BT549) and breast adenocarcinoma with ER(-) (SKBR3). Two primer sets were used to evaluate CK19 expression. Primer set I (hCK19/1) and primer set II (hCK19/2) were used to amplify the CK19 human gene at a 215bp and 384bp, respectively, whereas PBMC and RAW264.7 (mouse macrophage) no detectable PCR products were obtained. The sensitivity for detection was determined by two methods, i.e., cDNA dilution (the dilution of cDNA from RNA of breast cancer cells) and cell dilution (the dilution of breast cancer cells in PBMC). hCK19/2 was more sensitive than hCK19/1. In cDNA dilution, the lower limits of primer set II for detection were 400, 40 and 40 cells for SKBR3, BT549 and BT474 cells, respectively. While in cell dilution all of the 3 breast cancer cells could be detected at 1 cancer cell in 10(4), 10(6) and 10(5) PBMC, respectively. The data supported the possibility that CK19 could be detected and be the marker for breast cancer in patient blood.

Assessment of Cytokeratin-19 Gene Expression in Peripheral Blood of Breast Cancer Patients and Breast Cancer Cell Lines.

Detection of cytokeratin-19 (CK19) expression as an epithelial-specific marker in circulating tumor cells (CTCs) of breast cancer patients can be important for diagnostic purposes. Comparison of CK19 expression in breast cancer cell lines can indicate that expression of this marker is different in various breast cancer cell lines based on their category. Thirty-five breast cancer patients were evaluated for detection of CK19 mRNA in their peripheral blood using CK19-specific primers and a nested reverse transcriptase polymerase chain reaction (RT-PCR) technique. CK19 expression levels were detected in MCF7, T47D, SK-BR-3, and MDA-MB-231 cell lines by semiquantitative RT-PCR and Western blot analyses. Statistical analysis of our data indicates that there is no significant difference between CK19 expression and histopathological parameters and some molecular markers, including Ki-67, HER-2, and P53, but there are statistically significant correlations between estrogen receptor (P = 0.040) and progesterone receptor (P = 0.046) with CK19 expression. CK19 expression was detected in MCF7, T47D, and SK-BR-3 cell lines but not in MDA-MB-231 cell line. More studies are needed to determine the relationship between this marker and other markers in the diagnosis and treatment of breast cancer. On the other hand, the study of different markers using breast cancer cell lines as experimental models of breast cancer could have an impact on improving the health outcomes of patients with breast cancer.

Expression of CK19 is an independent predictor of negative outcome for patients with squamous cell carcinoma of the tongue.

OBJECTIVES: To explore the prognostic role of CK19 expression in squamous cell carcinomas within a well-defined cohort of oral tongue cancer patients. METHODS: In our retrospective study, we investigated 129 patients with tongue cancer that had suitable material for inclusion in a tissue microarray (TMA). Where possible, samples were taken from central and peripheral regions of the tumor to generate a representative sample of the tumor. The expression level of CK19 was assessed by immunohistochemical staining. RESULTS: Expression of CK19 in squamous cell carcinoma of the tongue was identified as a negative predictor for overall survival (OS; p<0.000) and disease specific survival (DSS; p=0.001). No significant difference could be shown for disease free survival (DFS) between patients with positive and negative CK19 staining (p=.094). CONCLUSION: This is the first description of the highly significant role of CK19 in a selective, organ specific head and neck cancer cohort. Our results are of special importance against the background that CK19 positive carcinomas revealed a significantly poorer prognosis and therefore emphasize its prognostic and possible diagnostic role in tongue cancer.

Prognostic value of cytokeratin 19 in hepatocellular carcinoma: A meta-analysis.

BACKGROUND: Although many studies have investigated the relationship between cytokeratin 19 (CK-19) and hepatocellular carcinoma (HCC), the prognostic value of CK-19 in HCC remains inconclusive. METHODS: Eligible studies were sought in PubMed, Embase, Web of Science, Cochrane Library and Wanfang databases. Pooled hazard ratios (HRs) and odds ratios (ORs) with corresponding 95% confidence intervals (CIs) were calculated. RESULTS: 17 studies with 2943 patients were included in this meta-analysis. Meta-analysis results showed that CK-19 over-expression was significantly associated with overall survival (OS) (HR=1.60, 95% CI: 1.32-1.93, univariate analysis; HR=2.25, 95% CI: 1.79-2.83, multivariate analysis) and disease-free survival (DFS) (HR=1.68, 95% CI: 1.35-2.10, univariate analysis; HR=1.97, 95% CI: 1.54-2.53, multivariate analysis). Meanwhile, CK-19 over-expression was also correlated with decreased 1-year OS rate (OR=0.32, 95% CI: 0.21-0.50), 5-year OS rate (OR=0.44, 95% CI: 0.14-0.87) and 1-year DFS rate (OR=0.51, 95% CI: 0.34-0.76), but not with 5-year DFS rate (OR=0.62, 95% CI: 0.35-1.10). These results suggested that CK-19 over-expression was significantly associated with poor survival rate and early tumor recurrence rate in HCC patients. CONCLUSIONS: CK-19 can serve as an indicator of poor prognosis as well as a novel target for treatment in HCC.

A differential intra-operative molecular biological test for the detection of sentinel lymph node metastases in breast carcinoma. An extended experience from the first U.K. centre routinely offering the service in clinical practice.

INTRODUCTION: One-Step Nucleic acid Amplification (OSNA) is a molecular biological assay of cytokeratin-19 (a breast epithelial marker) mRNA. It can be employed intra-operatively for detection of lymph node metastases in breast carcinoma. Patients with positive sentinel nodes may proceed to axillary lymph node dissection (ALND) level I or higher dependent upon the OSNA quantitative result, during the same surgical procedure, avoiding a second operation and eliminating the technical difficulties possibly associated with delayed ALND. AIMS: Our Breast Unit was the first in the UK to implement this novel technique in routine practice. This study reviews our first 44-month data following introduction of OSNA "live" on whole sentinel nodes following an extensive validation study (Snook et al.).(9) METHODS: Data was collected prospectively from the period of introduction 01/12/2008 to 30/08/2012. All patients eligible for sentinel node biopsy were offered OSNA and operations were performed by five consultant breast surgeons. On detection of macro-metastasis a level II/III and for a micro-metastasis a level I ALND was performed. RESULTS: A total of 859 patients (1709 sentinel lymph nodes) were analysed. All except one were females. The majority underwent wide local excision (73.4%, n = 631) or mastectomy 25% (n = 215) and 1.6% (13) underwent SLN biopsy alone. IDC was seen in 79% (n = 680) of the patients and 53.5% (n = 460) had grade II tumours. One-third (30.8%, n = 265) had positive sentinel nodes and had further axillary surgery at the time of SLN biopsy. Of these, 47% (n = 125/265) had macro-metastases, 38% (n = 101/265) had micro-metastases and 14.7% (n = 39/265) had "positive but inhibited" results. Positive non-sentinel lymph nodes (NSLN) were seen in 35% (44/125) of those with macro-metastases; 17.8% (18/101) of the patients with micro-metastases and 10.2% (4/39) of the "positive but inhibited" group. CONCLUSION: In our series over a third of our patients had positive lymph nodes detected with OSNA allowing them to proceed directly to axillary surgery at the same operation. This technique eliminates the need for a second operation in sentinel lymph node positive patients and avoids the anxiety waiting for histological results.