Serum CYFRA 21-1 and CK19-2G2 as Predictive Biomarkers of Response to Transarterial Chemoembolization in Hepatitis C-related Hepatocellular Carcinoma Among Egyptians: A Prospective Study.

Background and aim: Cytokeratin 19 (CK19)-positive HCC is a subtype of hepatocellular carcinoma (HCC) with poor biological behavior and resistance to different treatments including transarterial chemoembolization (TACE). The current study aimed to investigate the predictive value of serum CK 19 fragment 21-1 (CYFRA 21-1) and serum CK 19 fragment 2G2 (CK 19-2G2) for TACE response in patients with hepatitis C virus (HCV)-related HCC. Methods: This prospective study assessed the pretreatment serum CYFRA 21-1 and CK 19-2G2 levels in 64 patients with HCV-related naïve HCC who underwent TACE to predict 1-year overall survival (OS), progression-free survival (PFS), and objective response rate (ORR). Additionally, 40 healthy individuals were included as controls. Pretreatment alpha-fetoprotein (AFP) was also measured for comparison. Results: After exclusions, 60 patients completed TACE sessions, and the 1-year OS was 52%, and ORR post TACE was 71.8%. HCC patients with elevated levels of CYFRA 21-1, CK 19-2G2, or baseline AFP measuring ≥400 ng/ml have decreased 1-year OS and PFS after TACE. Serum CK19-2G2 was an independent predictor of 1-year OS using multivariate hazard regression analysis. Pretreatment normal serum CYFRA 21-1 levels (P = 0.047), serum AFP measuring <400 ng/ml (P = 0.016), and lower AST (P = 0.002) were independent predictors of ORR to TACE using multivariate logistic regression analysis. The predictive ability of pretreatment elevated serum CYFRA 21-1, AFP measuring ≥400 ng/ml, AFP + CYFRA 21-1, AFP + CK 19-2G2, or AFP + CYFRA 21-1+ CK19-2G2 to predict nonresponse (progressive disease) to TACE (area under the curve = 0.795, 0.690, 0.830, 0.725, and 0.850, respectively). Conclusions: This study demonstrated that incorporating the measurement of serum CYFRA 21-1 or CK19-2G2 levels, along with AFP, during the initial diagnosis can aid in predicting poor 1-year OS, PFS, and ORR to TACE in patients with HCV-related HCC.

Estudio de la citoqueratina- 18 como marcador no invasivo en pacientes con enfermedad de hígado graso no alcohólico

Introducción: La enfermedad por hígado graso no alcohólico es una de las principales causas de afección hepática. La citoqueratina 18 surge como marcador no invasivo para la valoración de fibrosis hepática. El objetivo del trabajo fue validar el uso de la citoqueratina 18 en sangre periférica en el diagnóstico y evolución de los pacientes con enfermedad por hígado graso no alcohólico. Metodología: Para validar la citoqueratina 18 en el diagnóstico se realizó un estudio de tipo caso-control. El grupo caso fueron los pacientes mayores de 18 años, de ambos sexos, con diagnóstico de enfermedad por hígado graso no alcohólico vinculado al síndrome metabólico, captados entre 2/2/2019 al 2/2/2020. El grupo control fueron personas donantes de sangre. Se parearon 1-1 por edad y sexo. Se cuantificó la citoqueratina 18 en sangre periférica de ambos grupos. Para validar la citoqueratina 18 en la evolución de los pacientes con enfermedad de hígado graso no alcohólico se realizó un trabajo prospectivo, longitudinal. El grupo de pacientes captados fueron seguidos durante un año bajo tratamiento estándar, finalizando el mismo se realizó la cuantificación de citoqueratina 18 en sangre periférica. Las variables continuas se expresan con la media y desvío estándar. Se analizó con test de t Student, error α < 5% Resultados: 13 pacientes integran el grupo caso (12 mujeres), de 53 ± 11 años, con IMC 35.01 ± 8.9 kg/m2. El valor de citoqueratina 18 pre-tratamiento fue de 1410 ± 120 UI, y el valor post-tratamiento fue de 117 ± 56, p < 0,005.El grupo control fueron 13 personas (12 mujeres), de 43,4 ± 8,1 años e IMC 28,10 ± 5,4 kg/m2 El valor de citoqueratina 18 fue de 193 ± 7.2 UI, p < 0.005 vs grupo caso pretratamiento. Conclusiones: La citoqueratina 18 es más elevada en los pacientes con enfermedad hígado graso no alcohólico, siendo estadísticamente significativa y disminuye con el tratamiento con significación estadística, pudiendo constituirse en un marcador útil en este grupo de pacientes.

Introduction: Nonalcoholic fatty liver disease is one of the main causes of liver disease. Cytokeratin 18 emerges as a non-invasive marker for the assessment of liver fibrosis. The objective of the work was to validate the use of cytokeratin 18 in peripheral blood in the diagnosis and evolution of patients with non-alcoholic fatty liver disease. Methodology: To validate cytokeratin 18 in the diagnosis, a case-control study was carried out. The case group was patients over 18 years of age, of both sexes, with a diagnosis of non-alcoholic fatty liver disease linked to metabolic syndrome, recruited between 2/2/2019 to 2/2/2020. The control group were blood donors. They were matched 1-1 for age and sex. Cytokeratin 18 was quantified in peripheral blood of both groups. To validate cytokeratin 18 in the evolution of patients with non-alcoholic fatty liver disease, a prospective, longitudinal study was carried out. The group of patients recruited were followed for one year under standard treatment, at the end of which cytokeratin 18 was quantified in peripheral blood. Continuous variables are expressed with the mean and standard deviation. It was analyzed with Student's t test, α error < 5%. Results: 13 patients make up the case group (12 women), 53 ± 11 years old, with BMI 35.01 ± 8.9 kg/m2. The pre-treatment cytokeratin 18 value was 1410 ± 120 IU, and the post-treatment value was 117 ± 56, p < 0.005. The control group was 13 people (12 women), 43.4 ± 8.1 years and BMI 28.10 ± 5.4 kg/m2 The cytokeratin 18 value was 193 ± 7.2 IU, p < 0.005 vs. pretreatment case group. Conclusions: Cytokeratin 18 is higher in patients with non-alcoholic fatty liver disease, being statistically significant, and decreases with treatment with statistical significance, and may become a useful marker in this group of patients.

Introdução: A doença hepática gordurosa não alcoólica é uma das principais causas de doença hepática. A citoqueratina 18 surge como um marcador não invasivo para avaliação de fibrose hepática. O objetivo do trabalho foi validar o uso da citoqueratina 18 no sangue periférico no diagnóstico e evolução de pacientes com doença hepática gordurosa não alcoólica. Metodologia: Para validar a citoqueratina 18 no diagnóstico, foi realizado um estudo caso-controle. O grupo caso foi composto por pacientes maiores de 18 anos, de ambos os sexos, com diagnóstico de doença hepática gordurosa não alcoólica ligada à síndrome metabólica, recrutados entre 02/02/2019 a 02/02/2020. O grupo controle eram doadores de sangue. Eles foram comparados em 1 a 1 por idade e sexo. A citoqueratina 18 foi quantificada no sangue periférico de ambos os grupos. Para validar a citoqueratina 18 na evolução de pacientes com doença hepática gordurosa não alcoólica, foi realizado um estudo prospectivo e longitudinal. O grupo de pacientes recrutados foi acompanhado durante um ano sob tratamento padrão, ao final do qual a citoqueratina 18 foi quantificada no sangue periférico. As variáveis ​​contínuas são expressas com média e desvio padrão. Foi analisado com teste t de Student, erro α < 5%. Resultados: Compõem o grupo caso 13 pacientes (12 mulheres), 53 ± 11 anos, com IMC 35,01 ± 8,9 kg/m2. O valor de citoqueratina 18 pré-tratamento foi de 1410 ± 120 UI e o valor pós-tratamento foi de 117 ± 56, p < 0,005. O grupo controle foi de 13 pessoas (12 mulheres), 43,4 ± 8,1 anos e IMC 28,10 ± 5,4 kg/m2 O valor da citoqueratina 18 foi de 193 ± 7,2 UI, p < 0,005 vs. grupo de casos pré-tratamento. Conclusões: A citoqueratina 18 é maior em pacientes com doença hepática gordurosa não alcoólica, sendo estatisticamente significativa, e diminui com o tratamento com significância estatística, podendo se tornar um marcador útil neste grupo de pacientes.

Immunophenotypic and molecular changes during progression of papillary urothelial carcinoma.

PURPOSE: Urothelial carcinoma has various molecular subtypes, each with different tumor characteristics. Although it is known that molecular changes occur during tumor progression, little is known about the specifics of these changes. In this study, we performed transcriptional analysis to understand the molecular changes during tumor progression. MATERIALS AND METHODS: Formalin-fixed, paraffin-embedded tumor tissues were obtained from 12 patients with muscle-invasive bladder cancer (MIBC). The invasive and non-invasive papillary areas were identified in papillary urothelial carcinoma specimens. Immunohistochemistry (IHC) and mRNA sequencing were performed for each tumor area. RESULTS: Patients with CK5/6-negative and CK20-positive non-invasive papillary areas were selected and classified into the IHC switch subgroup (CK5/6-positive and CK20-negative in the invasive area) and the IHC unchanged subgroup (CK5/6-negative and CK20-positive in the invasive area) according to the IHC results of the invasive area. We identified differences in the mRNA expression between the non-invasive papillary and invasive areas of the papillary MIBC tissue samples. In both the non-invasive papillary and invasive areas, the IHC switch subgroup showed basal subtype gene expression, while the IHC unchanged subgroup demonstrated luminal subtype gene expression. CONCLUSIONS: The non-invasive papillary area showed a gene expression pattern similar to that of the invasive area. Therefore, even if the non-invasive papillary area exhibits a luminal phenotype on IHC, it can have a basal subtype gene expression depending on the invasive area.

Expression and diagnostic value of serum free light chain in lung cancer. / è¡€æ¸ æ¸¸ç¦»è½»é“¾åœ¨è‚ºç™Œä¸­çš„è¡¨è¾¾åŠè¯Šæ–­ä»·å€¼.

OBJECTIVES: The expression of serum free light chain (FLC) is abnormal in various diseases, but its role in lung cancer remains unclear. This study aims to investigate the expression and diagnostic value of serum FLC in lung cancer. METHODS: A total of 80 lung cancer patients treated at Xiangdong Hospital, Hunan Normal University from January to December 2021 were selected as the lung cancer group. Another 80 healthy individuals undergoing routine physical examinations during the same period were chosen as the control group. General information and serum κFLC and λFLC levels were collected for all subjects. Clinical indicators such as serum carcinoembryonic antigen (CEA), cytokeratin fragment antigen 21-1 (CYFRA21-1) levels, tumor diameter, histological type, TNM stage, and lymph node metastasis status were recorded for lung cancer patients. The expression levels of serum FLC [κFLC, λFLC, and FLC (κ+λ)] were compared between the lung cancer group and the control group. Lung cancer patients were grouped based on gender, age, smoking history, tumor diameter, TNM stage, histological type, and lymph node metastasis to compare differences in serum κFLC and λFLC levels. Receiver operating characteristic (ROC) curves were used to evaluate the diagnostic value of serum FLC alone and in combination with other indicators in lung cancer. RESULTS: The expression levels of serum FLC (κ+λ) and κFLC were significantly higher in the lung cancer group than those in the control group (both P<0.001), while there was no significant difference in serum λFLC levels between the 2 groups (P>0.05). There were no significant differences in serum κFLC levels among lung cancer patients with different tumor diameters, histological types, or TNM stages (all P>0.05); however, serum κFLC levels were higher in lung cancer patients with lymph node metastasis than in those without, with statistical significance (P=0.033). There were no significant differences in serum λFLC levels based on tumor diameter or histological type (both P>0.05), but serum λFLC levels were higher in stage III+IV and lymph node metastatic lung cancer patients compared to stage I+II and non-metastatic patients, with statistical significance (P=0.033 and P=0.019, respectively). The area under the curve (AUC) for κFLC and CEA in diagnosing lung cancer showed no significant difference (P=0.333). The combination of κFLC+CYFRA21-1 had the highest diagnostic efficacy (AUC=0.875) and sensitivity (71.3%). The AUC for the combined diagnosis of κFLC+λFLC+CEA+CYFRA21-1 was 0.915 (95% CI 0.860 to 0.953, P<0.001). CONCLUSIONS: Serum FLC is highly expressed in lung cancer and is associated with its invasion and metastasis. Serum FLC, particularly κFLC, has diagnostic value for lung cancer, and the combined detection of FLC, CEA, and CYFRA21-1 offers the best diagnostic efficacy.

CYFRA 21-1 predicts efficacy of combined chemoimmunotherapy in patients with advanced non-small cell lung cancer: a prospective observational study.

Background: Tumor markers such as serum carcinoembryonic antigen (CEA) and cytokeratin fragment 19 (CYFRA 21-1) are utilized for assessing the effectiveness of chemotherapy in non-small cell lung cancer (NSCLC) patients. Yet, it remains uncertain whether these markers can reliably forecast responses to combined chemoimmunotherapy. Our study aimed to examine the significance and effectiveness of these markers in predicting responses among NSCLC patients undergoing combined chemoimmunotherapy. Methods: This two-part observational study involved patients with NSCLC who were treated with combined chemoimmunotherapy in Japanese hospitals. An initial retrospective study of these patients, with serum CEA and CYFRA 21-1 as prognostic factors for combined chemoimmunotherapy outcomes, served as a discovery cohort. Patients in a subsequent prospective study served as a validation cohort, where we assessed the prognostic accuracy of CEA and CYFRA 21-1 cut-off points determined by the discovery cohort. Results: In total, 121 patients treated with combined chemoimmunotherapy were included, with 44 and 77 patients in the discovery and validation cohorts, respectively. Serum CYFRA 21-1 levels >3.0 ng/mL were significantly associated with progression-free survival (PFS) in both the discovery and validation cohorts (P=0.01, P=0.04, respectively). PFS did not differ significantly by CEA levels (P=0.21). Conclusions: After combined chemoimmunotherapy treatment, serum CYFRA 21-1 stands out as a potentially valuable biomarker for predicting the prognosis of NSCLC.

Cytokeratin-18 is Elevated Prior to Conventional Measures of Liver Disease in Fontan-Associated Liver Disease.

The Fontan procedure is used to palliate complex forms of congenital heart disease. This results in adverse hepatic sequelae now known as Fontan-associated liver disease (FALD). Conventional laboratory measures of liver disease do not correlate well with FALD severity. Cytokeratin-18 (CK-18) is a measure of cell death and is sensitive in detecting other causes of liver disease. Our aim was to assess the use of a novel measure of liver disease, CK-18, in Fontan patients. This is a single-center, prospective, cross-sectional study of Fontan patients aged 8-21 years old. We performed ultrasound elastography, echocardiography, magnetic resonance imaging, and serum laboratory testing. Novel laboratory test CK-18 levels in Fontan subjects were compared to healthy age-matched controls. Thirteen Fontan patients were evaluated with a median age 15 years (10, 14), 4 Hypoplastic left heart syndrome, 11 were male, and 5 were symptomatic. Fontan patients had normal AST/ALT, but a significantly elevated liver stiffness by elastography (median 13.4 kPa). Hepatic stiffness by elastography was associated with diastolic-indexed (rho = 0.58, p = 0.04) ventricular volumes. Compared to 10 aged-matched controls, CK-18 was higher in the Fontan group-cleaved CK-18 protein (p < 0.01) and full CK-18 protein, (p = 0.02). CK-18 was positively associated with AST and ALT. Elevated CK-18 levels were found in Fontan patients compared to controls suggesting hepatic cell death even in these relatively healthy Fontan patients. CK-18 was elevated prior to changes in traditional testing. CK-18 may be a useful sensitive marker of liver disease in FALD.

Keratin 8/18a.1 Expression Influences Embryonic Neural Crest Cell Dynamics and Contributes to Postnatal Corneal Regeneration in Zebrafish.

A complete understanding of neural crest cell mechanodynamics during ocular development will provide insight into postnatal neural crest cell contributions to ophthalmic abnormalities in adult tissues and inform regenerative strategies toward injury repair. Herein, single-cell RNA sequencing in zebrafish during early eye development revealed keratin intermediate filament genes krt8 and krt18a.1 as additional factors expressed during anterior segment development. In situ hybridization and immunofluorescence microscopy confirmed krt8 and krt18a.1 expression in the early neural plate border and migrating cranial neural crest cells. Morpholino oligonucleotide (MO)-mediated knockdown of K8 and K18a.1 markedly disrupted the migration of neural crest cell subpopulations and decreased neural crest cell marker gene expression in the craniofacial region and eye at 48 h postfertilization (hpf), resulting in severe phenotypic defects reminiscent of neurocristopathies. Interestingly, the expression of K18a.1, but not K8, is regulated by retinoic acid (RA) during early-stage development. Further, both keratin proteins were detected during postnatal corneal regeneration in adult zebrafish. Altogether, we demonstrated that both K8 and K18a.1 contribute to the early development and postnatal repair of neural crest cell-derived ocular tissues.

Cytokeratin expression by sarcomas does not indicate synovial origin in dogs.

Sarcoma samples from 33 dogs, 25 subcutaneous and 8 articular, were submitted for cytokeratin immunohistochemistry. Eight of the 25 subcutaneous sarcomas (32%) expressed cytokeratin in 1% to 50% of the neoplastic cells. Of the 7 articular sarcomas evaluated, 1 (14%) expressed cytokeratin in 10% of neoplastic cells. The Kaplan-Meier survival analysis showed that the mean overall survival of dogs with subcutaneous sarcomas (28.1 months [confidence interval [CI]:17.8, 38.4]) did not significantly differ from those with articular sarcomas (24.8 months [CI = 0.5, 29.0]). Overall survival of dogs with sarcomas (both locations combined) immunoreactive for cytokeratin (31.2 months [CI = 17.8, 44.6]) did not differ from those not immunoreactive for cytokeratin (22.0 months [CI = 8.4, 35.6]). Therefore, cytokeratin expression does not indicate synovial origin (P = .64) and neither sarcoma location (P = .76) nor cytokeratin expression (P = .53) affects patient overall survival in this small study. The use of cytokeratin immunohistochemistry is not helpful to determine synovial origin of sarcomas in dogs.

Cytokeratin 18 as a Novel Biomarker in Patients with Hypertrophic Cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a heart muscle disease associated with an increased risk for sudden cardiac death (SCD). Cytokeratin 18-based proteins, such as M30 and M65 antigens, are known cell-death biomarkers. M30 antigen is released from cells during apoptosis, and M65 antigen is released during cell death from any cause, such as apoptosis or necrosis. We aimed to study the expression of M30 and M65 antigens in peripheral blood obtained by 46 HCM patients and compare with 27 age- and sex-matched patients without HCM. We also investigated the CK18 expression in myocardium from postmortem HCM hearts. M30 and M65 antigens were significantly increased in the HCM vs. non-HCM group (Μ30: 338 ± 197 U/uL vs. 206 ± 166 U/uL, p = 0.003; M65: 428 ± 224 U/uL vs. 246 ± 214 U/uL, p = 0.001), and HCM patients with a higher expression of these markers (M30: 417 ± 208 vs. 271 ± 162 U/uL, p = 0.011; M65: 518 ± 242 vs. 351 ± 178 U/uL, p = 0.011) had a higher risk for SCD. In HCM, both apoptosis and necrosis are increased, but particularly necrosis (M30/M65 ratio: 0.75 ± 0.09 vs. 0.85 ± 0.02, p < 0.001). CK18 is expressed in the HCM myocardium (1.767 ± 0.412 vs. 0.537 ± 0.383, % of area, p = 0.0058). Therefore, M30 and M65 antigens may be novel biomarkers in HCM.

Analysis of variation of serum CEA, SCC, CYFRA21-1 in patients with lung cancer and their diagnostic value with EBUS-TBNA.

Background: To explore the variation of serum carcinoembryonic antigen (CEA), cytokeratin 19 fragment (CYFRA21-1), and squamous cell carcinoma (SCC) antigen in patients with lung cancer (LC) and their diagnostic value with endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA). Methods: This study examined the diagnostic value of serum tumor marker testing and EBUS-TBNA joint detection for LC in 150 patients with suspected LC. Results: Compared to benign patients, the serum levels of CYFRA21-1, SCC, and CEA in LC were higher (P<0.05). In patients with squamous cell carcinoma (LSCC), small cell lung cancer (SCLC), and lung adenocarcinoma, lung adenocarcinoma had higher serum CEA levels (P<0.05). In comparison, LSCC patients had higher serum SCC and CYFRA21-1 levels (P<0.05). As compared to each index detected alone, the AUC of combined detection of each index to diagnose LC and identify pathological types of LC was elevated. Conclusions: The clinical significance of serum CYFRA21-1, SCC, and CEA conjugated with EBUS-TBNA is demonstrated for diagnostic purposes and identification of LC pathological types.

Pilomatricoma in the infraorbital region.

Key Clinical Message: Pilomatricoma, a rare benign skin tumor arising from hair follicle matrix cells, warrants consideration in the evaluation of subcutaneous nodules or masses, especially when presenting as painless and firm lesions. Accurate diagnosis hinges on histopathological examination, underscoring the significance of clinician vigilance and prompt intervention. Abstract: Pilomatricoma, also referred to as Pilomatrixomas or Calcifying epithelioma of Malherbe, is a rare benign skin tumor derived from hair follicle matrix cells, presents a diagnostic challenge due to its diverse clinical manifestations. Females are more commonly affected by Pilomatricoma. This condition typically manifests as a painless, firm, and slowly progressive lesion. Histopathological analysis shows characteristic findings, such as basaloid cells at the periphery and shadow cells centrally. Immunohistochemical studies assess the expression of cytokeratin's associated with hair matrix differentiation. Complete surgical excision remains the cornerstone of treatment, ensuring favorable outcomes and minimizing the risk of recurrence. Awareness of this entity among clinicians is essential for timely recognition and appropriate intervention. In this specific case-study, we present a case of Pilomatricoma situated in the lower left orbital region of a 32-year-old male individual who had been noticing swelling in that vicinity over the preceding 7 months.

Cytokeratin AE1/AE3 immunolabeling in epithelioid hemangiosarcoma.

Epithelioid hemangiosarcoma (EH), a rare histological variant of hemangiosarcoma, is reported in various animal species, including humans, dogs, cows, horses, and cats. Epithelioid hemangiosarcomas are composed of highly pleomorphic epithelioid cells arranged in cords, islands, nests, or solid cellular areas, similar to epithelial neoplasms. Moreover, in humans, approximately 50% of EHs have cytoplasmic immunolabeling for cytokeratin AE1/AE3 (CK AE1/AE3), making it challenging to distinguish them from carcinomas. This retrospective study assessed the CK AE1/AE3 immunolabeling in canine EH cases from 5 veterinary institutions. Immunohistochemistry for CD31 and CK AE1/AE3 was performed on 30 cases. CK AE1/AE3 immunolabeling was detected in 43% (13/30) of cases, with cytoplasmic labeling ranging from 5% to 100% of neoplastic cells. All tumors consistently had membranous immunolabeling for CD31. The CK AE1/AE3 immunolabeling pattern in canine EHs closely resembled those documented in humans, indicating a similar diagnostic challenge. Therefore, it is recommended to include a vascular immunohistochemistry marker, such as CD31, whenever EH is suspected, particularly in small incisional cutaneous and subcutaneous biopsies.

Association Between Cytokeratin 19-Specific IgG and Neutrophil Activation in Asthma.

PURPOSE: Patients with non-eosinophilic asthma (NEA) are less responsive to anti-inflammatory drugs and suffer from frequent asthma exacerbations. The pathogenic mechanism of NEA is not fully understood; however, the roles of monocytes and autoimmune mechanisms targeting airway epithelial cell (AEC) antigens have been proposed. METHODS: The effects of monocyte extracellular traps (MoETs) on cytokeratin 19 (CK19) production in AECs, as well as the impact of CK19-specific immunoglobulin (Ig) G on neutrophil and monocyte activation, were investigated both in vivo and in vitro. Sixty asthmatic patients and 15 healthy controls (HCs) were enrolled, and the levels of serum immune complexes containing CK19-specific IgG and neutrophil extracellular trap (NET)-specific IgG were measured using enzyme-linked immunoassay. RESULTS: MoETs induced CK19 and CK19-specific IgG production. Furthermore, the levels of serum CK19-specific IgG were significantly higher in the NEA group than in the eosinophilic asthma group. Among patients with NEA, asthmatics with high levels of CK19-specific IgG had higher levels of myeloperoxidase and NET-specific IgG than those with low levels of CK19-specific IgG (P = 0.020 and P = 0.017; respectively). Moreover, the immune complexes from asthmatics with high CK19-specific IgG enhanced NET formation and reactive oxygen species production (neutrophil activation), which were suppressed by N-acetylcysteine and anti-CD16 antibody treatment. CONCLUSIONS: These findings suggest that circulating CK19 and CK19-specific IgG may contribute to NET formation, leading to airway inflammation and steroid resistance in NEA.

A multiparametric analysis including single-cell and subcellular feature assessment reveals differential behavior of spheroid cultures on distinct ultra-low attachment plate types.

Spheroids have become principal three-dimensional models to study cancer, developmental processes, and drug efficacy. Single-cell analysis techniques have emerged as ideal tools to gauge the complexity of cellular responses in these models. However, the single-cell quantitative assessment based on 3D-microscopic data of the subcellular distribution of fluorescence markers, such as the nuclear/cytoplasm ratio of transcription factors, has largely remained elusive. For spheroid generation, ultra-low attachment plates are noteworthy due to their simplicity, compatibility with automation, and experimental and commercial accessibility. However, it is unknown whether and to what degree the plate type impacts spheroid formation and biology. This study developed a novel AI-based pipeline for the analysis of 3D-confocal data of optically cleared large spheroids at the wholemount, single-cell, and sub-cellular levels. To identify relevant samples for the pipeline, automated brightfield microscopy was employed to systematically compare the size and eccentricity of spheroids formed in six different plate types using four distinct human cell lines. This showed that all plate types exhibited similar spheroid-forming capabilities and the gross patterns of growth or shrinkage during 4 days after seeding were comparable. Yet, size and eccentricity varied systematically among specific cell lines and plate types. Based on this prescreen, spheroids of HaCaT keratinocytes and HT-29 cancer cells were further assessed. In HaCaT spheroids, the in-depth analysis revealed a correlation between spheroid size, cell proliferation, and the nuclear/cytoplasm ratio of the transcriptional coactivator, YAP1, as well as an inverse correlation with respect to cell differentiation. These findings, yielded with a spheroid model and at a single-cell level, corroborate earlier concepts of the role of YAP1 in cell proliferation and differentiation of keratinocytes in human skin. Further, the results show that the plate type may influence the outcome of experimental campaigns and that it is advisable to scan different plate types for the optimal configuration during a specific investigation.

Cytokeratin 8 depicts nodal metastasis in head and neck squamous cell carcinoma.

Background: Nodal involvement in squamous cell carcinoma is an important feature directly associated with the poor prognosis in patients with head and neck squamous cell carcinoma (HNSCC). There are no clear cut indicators available currently to identify the lymph node metastases and overall prognosis in HNSCC. Thus, the current study was conducted to correlate the immunoexpression of cytokeratins (CK) 8, 10, and 14 with lymph node metastases and tumour differentiation in patients with HNSCC. Material and Methods: The study population included 61 retrospective cases of HNSCC with lymph node metastases (n = 31) and without lymph node metastases (n = 30). Expression of CK 8, 10, and 14 was assessed by immunohistochemical staining procedure. Using Pearson's Chi-square test and Spearman's correlation coefficient, the correlation of these markers with lymph node metastases and tumour differentiation was statistically analysed. Results: The expression of CKs in HNSCC cases was higher than in controls. In nodal metastasis cases, CK 8 expression was noted in >50% of the tumour cells at the invasive tumour front (ITF) (P value 0.008), and in cases without nodal metastasis, <1% or negative expression was noted. CK 10 expression gradually decreased as the tumour grade increased. Association of CK 10 expression and tumour differentiation exhibited statistically significant results (P value 0.03). CK 14 expression was noted in the entire epithelium and at the ITF, strongly in most cases; however, CK 14 did not correlate with the lymph node metastasis and tumour differentiation as well. Conclusion: We found a strong correlation of CK 8 expression with nodal metastasis in HNSCC, and it can be utilised as a reliable prognostic indicator.

Identification and culture of functional salivary gland ductal epithelial cells.

Sialadenitis is a prevalent salivary gland disease resulting in decreased salivary flow rate. To date, little is known about the exact changes and mechanism of ductal cells in sialadenitis. This study aims to establish an efficient method to identify and isolate ductal cells, thereby facilitating further research on this specific cell type. Immunofluorescence for cytokeratin 13 and cytokeratin 19 was conducted in salivary glands to confirm their specificity as ductal cell markers. The dissected ducts were assessed through PCR and Western blot of cytokeratin 19 and digested by dispase and collagenase. The functionality of the isolated ductal cells was determined by measuring intracellular calcium. Cytokeratin 19 and cytokeratin 13 were expressed in all segments of human ducts. Cytokeratin 19 was limited to ducts excluding granular convoluted tubules in rat and mouse. The purities of the obtained ductal cells were approximately 98% in humans and 93% in rats. Furthermore, intracellular free calcium increased with time and concentration of carbachol treatment. Cytokeratin 19 serves as a dependable marker for identifying ductal cells in salivary glands, except for granular convoluted tubules. Moreover, we have successfully developed an efficient method for isolating ductal cells from salivary glands.

Nodular regenerative hyperplasia: The role of the CK7 immunohistochemistry pattern of expression in diagnosis.

OBJECTIVES: Nodular regenerative hyperplasia (NRH) is a rare vascular disorder of the liver. Clinically, patients present with portal hypertension with or without a cholestatic pattern of injury. Histologically, the liver parenchyma is composed of small nodules of hypertrophic hepatocytes surrounded by atrophic hepatocytes without significant fibrosis. Nodular regenerative hyperplasia is a difficult diagnosis on biopsy specimens, but biopsy remains the gold standard for diagnosis. In this retrospective review, cytokeratin 7 (CK7) immunohistochemistry (IHC) was used to aid in the diagnosis and further characterization of NRH and NRH-like changes. METHODS: The H&E-stained slides, reticulin, and CK IHC were reviewed for 22 cases. The percentage of hepatocytes staining for CK7 (0%-100%), the location of staining (centrilobular hepatic progenitor cells vs periportal/bile ductular reaction), and the pattern of staining distribution (patchy or diffuse) were recorded for comparison. RESULTS: Of the 22 cases, 9 were CK7 positive. Cases of NRH, however, expressed various degrees of CK7 positivity in centrilobular hepatic progenitor cells, unlike NRH-like changes, which were either CK7 negative or CK7 positive in periportal hepatocytes or in areas of bile ductular reaction. CONCLUSIONS: In cases with the appropriate clinical history and histology, CK7 immunohistochemistry can be performed to distinguish nodular regenerative hyperplasia (primary) and NRH-like changes (secondary). In difficult cases, CK7 positivity in centrilobular hepatic progenitor cells can help confirm the diagnosis of NRH. These data support NRH as a true entity with a distinct pathophysiology from NRH-like changes.

Reproducibility of Assessment of Lepidic (Noninvasive) Patterns in Lung Adenocarcinoma With Cytokeratin Immunostain Compared With Hematoxylin and Eosin and the Proposed New International Association for the Study of Lung Cancer (IASLC) Algorithm.

Introduction: Lepidic growth is considered noninvasive in lung nonmucinous adenocarcinoma, whereas other patterns are invasive. Considerable interobserver variability in assessing "invasion" has been reported. We assessed the utility of cytokeratin 7 (CK7) stain and recently proposed International Association for the Study of Lung Cancer criteria to improve assessment of noninvasion in lung adenocarcinoma. Methods: Four pathologists (two staff, two trainees) assessed 158 hematoxylin and eosin (HE)- and CK7-stained slides of 108 pT1N0-2 nonmucinous lung adenocarcinoma cases. Scoring took place in four rounds. First, sections were independently scored for percentage of noninvasive or probable noninvasive and invasive or probable invasive patterns. Second, after a consensus scoring algorithm for CK7 was formulated, the slides were rescored. Subsequent third-round scoring was conducted only on HE slides using the 2023 International Association for the Study of Lung Cancer proposed criteria, and fourth-round scoring on both HE and CK7 slides simultaneously. Intraclass correlation coefficient (ICC) was calculated for each round. Recurrence-free survival was assessed using Cox proportional hazards regression methods. Results: In the first two rounds, interobserver concordance was consistently higher with CK7 (ICC range = 0.44-0.6) than HE (range = 0.24-0.49) scores. The IASLC proposed algorithm improved ICC of HE scores to 0.60 (95% confidence interval: 0.52-0.67), and round 4 HE and CK7 combined improved ICC to 0.75 (95% confidence interval: 0.70-0.80). Continuous measures of averaged noninvasive and probable noninvasive scores on HE were associated with improved recurrence-free survival (hazard ratio: 0.83-0.86). Conclusions: CK7 staining consistently increased interobserver concordance in assessment of invasive versus noninvasive patterns than HE. Combining CK7 with the 2023 IASLC criteria for morphologic features of invasion may further improve the interobservers' concordance for the recognition of lepidic growth in nonmucinous lung adenocarcinoma.

A Mouse Model for Conditional Expression of Activated ß-Catenin in Epidermal Keratinocytes.

We report the generation and characterization of the K5: CAT bigenic mouse in which the constitutively activated form of ß-catenin (ΔN89 ß-catenin) is conditionally expressed in cytokeratin-5 (K5) positive epidermal keratinocytes. Following short-term doxycycline intake during the telogen resting phase, the adult K5: CAT bigenic develops enlarged pilosebaceous units that expand deep into the dermis, an expansion usually observed during the anagen growth phase. Prolonged doxycycline treatment results in significant thickening and folding of the K5: CAT epidermis. During this persistent induction period, there is clear evidence of increased keratinocyte proliferation, particularly in the epidermal basal cell layer and the outer root sheath of the hair follicle. This unscheduled increase in cellular proliferation likely explains the decrease in hair density observed in the K5: CAT mouse following persistent doxycycline intake. Numerous hyperplastic endometrioid cysts, which display cornification toward their lumens, are also observed during this treatment period. Remarkably, de-induction of ΔN89 ß-catenin expression through doxycycline withdrawal results in a marked reversal of the skin phenotype, suggesting that these morphological changes are dependent on continued signaling by ß-catenin and/or its downstream molecular mediators. Joining a small group of mouse models for conditional ß-catenin signaling, our K5: CAT mouse model will be particularly useful in identifying those molecular mediators of ß-catenin that are responsible for initiating and maintaining these phenotypic responses in the K5: CAT skin. Such studies are predicted to shed more light on ß-catenin signaling in epidermal epithelial morphogenesis, hair follicle cycling, and hair growth pathologies.

Expression of cytokeratin 7/20 and Ki67 in Helicobacter pylori-associated gastritis and intestinal metaplasia.

INTRODUCTION: Cytokeratins (CKs) have been associated with precancerous and cancerous gastric lesions in patients with Helicobacter pylori-associated chronic gastritis, making them useful for diagnosing epithelial tumors. METHODOLOGY: A retrospective study was conducted utilizing 200 formalin-fixed paraffin-embedded gastric biopsy samples collected from the lesser curvature of the stomach. Samples from the control group, patients with H. pylori infection, and patients with H. pylori-associated gastritis, with complete and incomplete intestinal metaplasia (IM) were immunostained. Monoclonal antibodies were utilized to determine the expression of CK7, CK20, and Ki-67. RESULTS: Patients infected with H. pylori had strong CK20 expression on the surface, and weak CK7 expression on the surface and deep glands; while non-specific chronic gastritis patients had weak focal CK7 expression and strong CK20 expression. The normal gastric mucosa of patients in the control group had relatively weak CK7 expression, restricted to a few cells in the neck and deep glands. CK20 showed diffuse strong reactivity on the surface. On the other hand, patients with complete IM showed a CK7 staining pattern that was either negative or weakly focal on the surface and crypts associated with diffuse surface CK20 and focal crypt staining corresponding to gastric type IM. The Ki67 proliferating index was low (≤ 15%) in H. pylori infected patients, high (> 30%) in patients with incomplete IM, and intermediate (16-30%) in patients with complete IM. CONCLUSIONS: These results indicate a significant link between the expressions of CK7/CK20 and Ki67 in patients afflicted with H. pylori and IM.