Cytokeratin Research Articles

Evaluating CK20 and MCPyV Antibody Clones in Diagnosing Merkel Cell Carcinoma

Merkel cell carcinoma (MCC) is diagnosed through histopathological and immunohistochemical examination of biopsies from skin or other organs. Its distinguishing features include perinuclear dot-like staining with Cytokeratin 20 (CK20) and detection of Merkel cell polyomavirus (MCPyV) using various methods. However, CK20 and MCPyV negative MCC cases have been reported at varying rates. In this single center cross-sectional study, we aimed to determine which clones are more effective in diagnosing MCC by comparing the performance of CK20 antibody clones Ks20.8 and SP33, as well as MCPyV antibody clones Ab3 and CM2B4. Fifty-four patients diagnosed with MCC were included. Among these, 42 cases were primary cutaneous, and 12 cases were nodal MCC. Fifty-two (96.3%) cases were positive with both CK20 clones, while two cases were negative. Clone SP33 stained areas of necrosis, whereas Ks20.8 showed no aberrant staining. MCPyV was detected in 44 cases (81.5%) using clone Ab3 and 39 cases (72.2%) using clone CM2B4. Staining with MCPyV clone Ab3 was diffuse and strong in most cases, while approximately 30% of CM2B4-positive cases exhibited low percentages and/or weak staining, complicating the evaluation. The two CK20-negative cases were also negative with both MCPyV clones. Our data demonstrated that CK20 clone Ks20.8 may be preferred for MCC diagnosis due to its consistent performance and lack of aberrant staining. Similarly, MCPyV clone Ab3 appears superior to CM2B4 for identifying MCPyV-positive cases.

Detection and Characterization of Circulating Tumor Cells in Colorectal Cancer Patients via Epithelial-Mesenchymal Transition Markers.

Background/Objectives: Liquid biopsy methods have gained prominence as minimally invasive tools to improve cancer treatment outcomes. Circulating tumor cells (CTCs) offer valuable insights into both primary and metastatic lesions. However, validating the CTC test results requires confirmation that the detected cells originate from cancer tissue. While studies have identified CTCs in colorectal cancer (CRC) patients using molecular markers, simultaneous validation of their cancer tissue origin remains unexplored. Methods: This study introduces a simple approach to detect adenomatous polyposis coli (APC) gene abnormalities alongside established CTC markers using a molecular imaging flow cytometer (MI-FCM). Given that APC gene abnormalities occur in 60-70% of CRC patients, their detection serves as strong evidence of cancer origin. Results: Our method achieved 92% concordance with DNA sequence analysis of tumor-derived cells. In a proof-of-concept study using 5 mL of whole blood from CRC patients, we observed a high frequency of cells exhibiting APC abnormalities, cytokeratin (CK), and vimentin (Vim) expression. Extending the study to 80 CRC patients across pathological stages I-IV confirmed CK and Vim as valid CTC markers. Three distinct cell populations were identified in blood: CK+/Vim-, CK+/Vim+, and CK-/Vim+. CTC number and frequency increased progressively with cancer stage. Conclusions: This is the first report demonstrating CK and Vim as effective markers for direct CTC detection in CRC patients. Our findings provide evidence-based validation of CTC markers, offering new insights and advancing approaches for patient care.

Predictive value of preoperative circulating tumor cells combined with hematological indexes for liver metastasis after radical resection of colorectal cancer.

Colorectal cancer is one of the most common malignant tumors in the world, and about 50% of its advanced patients will have liver metastasis. Preoperative assessment of the risk of liver metastasis in patients with colorectal cancer is of great significance for making individualized treatment plans. Traditional imaging examinations and tumor markers have some limitations in predicting the risk of liver metastasis. Therefore, it is of great clinical value to explore more sensitive and specific predictive indicators for improving early detection and treatment effect. In recent years, circulating tumor cells (CTCs), as a new biomarker, have attracted much attention because of their close relationship with tumor metastasis and prognosis. The purpose of this study is to collect and analyze the data of colorectal cancer patients treated in our hospital, so as to determine the predictive value of circulating tumor cells before operation and related hematological indexes for liver metastasis after radical resection of colorectal cancer, and to establish the corresponding prediction model to provide gastrointestinal surgeons with more accurate identification of high-risk patients and guidance for treatment. A total of 88 patients were included in this study, and 26 of whom developed liver metastasis after colorectal cancer surgery. The possible related factors are included in the single factor logistic regression, and the results are obtained after analysis. Body mass index, carcinoembryonic antigen (CEA), carbohydrate antigen 19-9, tumor marker CA72-4 (CA72-4), cytokeratin-7 (CK-7), CTC count, and neutrophil-to-lymphocyte ratio (P < .2) are risk factors for liver metastasis after radical resection of colorectal cancer. Furthermore, the data obtained were included in multivariate regression analysis, and CEA, CA72-4, CK-7, and CTC counts were independent risk factors for liver metastasis after radical resection of colorectal cancer (P < .05). This study confirmed that CEA, CA72-4, CK-7, and CTC counts are independent risk factors for liver metastasis after radical resection of colorectal cancer. In addition, the prediction model of this study can help gastrointestinal surgeons accurately identify patients who are prone to liver metastasis after colorectal cancer surgery.

Primary intraosseous squamous cell carcinoma with pagetoid spread arising in periapical odontogenic epithelium of the maxilla.

To date, pagetoid spread-the proliferation of pagetoid cells in intraepidermal lesions, as observed in secondary extramammary Paget's disease-has not been reported in squamous epithelium derived from the extension of head and neck carcinomas. Herein, we report a case of pagetoid squamous cell proliferation associated with a primary intraosseous carcinoma (PIOC) arising in the periapical lesion of the maxilla, a finding not reported previously. A 60-year-old man presented with prostate adenocarcinoma and bilateral pubic bone, ilium bone, and sacral bone metastases. Radiological examination revealed a cyst that enveloped the apices of the left maxillary first molar roots. Histopathological examination of the cyst specimen indicated squamous cell carcinoma; hence, PIOC was suspected and partial left maxillectomy was performed. Histopathology results showed distant epitheliotrophic spread of atypical clear cells regarding the tumor. The epitheliotrophic cells were positive for cytokeratin (CK)19 and CK7 as odontogenic markers. These phenotypes were similar to those of tumor cells, suggesting pagetoid squamous cell proliferation associated with a PIOC arising. Although diagnosis of this condition is challenging, early detection is vital to ensure prompt treatment and improve patient prognosis.

Cytokeratin 18 fragment in liver inflammation and fibrosis: Systematic review and meta-analysis.

This meta-analysis aimed to summarize the diagnostic accuracy and cut-off values of cytokeratin (CK) 18 measurements, specifically M30 and M65, as candidate biomarkers for the pathological evaluation of biopsy specimens used to stage liver inflammation and fibrosis in patients with chronic liver diseases. Databases were searched for studies collected up to January 11th, 2025. Pooled sensitivity, specificity, area under the receiver-operating characteristic curves, and mean cut-off values were calculated using random-effects models regardless of heterogeneity. A meta-regression analysis and subgroup analysis were performed to explore heterogeneity. Sixty-three studies comprising 9137 patients were included. The summarized AUROC curve of CK18 M30 for the diagnosis of significant liver inflammation, fibrosis ≥F1, ≥F2, ≥F3, and =F4 according to the METAVIR score system were 0.82, 0.75, 0.78, 0.78 and 0.76, with mean cut-off values of 264.3, 188.0, 276.9, 322.8 and 169.4 U/L. For M65, the summarized AUROC curve for detecting significant liver inflammation, fibrosis ≥F1, ≥F2, and =F4 were 0.79, 0.70, 0.76, 0.64 and 0.72, with mean cut-off values of 541.1, 417.6, 500.1, 424.6 and 674.0 U/L. The subgroup analyses implied that ethnicity may be the primary factor related to heterogeneity in CK18 M30 when applied to detect significant inflammation. Asian patients had values 79.7 U/L higher than those of non-Asian patients (p = 0.0157). CK18 M30 and M65 have clinically meaningful accuracy as alternative diagnostic tools for determining liver inflammation and fibrosis using biopsy specimens of patients with steatotic liver disease or viral hepatitis. PROSPERO registration number: CRD42022364598.

Establishment of a visualized mouse orthotopic xenograft model of nasopharyngeal carcinoma

ABSTRACT Mouse orthotopic xenograft tumor models are commonly employed in studies investigating the mechanisms underlying the development and progression of tumors and their preclinical treatment. However, the unavailability of mature and visualized orthotopic xenograft models of nasopharyngeal carcinoma limits the development of treatment strategies for this cancer. The aim of this study was to provide a simple and reliable method for building an orthotopic xenograft model of nasopharyngeal carcinoma. Human nasopharyngeal carcinoma (C666–1-luc) cells, stably expressing the firefly luciferase gene, were injected subcutaneously into the right axilla of BALB/C nude mice. Four weeks later, the resulting subcutaneous tumors were cut into small blocks and grafted into the nasopharynx of immunodeficient BALB/C nude mice to induce tumor formation. Tumor growth was monitored by bioluminescence imaging and small animal magnetic resonance imaging (MRI). The expression of histological and immunological antigens associated with orthotopic xenograft nasopharyngeal carcinoma was analyzed by tissue section analysis and immunohistochemistry (IHC). A visualized orthotopic xenograft nasopharyngeal carcinoma model was successfully developed in this study. Luminescence signal detection, micro-MRI, and hematoxylin and eosin staining revealed the successful growth of tumors in the nasopharynx of the nude mice. Moreover, IHC analysis detected cytokeratin (CK), CK5/6, P40, and P63 expression in the orthotopic tumors, consistent with the reported expression of these antigens in human nasopharyngeal tumors. This study established a reproducible, visual, and less lethal orthotopic xenograft model of nasopharyngeal carcinoma, providing a platform for preclinical research.

Serum biomarkers in the metabolic dysfunction-associated steatotic liver fibrosis diagnosis in children

Background. The COVID-19 epidemic and the war in Ukraine have led to a significant increase in the number of children suffering from metabolic dysfunction-associated steatotic liver disease (MASLD). One of the unresolved problems associated with MASLD is the identification of individuals at risk of rapid disease progression and development of irreversible liver changes. The search for alternative noninvasive markers suitable for the early detection of liver fibrosis in children remains extremely relevant. The aim of the study was to determine the diagnostic value of serum fibrosis markers and their relationship with sonographic and body composition parameters in children with MASLD. Materials and methods. The case-control study included 80 children aged 6 to 17 years (mean of (12.15 ± 2.51) years). The presence of steatosis and liver fibrosis was determined by transient elastography (Fibro­Scan® 502 touch F60156, Echosens, France). All subjects underwent anthropometric studies to determine body mass index. If it was within one-two Z-score, overweight was diagnosed. If the body mass index exceeded two Z-score, obesity was diagnosed. According to transient elastography and body mass index, all children were divided into four groups: group I — 27 children with MASLD and fibrosis ≥ F1, group II — 35 children with MASLD without fibrosis, group III — 18 obese or overweight children without MASLD and without fibrosis. The control group IV consisted of 14 children with normal weight without MASLD and without fibrosis. The groups had no significant differences in age and gender distribution. The study of body composition was performed by bioimpedance analysis using a TANITA MC-780MA analyzer (manufactured by Maeno-cho, Itabashi-ku, Tokyo, Japan). Quantitative determination of the serum concentration of vascular endothelial growth factor (VEGF) was performed by enzyme-linked immunosorbent assay (ELISA) using test systems from Wuhan Fine Biotech Co., Ltd (China) according to the manufacturer’s recommendations. The level of serum cytokeratin 18 (CK-18) was evaluated with IDL Biotech AB kits (Sweden) for ELISA. Serum content of transforming growth factor beta 1 (TGF-β1) was studied using an ELISA test system from IBL International (Germany). Fibrogenesis processes were evaluated by the serum content of free hydroxyproline (HPf), protein-bound hydroxyproline (HPp/b) and glycosaminoglycans (GAG). Results. The study revealed a significant increase in the level of CK-18 and TGF-β1 in children with MASLD-associated liver fibrosis. In children with liver fibrosis, an increase in the ratio of HPf/HPp/b and the level of GAG in the blood serum was observed compared to patients with MASLD without fibrosis and with overweight and obese children. The threshold value of CK-18 for liver fibrosis diagnosis was 90.3 U/l (sensitivity 81.3 %, specificity 76.9 %, AUC 0.843, p &lt; 0.001). The sensitivity of the threshold value of serum TGF-β1 (96.8 pg/mL) in children with MASLD was 80.0 %, specificity 65.7 %, AUC 0.787 (p &lt; 0.001). Threshold value of serum GAG (4.24 mmol/L) demonstrated a sensitivity of 70.6 % and a specificity of 69.6 %, AUC 0.743 (p &lt; 0.01). CK-18, TGF-β1, GAG shown a positive correlation with liver stiffness and elasticity, body composition of MASLD children and had high levels of diagnostic accuracy, which allows them to be used in children when screening for MASLD-associated liver fibrosis. Conclusions. Children with liver fibrosis are characterized by elevated serum levels of CK-18, VEGF, TGF-β1, HPp/b and GAG. The threshold values of CK-18 (more than 90.3 U/l), TGF-β1 (above 96.8 pg/mL) and GAG (more than 4.24 mmol/l) have high sensitivity and specificity, which allows them to be used for the diagnosis of liver fibrosis in children with MASLD.

Molecular Detection of Lymph Node Metastases with One-Step Nucleic Acid Amplification (OSNA) Pooling in Prostate Cancer: The POPCORN Study.

Pelvic lymph node dissection (PLND) is the most accurate procedure for lymph node (LN) staging in prostate cancer (PCa) patients. LN sectioning and hematoxylin and eosin (H&E) staining of at least one slice remains the gold standard for LN evaluation, potentially leading to misdetection of small metastatic focus. Entire LN analysis is possible with One-Step Nucleic Acid Amplification (OSNA) by detecting cytokeratin 19 (CK19) mRNA as a surrogate for LN invasion. This study aimed to compare postoperative performance of OSNA pooling with conventional H&E staining for pathological LN detection in PCa patients. POPCORN was an observational, prospective, and multicenter study of patients with PCa who underwent PLND. Dissected LNs were analyzed by both methods. This study included 2503 LNs from 131 patients, showing no statistically significant differences in pathological LN detection. Concordance between methods was high (93.9%), as were specificity (96.6%) and negative predictive value (96.6%) of OSNA pooling. The measure of agreement (Cohen's Kappa [κ]) was 0.70. Only eight (6.1%) discordances were observed, including four misdetections from each method. Results showed a high concordance between OSNA pooling and H&E staining, suggesting that OSNA pooling may be a good alternative to H&E staining to detect LN metastases in PCa patients.

A preoperative nomogram with MR elastography in identifying cytokeratin 19 status of hepatocellular carcinoma.

Developing a nomogram integrating MR elastography (MRE)-based tumor stiffness and contrast-enhanced MRI in identifying cytokeratin 19 (CK19) status of hepatocellular carcinoma (HCC) preoperatively. 120 CK19-negative HCC and 39 CK19-positive HCC patients undergoing curative resection were prospectively evaluated. All received MRE and contrast-enhanced MRI. Clinical and MRI tumor features were compared. Univariate and multivariate logistic regression analyses identified independent predictors for CK19 status. Receiver operating characteristic curve analysis evaluated diagnostic performance. A nomogram was established with calibration and decision curve analysis. Multivariate analysis revealed serum alpha fetoprotein (AFP) level (P < 0.001), targetoid appearance (P = 0.007), and tumor stiffness (P = 0.011) as independent significant variables for CK19-positive HCC. The area under the curve for tumor stiffness was 0.729 (95% CI 0.653, 0.796). Combining these features, a nomogram-based model achieved an area under the curve value of 0.844 (95% CI 0.778, 0.897), with sensitivity, specificity, and accuracy of 76.92%, 85.00%, and 83.02%, respectively. Calibration and decision curve analyses demonstrated good agreement and optimal net benefit. MRE-measured tumor stiffness aids in predicting CK19 status in HCC. The combined nomogram incorporating tumor stiffness, targetoid appearance, and AFP provides a reliable biomarker for CK19-positive HCC. MRE-measured tumor stiffness can be used to predict CK19 status in HCC. The nomogram, which integrates tumor stiffness, targetoid appearance, and AFP levels, has shown enhanced diagnostic performance. It offers a comprehensive preoperative tool for clinical decision-making, further advancing personalized treatment strategies in HCC management.

Characterization of Epithelial-Mesenchymal and Neuroendocrine Differentiation States in Pancreatic and Small Cell Ovarian Tumor Cells and Their Modulation by TGF-β1 and BMP-7.

Pancreatic ductal adenocarcinoma (PDAC) has an extremely poor prognosis, due in part to early invasion and metastasis, which in turn involves epithelial-mesenchymal transition (EMT) of the cancer cells. Prompted by the discovery that two PDAC cell lines of the quasi-mesenchymal subtype (PANC-1, MIA PaCa-2) exhibit neuroendocrine differentiation (NED), we asked whether NED is associated with EMT. Using real-time PCR and immunoblotting, we initially verified endogenous expressions of various NED markers, i.e., chromogranin A (CHGA), synaptophysin (SYP), somatostatin receptor 2 (SSTR2), and SSTR5 in PANC-1 and MIA PaCa-2 cells. By means of immunohistochemistry, the expressions of CHGA, SYP, SSTR2, and the EMT markers cytokeratin 7 (CK7) and vimentin could be allocated to the neoplastic ductal epithelial cells of pancreatic ducts in surgically resected tissues from patients with PDAC. In HPDE6c7 normal pancreatic duct epithelial cells and in epithelial subtype BxPC-3 PDAC cells, the expression of CHGA, SYP, and neuron-specific enolase 2 (NSE) was either undetectable or much lower than in PANC-1 and MIA PaCa-2 cells. Parental cultures of PANC-1 cells exhibit EM plasticity (EMP) and harbor clonal subpopulations with both M- and E-phenotypes. Of note, M-type clones were found to display more pronounced NED than E-type clones. Inducing EMT in parental cultures of PANC-1 cells by treatment with transforming growth factor-β1 (TGF-β1) repressed epithelial genes and co-induced mesenchymal and NED genes, except for SSTR5. Surprisingly, treatment with bone morphogenetic protein (BMP)-7 differentially affected gene expressions in PANC-1, MIA PaCa-2, BxPC-3, and HPDE cells. It synergized with TGF-β1 in the induction of vimentin, SNAIL, SSTR2, and NSE but antagonized it in the regulation of CHGA and SSTR5. Phospho-immunoblotting in M- and E-type PANC-1 clones revealed that both TGF-β1 and, surprisingly, also BMP-7 activated SMAD2 and SMAD3 and that in M- but not E-type clones BMP-7 was able to dramatically enhance the activation of SMAD3. From these data, we conclude that in EMT of PDAC cells mesenchymal and NED markers are co-regulated, and that mesenchymal-epithelial transition (MET) is associated with a loss of both the mesenchymal and NED phenotypes. Analyzing NED in another tumor type, small cell carcinoma of the ovary hypercalcemic type (SCCOHT), revealed that two model cell lines of this disease (SCCOHT-1, BIN-67) do express CDH1, SNAI1, VIM, CHGA, SYP, ENO2, and SSTR2, but that in contrast to BMP-7, none of these genes was transcriptionally regulated by TGF-β1. Likewise, in BIN-67 cells, BMP-7 was able to reduce proliferation, while in SCCOHT-1 cells this occurred only upon combined treatment with TGF-β and BMP-7. We conclude that in PDAC-derived tumor cells, NED is closely linked to EMT and TGF-β signaling, which may have implications for the therapeutic use of TGF-β inhibitors in PDAC management.

Walthard Cell Nests/Transitional Cell Metaplasia in Distal Fallopian Tubes and Pelvic Peritoneum Derived From Reserve Cells.

Transitional cell metaplasia (TCM) resembling benign urothelium is commonly seen around the distal fallopian tube and/or neighboring mesothelial surface; however, its histogenesis remains largely unknown. We observed the emergence of a cytokeratin (CK) 17-positive reserve cell layer in early TCM foci beneath the tubal epithelium, leading us to hypothesize that TCM could be derived from reserve cells. To elucidate the histogenetic process of TCM, we analyzed the histomorphologic features and immunoprofiles for CK17, CK5/6, p63, GATA-3, estrogen receptor (ER), and androgen receptor (AR) in TCM foci arising in the tubal epithelium (31 foci) and pelvic mesothelium (35 foci). Overall, the histologic features and immunoprofiles of TCM in the tubal epithelium and pelvic mesothelium were similar, but distinct differences appeared during TCM development. A single-layered CK17-expressing reserve cells became apparent beneath the tubal epithelium, and the CK17 expression disappeared as these cells multiplied. In contrast, a short segment of normal mesothelium next to the tubo-peritoneal junction expressed CK17 even before the emergence of a single-layered reserve cells beneath the mesothelium, suggesting a potential reserve/stem cell function within the mesothelium itself. Then, the single-layered cells in both areas multiplied and differentiated to display urothelial characteristics, including nuclear grooves and clear cytoplasm. Strong CK5/6, p63, and GATA-3 expression appeared in the single-layered reserve cell stage and was maintained thereafter to the fully differentiated TCM. AR was expressed in both normal tubal epithelium and pelvic mesothelium, and the intensity of AR and ER were reciprocal during the entire histogenetic process of TCM in most reserve cell-derived populations (98.5%), AR expression being significantly stronger than ER. The histogenesis of TCM was initiated from the emergence of reserve cells beneath the tubal epithelium and pelvic mesothelium, which then multiplied and differentiated into urothelium. AR might have an important role during the histogenesis of TCM.

Early Alterations of Cytoskeletal Proteins Induced by Radiation Therapy in the Parenchymal Cells of Rat Major Salivary Glands: A Comparative Immunohistochemical Analysis.

Head and neck malignancies (HNMs) encompass a variety of cancers that affect the oral and para-oral tissues, the most common of which are squamous cell carcinomas. Radiotherapy is commonly used to treat these cancers, often involving radiation exposure to the salivary glands. This study aims to investigate the early impacts of radiotherapy on the internal microstructure of the salivary gland cells and identify which gland exhibits the highest level of radiosensitivity. Twelve male albino rats were divided into two groups (control group and experimental group subjected to radiotherapy). The experimental group underwent adaily dose of 5 Grays of radiotherapy for six days with a total dose of 30 Grays, targeting the salivary glands. One month later, the salivary gland complex was dissected and processed for histological analysis and cytokeratin 17 (CK17) immunostaining. Histological examination of the irradiated salivary glands revealed atrophic changes in the gland parenchyma, accompanied by the proliferation of dense fibrous stroma. The parenchymal components consisted of small serous acini with poorly defined lumens, many of which had been replaced by fatty tissue with the formation of duct-like structures. Immunohistochemical findings of control glands exhibited weak to mild CK17 expression in duct cells, with the staining pattern typically diffuse or localized to the basal region of the cell. Some serous acini and serous demilunes within mixed acini exhibited diffuse, weak to mild CK17 expression, whereas the mucous acinar cells showed no expression. Radiated major glands revealed moderate to strong CK17 expression in the duct and serous acinar cells (p<0.05). Two distinct expression patterns were identified: the first exhibited diffuse expression across the cells, while the second showed intense expression at the apical region with mild expression at the basal part. The positive immunostaining in control salivary glands suggests that small amounts of intermediate filaments are essential for saliva secretion. Radiotherapy significantly disrupts the cytokeratin arrangement and density in major salivary glands, impairing saliva production. The most prominent changes occurred in the submandibular gland, followed by the parotid gland, with the sublingual gland showing the least impact. However, CK17 intensity in acinar cells was highest in the parotid, then submandibular, and lowest in the sublingual gland. Further research is needed to assess the short- and long-term effects and potential for recovery over time.

Syringocystadenocarcinoma of the perianal region: a case report

IntroductionSyringocystadenocarcinoma papilliferum is an extremely rare malignant adnexal tumor that typically arises from a papilliferous syringocystadenoma (World Health Organization classification of skin tumors, 2018.). This tumor predominantly occurs in the cephalic region.Case presentationWe present the case of a 68-year-old Moroccan male with no significant medical history who presented with a slowly progressing skin mass in the perianal region. Despite the chronic nature of the swelling, the patient remained in overall good health. Physical examination revealed a firm 2.3-cm mass in the perianal area, with no other remarkable findings. The patient underwent surgical excision of the mass. Macroscopic examination showed a solid-cystic, rounded mass. Microscopic examination revealed a malignant adnexal tumor with apocrine differentiation and papillary architecture. Immunohistochemistry was positive for p63, cytokeratin 7 (CK7), and smooth muscle actin, with a Ki67 labeling index in 60% of tumor cells, and negative for p16. The diagnosis of syringocystadenocarcinoma papilliferum was confirmed. A further surgical resection was performed, and the patient’s postoperative course was unremarkable.ConclusionSyringocystadenocarcinoma papilliferum is an extremely rare tumor, with its occurrence in the perianal region being particularly uncommon. This case contributes to the limited literature on this malignancy, highlighting its clinical and pathological features.

Expression of CK17 and SOX2 in Vulvar Intraepithelial Neoplasia: A Comprehensive Analysis of 150 Vulvar Lesions.

Recently, the immunohistochemical markers cytokeratin 17 (CK17) and SRY-box2 (SOX2) have been evaluated as adjuncts for the diagnosis of high-grade vulvar intraepithelial neoplasia (VIN). In the present study, the aim was to assess CK17 and SOX2 expression in VIN by studying 150 vulvar lesions, originally reported as high-grade VIN and to assess the diagnostic accuracy. All slides (H&E, p16INK4a, p53, Ki-67, CK17, and SOX2 stains) were independently assessed by six pathologists and the final diagnosis was reached in consensus meetings, as follows: 46 human papillomavirus (HPV)-independent VIN (including 30 p53 mutant and 16 p53 wild-type lesions), 58 high-grade squamous intraepithelial lesions (HSILs), 4 low-grade SILs (LSILs), 37 non-dysplastic lesions, and 5 lesions where the histology was inconclusive. CK17 positivity was observed in 100% p53 wild-type HPV-independent VIN, compared to 73% p53 mutant HPV-independent VIN, 14% HSILs, 0% LSILs, and 24% non-dysplastic lesions. SOX2 positivity was observed in 13% p53 wild-type HPV-independent VIN, 43% p53 mutant HPV-independent VIN, 2% HSILs, 0% LSILs, and 3% non-dysplastic lesions. The highest diagnostic accuracy (89%) for HPV-independent VIN was obtained when combining p53 and CK17 immunohistochemistry. The addition of SOX2 did not further increase diagnostic accuracy. To conclude, aside from p53, both CK17 and SOX2 can be of value for reaching an accurate diagnosis of HPV-independent VIN.

Anorectal Remodeling in the Transitional Zone with Increased Expression of LGR5, SOX9, SOX2, and Keratin 13 and 5 in a Dextran Sodium Sulfate-Induced Mouse Model of Ulcerative Colitis.

Although hyperplasia of the anorectal transitional zone (TZ) has been reported in mouse models of ulcerative colitis, the mechanisms underlying this phenomenon are not fully understood. We characterized keratin subtypes and examined the expression of stem cell markers in the TZ. Dextran sodium sulfate-treated mice showed abnormal repair of the anorectal region, which consisted of mixed hyperplastic TZ and regenerating crypts. Liquid chromatography-tandem mass spectrometry from the paraffin-embedded TZ in the treated mice revealed that the major keratins were type I cytokeratin (CK)13 and type II CK5, but notable expression of type I CK10 and CK42 and type II CK1, CK4, CK6a, CK8, and CK15 was also detected. Hyperplastic TZ was characterized by the expression of tumor protein 63, sex-determining region Y-box 2 (SOX2), SOX9, and leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5). Lgr5 was highly expressed in the TZ in the early stages of colitis, followed by higher expression levels of SOX2. The TZ-derived organoids expressed LGR5, SOX2, and SOX9. The present study suggests that transitional zones showing abnormal keratin assembly and stem cell activation may interfere with rectal crypt regeneration, leading to pathological anorectal remodeling in severe colitis.

Gastroesophageal varices in primary biliary cholangitis with anti-centromere antibody positivity: Early onset?

Primary biliary cholangitis (PBC) is an autoimmune liver disease. During the diagnostic process, the patient's autoimmune antibodies are routinely examined. Approximately 20% of PBC patients have positive anti-centromere antibody (ACA). We evaluated the clinical characteristics of ACA-positive and ACA-negative PBC patients to explain the differences in disease progression between these two groups. Retrospective data from 961 PBC patients at Beijing Youan Hospital from 2010 to 2019 were gathered and separated into two groups based on ACA positivity. We collected and evaluated clinical laboratory indices, gastroscopy findings, and liver function assessments. In addition, 60 liver biopsies were available for comparison between the 2 groups. Pathologists staged the histological findings using the Ludwig staging criteria and Nakanuma staging and grading. Immunohistochemical staining was also performed on liver biopsies to examine the expression of cytokeratin 7 (CK7) in the tissue. A synthesis of clinical indicators in the large cohort showed that alanine transaminase, aspartate aminotransferase, total bilirubin, IgG, white blood cell, and platelet were significantly lower in the ACA-positive group, indicating that the overall status of liver injury was more moderate in the ACA-positive group. Additionally, ACA-positive patients in the non-cirrhotic group were more likely to present with gastroesophageal varices related to portal hypertension. Finally, analysis of pathologic findings showed that parameters were mostly comparable in the two groups, but CK7 differed and was more significantly lower in the ACA-positive group in albumin-bilirubin grade 2 and 3 patients. In summary, we characterized and compared the clinical features of ACA-positive and ACA-negative PBC patients, corroborating previous studies on the relationship between ACA positivity and portal hypertension cross-sectionally. It suggested that gastroesophageal varices might happen in the earlier course of PBC natural progression in the ACA-positive group.

Glypican-3 and Cytokeratin-19 Expression in Pancreatic Cancer in a Canadian Population.

Background/Objectives: One study of pancreatic ductal adenocarcinoma has found expression of glypican-3 (GPC3) and cytokeratin-19 (CK19) determined by immunohistochemistry to be associated with higher stage and grade disease, with a more adverse prognosis. The reported 44% rate of GPC3 expression in pancreatic cancer raises the important possibility that targeted immunotherapies currently in development for hepatocellular carcinoma may also prove useful for GPC3-expressing pancreatic cancers. The present study aims to determine if a similar expression pattern of these markers and stage/grade/prognostic associations is present in our Canadian patient population. Methods: Patients with a pancreatic surgical resection for adenocarcinoma or neuroendocrine tumor (NET) were identified from pathology records over a 5-year period. Immunohistochemistry for GPC3 and CK19 was performed on archived tumor tissue and the proportion of positive cells and intensity of staining were recorded. Grade, stage, and overall survival were compared in patients with NETs that were CK19-positive versus -negative. Results: All 72 pancreatic adenocarcinomas and 20 NETs tested were negative for GPC3, apart from a single case of pancreatic adenocarcinoma. All 72 adenocarcinomas were positive for CK19 expression. Half of the NETs were positive for CK19. There was no correlation between CK19 expression in NETs and tumor grade, lymph node metastasis, distant metastasis, or overall survival. Conclusions: We are skeptical of the reported prognostic value of GPC3 and CK19 in pancreatic adenocarcinomas. CK19 as a prognostic marker in NETs has potential for further study. The results with our protocol for GPC3 immunohistochemistry suggest that pancreatic cancer may be a less promising target for GPC3-targeted immunotherapies than previously thought.

Abstract A001: Analytical validation of a novel multiomic metastatic breast cancer liquid biopsy test that combines ctDNA analysis with CTC HER2-ultralow and ER co-expression, as well as single-cell chromosomal instability

Abstract Background: Metastatic breast cancer (MBC) subtype characterization is critical for effective treatment. The DefineMBC™ multiomic blood biopsy clinical diagnostic combines ctDNA and circulating tumor cell (CTC) analyses to characterize MBC patients when a tissue biopsy is not feasible. Innovation in the DefineMBC 2.0 5-channel immunofluorescent (IF) CTC characterization assay now measures ultralow (UL) levels of HER2 protein expression, ER protein expression, and chromosomal instability (CI) as well as ERBB2 amplification via single- cell sequencing. Cell-level HER2/ER co-detection enables monitoring of receptor conversion during a patient’s MBC treatment journey. As trastuzumab-deruxtecan (T-DxD) has become standard of care for HER2-low disease, distinguishing HER2-UL from HER2[-] by DefineMBC’s liquid biopsy approach may identify additional patients who can benefit from such therapy. This addresses an unmet need given the known shortcomings of tissue biopsy IHC in the metastatic setting as recently noted by communications from NCCN, ASCO, and the College of American Pathologists. Methods: Assay development including analytical validation studies utilized biologically relevant cell lines of epithelial origin and known expression levels of HER2 and ER (MDA-MB-453: HER2[+] &amp; ER[-]; MCF-7: HER2-low &amp; ER[+]; and MCF-7/ERBB2 knockout: HER2[-]) that were spiked into healthy donor blood. Additionally, patients with various forms of MBC were characterized with the new assay. As with all samples, following red blood cell lysis, nucleated cells were deposited on glass slides at high density (∼3E6 WBC/slide). Slides underwent IF staining for cytokeratins (CK), CD31, CD45, HER2, ER, and Hoechst to localize nuclear DNA, followed by scanning and machine learning-based rare cell detection. CK[+], CD31[-]/CD45[-] cells were classified as CTC candidates and assessed for HER2/ER expression. Pathologist-selected CTCs were isolated for single-cell whole genome sequencing and analyzed using a proprietary CTC DNA copy number pipeline to detect CI and amplification at the ERBB2 locus. Results: Analytical validation studies demonstrated the limit of detection at 1 CTC per 12E6 WBC (∼1.6 mL of blood) with a linear range of 1- 300 CTCs per slide. Sensitivity, specificity, accuracy, and precision metrics of the HER2 and ER IF assays were 96%, 96%, 96%, 2% CV; 91%, 95%, 93%, 3% CV; respectively. CTC enumeration precision was validated at greater than 80% for enumeration bins of 0, 1-10, 11-50, and&amp;gt;50 CTCs per sample. During patient testing 7 patients previously called HER2[-] are now designated as consistent with HER2-low by the pathologist, suggesting improved test performance in future clinical validation studies is likely. Conclusions: Advancement of our comprehensive cancer profiling platform now includes ER/HER2-UL/CI co-detection on enumerated CTCs in MBC patients. Clinical studies currently in progress will reveal whether HER2-UL/HER2[+] detection on CTCs will identify patients better suited for T-DxD or trastuzumab, respectively. Citation Format: Raj Srikrishnan, Jordan Ritchie, Alessandra Cunsolo, Andrew Kunihiro, Brandon Guillory, Sara Tin, Zhuo Meng, Ernest T Lam, Bharat Annaldas, Evan Schwab, Nilesh Dharajiya, Timothy Pluard, Martin Blankfard, David Bourdon. Analytical validation of a novel multiomic metastatic breast cancer liquid biopsy test that combines ctDNA analysis with CTC HER2-ultralow and ER co-expression, as well as single-cell chromosomal instability [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A001.

Linking Antibodies Against Apolipoprotein A-1 to Metabolic Dysfunction-Associated Steatohepatitis in Mice.

Metabolic dysfunction-associated fatty liver disease (MASLD) is a common liver and health issue associated with heightened cardiovascular disease (CVD) risk, with Cytokeratin 18 (CK-18) as a marker of liver injury across the MASLD to cirrhosis spectrum. Autoantibodies against apolipoprotein A-1 (AAA-1s) predict increased CVD risk, promoting atherosclerosis and liver steatosis in apoE-/- mice, though their impact on liver inflammation and fibrosis remains unclear. This study examined AAA-1s' impact on low-grade inflammation, liver steatosis, and fibrosis using a MASLD mouse model exposed to AAA-1s passive immunization (PI). Ten-week-old male C57BL/6J mice under a high-fat diet underwent PI with AAA-1s or control antibodies for ten days. Compared to controls, AAA-1-immunized mice showed higher plasma CK-18 (5.3 vs. 2.1 pg/mL, p = 0.031), IL-6 (13 vs. 6.9 pg/mL, p = 0.035), IL-10 (27.3 vs. 9.8 pg/mL, p = 0.007), TNF-α (32.1 vs. 24.2 pg/mL, p = 0.032), and liver steatosis (93.4% vs. 73.8%, p = 0.007). Transcriptomic analyses revealed hepatic upregulation of pro-fibrotic mRNAs in AAA-1-recipient mice, though histological changes were absent. In conclusion, short-term AAA-1 PI exacerbated liver steatosis, inflammation, and pro-fibrotic gene expression, suggesting that AAA-1s may play a role in MASLD progression. Further research with prolonged AAA-1 exposure is warranted to clarify their potential role in liver fibrosis and associated complications.

Biological and clinical characteristics of non-small cell lung cancer non-specific subtype

BackgroundNon-small cell lung cancer-not otherwise specified (NSCLC-NOS) is a rare subtype of NSCLC that cannot be classified specifically based on morphology and/or special staining. This study aimed to explore the clinical features, biological and pathological characteristics, treatment, and prognosis of NSCLC-NOS. MethodsThis retrospective study included NSCLC-NOS patients diagnosed and treated between 2010 and 2022. Clinical features, gene expression, first-line treatment, and prognosis were analyzed. Kaplan-Meier methods were calculated and log-rank tests and univariable and multivariable Cox regression analyses were performed to determine the relationship between prognostic factors and survival. ResultsOf 105 NSCLC-NOS patients, most were male (92.4 %), smokers (78.1 %), with a median age of 64 years, and advanced stage (IIIc-IV, 72.4 %). Immunohistochemical analysis showed minimal expression of p40, NapsinA, and TTF-1, whereas cytokeratin (CK) was expressed in 100 % of cases. 20.5 % of 39 patients who underwent genetic testing had driver gene mutations, including EGFR, KRAS, and ROS1. Among 69 patients with complete treatment information, 58 received platinum-based chemotherapy, with paclitaxel being the most commonly used combination chemotherapy drug (n = 25), followed by pemetrexed (n = 21). The objective response rate (ORR) of paclitaxel was found to be higher compared to pemetrexed (83.3 % vs. 54.5 %, P = 0.296). Furthermore, the combination of paclitaxel with immunotherapy demonstrated superior benefits in comparison to pemetrexed (76.9 % vs. 50.0 %, P = 0.367). The median progression-free survival (PFS) for patients treated with monotherapy paclitaxel, the paclitaxel-immunotherapy combination, and the pemetrexed-immunotherapy combination were 6.6 months (95 % CI: 1.508–11.692; P = 0.017), 15.7 months (95 % CI: 14.071–17.329; P = 0.017), and 11.8 months (95 % CI: 10.279–13.321; P = 0.324), respectively. The median overall survival (OS) was 13.6 months. Anatomic location (P = 0.026) and immunotherapy use (P = 0.003) were associated with OS. Multivariate analysis confirmed that anatomical location and immunotherapy use were factors influencing the prognosis. ConclusionNSCLC-NOS is common in male smokers and often diagnosed at an advanced stage with low mutation rate. Paclitaxel with immunotherapy may have better benefits as a first-line treatment. Anatomic location and immunotherapy use are prognostic factors.