Cytopathologic features of human papillomavirus-independent, gastric-type endocervical adenocarcinoma.

BACKGROUND: Gastric-type endocervical adenocarcinoma (GEA) is unrelated to human papillomavirus (HPV) infection and is clinically aggressive compared with HPV-associated usual-type endocervical adenocarcinoma (UEA). The cytological diagnosis falls short of a definitive diagnosis of GEA and is often categorized as atypical glandular cells (AGCs). To improve cytologic recognition, cytological findings of HPV-independent GEA were analyzed and the results compared with HPV-associated UEA. METHODS: Cervical Papanicolaou (Pap) smears from eight patients with a histopathologic diagnosis of GEA and 12 control cases of UEA were reviewed. All slides were conventionally prepared and/or liquid-based prepared (ThinPrep) and stained following the Pap method. A mucinous background, architectural, nuclear, and cytoplasmic features were analyzed and compared with UEA. RESULTS: Preoperative cytologic diagnoses of the eight GEA cases were AGCs, favor neoplastic in three cases, adenocarcinoma in situ in one case, and adenocarcinoma in four cases. Cytologically, monolayered honeycomb-like sheets (p = .002) of atypical endocervical cells with vacuolar granular cytoplasm (p = .001) were extensive in GEA, and three-dimensional clusters (p = .010) were extensive in UEA. Although the differences were not statistically significant, background mucin (p = .058), vesicular nuclei (p = .057), and golden-brown intracytoplasmic mucin (p = .089) were also discriminatory findings for GEA versus UEA. CONCLUSIONS: Although GEA is difficult to diagnose on cytologic screening, GEA can be recognized based on cytologic features of monolayered honeycomb sheets of atypical endocervical cells with abundant vacuolar cytoplasm and some golden-brown intracytoplasmic mucin. UEA cases are characterized by three-dimensional clusters.

Alteration of CYP4A11 expression in renal cell carcinoma: diagnostic and prognostic implications.

Background: Cytochrome P-450 4A11 (CYP4A11) and peroxisome proliferator-activated receptor-α (PPARα) are expressed at high levels in renal proximal tubules, and upregulation of CYP4A11 protein levels is known to be influenced by PPAR agonists. The goal of this study was to evaluate the clinicopathological role of CYP4A11 expression in renal cell carcinoma (RCC). Methods: We performed immunohistochemical analysis of CYP4A11, CYP4A22 and PPARα and correlated the results with the clinicopathological features of RCC (n=139). Reverse transcription digital droplet polymerase chain reaction (RT-ddPCR) against CYP4A11 and CYP4A22 was also performed. Results: CYP4A11 mRNA expression levels were higher in non-neoplastic kidney tissues than in matched tumor tissues in 12 matched pairs of freshly frozen primary clear-cell RCC (ccRCC) and nontumor tissue (p=0.002). Immunohistochemical staining showed that CYP4A11 expression was significantly lower in ccRCC than in non-ccRCCs, including papillary, chromophobe, and unclassified RCCs (p<0.001). CYP4A11 expression was associated with PPARα expression, males and high nuclear histologic grades (p=0.001, p=0.018 and p<0.001). Univariate and multivariate analyses revealed that CYP4A11 expression was correlated with short overall survival (p=0.007 and p=0.010). Conclusion: These findings suggest that CYP4A11 expression is a potential poor prognostic factor of RCC. The considerable decrease in CYP4A11 expression is a predictive diagnostic factor of ccRCC, and CYP4A11 metabolism in ccRCC might be different from that in non-ccRCCs.

High nuclear NADPH oxidase 4 expression levels are correlated with cancer development and poor prognosis in hepatocellular carcinoma.

NADPH oxidase (NOX) is a key source of reactive oxygen species (ROS). This study aimed to verify NOX2 and NOX4 expression levels in hepatocellular carcinoma (HCC). A total of 134 matched pairs of HCC cells and non-tumour hepatocytes from 134 patients were examined by immunohistochemical staining, and the association of NOX2 and NOX4 expression with clinicopathological parameters was analysed. Western blotting in four HCC cell lines and reverse transcription digital droplet polymerase chain reaction (RT-ddPCR) in 20 pairs of HCC and non-tumour tissue samples were also performed to detect NOX4. Cytoplasmic NOX2 and nuclear NOX4 expression levels were shown by immunohistochemistry to be higher in HCC cells than in non-tumour hepatocytes (p<0.001 each). The western blotting results for NOX4 in four HCC cell lines were consistent with the immunohistochemical results. Increased cytoplasmic expression of NOX2 and NOX4 in HCC cells was significantly correlated with liver cirrhosis (p<0.001 and p<0.031, respectively). However, decreased cytoplasmic expression of NOX2 and NOX4 was significantly correlated with advanced pathological TNM stage (p<0.029 and p<0.007, respectively). Multivariate analysis with clinicopathological parameters showed that high nuclear and low cytoplasmic NOX4 expression levels are correlated with short overall survival (p=0 .021). Our findings imply that cytoplasmic NOX2 and nuclear NOX4 expression is upregulated during HCC development. In particular, NOX4 translocation into the nucleus may affect the development and progression of HCC. NOX2 and NOX4 could be diagnostic markers and have therapeutic implications in HCC.

Immunohistochemical expression of CD44, matrix metalloproteinase2 and matrix metalloproteinase9 in renal cell carcinomas.

PURPOSE: The aim of our study was to investigate the clinicopathologic values of the expression of CD44, matrix metalloproteinase (MMP)2, and MMP9 in renal cell carcinoma (RCC). PATIENTS AND METHODS: A total of 107 clear cell RCCs (ccRCCs) and 32 nonclear cell RCCs (non-ccRCCs) were examined for CD44, MMP2, and MMP9 expression by immunohistochemistry. The membrane and cytoplasmic expression levels of the 3 proteins were scored by semiquantitative methods, and the correlations of the 3 proteins with clinicopathological parameters were verified. RESULTS: The expression levels of CD44, MMP2, and MMP9 were positively correlated with nuclear grade (grade 1-2 vs. grade 3-4) (P = 0.003, P < 0.001 and P < 0.001, respectively) in the ccRCCs, while in the non-ccRCCs, only CD44 expression was correlated with higher nuclear grade (grade 1-3 vs. grade 4) (P = 0.001). Furthermore, CD44 expression in ccRCCs and non-ccRCCs was correlated with shorter overall survival in the univariate analyses (P < 0.001 and P = 0.015, respectively). In the multivariate analysis, which accounted for age, sex, nuclear grade, and pathologic stage, CD44 expression was an independent predictor of shorter overall survival only in ccRCCs. Correlations among the 3 proteins were all positive in ccRCCs, but in non-ccRCCs, only MMP2 and MMP9 were positively correlated. CONCLUSION: CD44 expression may play an important role in the progression of both ccRCC and non-ccRCC. CD44 expression in ccRCC may be associated with elevated MMP2 and MMP9 expression levels, which is in contrast to non-ccRCC. The different correlations between CD44, MMP2, and MMP9 in ccRCC and non-ccRCC can be useful in understanding the mechanisms of carcinogenesis and stratifying patients for therapeutic purposes.

Profiling cytochrome P450 family 4 gene expression in human hepatocellular carcinoma.

Cytochrome P450 family 4 (CYP4) enzymes are known as microsomal omega (ω)-hydroxylases that metabolize fatty acids, eicosanoids, vitamin D and carcinogens. Thus, CYP4 enzymes may influence tumor development and progression. The aim of the present study was to evaluate the CYP4 expression profile in hepatocellular carcinoma (HCC) and its clinical relevance. The present study obtained CYP4 mRNA expression data for 377 HCC cases from The Cancer Genome Atlas cohort and performed Kaplan­Meier survival, Gene Ontology functional enrichment, and gene set enrichment analysis (GSEA). In addition, the level of CYP4F2 protein expression was evaluated in matched pairs of HCC and non­tumor tissue samples and the results were correlated with the clinicopathological characteristics of HCC (n=113). HCC survival analyses indicated better overall survival in patients with high CYP4F2, CYP4F12 and CYP4V2 mRNA expression levels; the results for histological grade and Tumor­Node­Metastasis stage supported these results. GSEA revealed high levels of CYP4F2, CYP4F12 and CYP4V2 mRNA expression to be negatively correlated with the expression of cell cycle­associated genes. CYP4F2 protein expression was higher in non­neoplastic liver tissue than in HCC tissue and positively correlated with favorable pathological tumor stage (I vs. II­IV; P=0.022) and was a good independent prognostic factor for overall survival (P=0.004). These results demonstrate that the expression levels of the genes CYP4F2, CYP4F12 and CYPV2 are favorable prognostic factors in HCC and suggest the potential predictive diagnostic and prognostic roles of CYP4F2, CYP4F12 and CYPV2 gene expression in HCC.

Nuclear Expression of CD133 Is Associated with Good Prognosis in Patients with Colorectal Adenocarcinoma.

BACKGROUND/AIM: Prominin-1 (CD133) has been suggested as a potential marker of cancer stem cells (CSCs) in various cancer types. The aim of this study was to compare CD133 expression between adenoma cells, colorectal adenocarcinoma (CRAC) cells, and tumor microenvironment cells (TMEs) in terms of the adenoma-carcinoma sequence and to investigate the clinicopathological value of CD133 expression in CRACs as a prognostic marker. MATERIALS AND METHODS: A total of 58 adenomas and 132 primary and 27 metastatic CRACs were examined for CD133 expression by immunohistochemistry. The cytoplasmic and nuclear expression levels of CD133 were scored by semi-quantitative methods. RESULTS: Adenomas showed significantly lower cytoplasmic and nuclear CD133 expression than CRACs (p<0.001). Among the CRACs, primary CRACs demonstrated higher cytoplasmic and nuclear expression levels of CD133 than metastatic CRACs (p<0.001). Furthermore, decreased nuclear CD133 expression in CRACs was correlated with a poor outcome, including disease-free survival (DFS), by univariate and multivariate analyses (p=0.012 and p=0.039). For TMEs, adenomas showed a significantly higher CD133 expression than CRACs (p<0.001), and decreased expression of CD133 in CRACs was correlated with shorter DFS by univariate and multivariate analyses (p=0.004 and p=0.042). CONCLUSION: Cytoplasmic and nuclear CD133 expression in CRAC cells and TMEs may play an important role in early CRAC carcinogenesis, while decreased CD133 nuclear expression in CRAC cells may contribute to CRAC metastasis. Further prognostic and therapeutic stratification may be performed according to CD133 localization.

Phosphorylated Protein Kinase C (Zeta/Lambda) Expression in Colorectal Adenocarcinoma and Its Correlation with Clinicopathologic Characteristics and Prognosis.

Background: Protein kinase C zeta/lambda (PKCζ/λ) is a family of protein kinase enzymes that contributes to cell proliferation and regulation, which are important for cancer development. PKCζ/λ has been shown to be an important regulator of tumorigenesis in intestinal cancer. The phosphorylated form of PKCζ/λ, p-PKCζ/λ, is suggested as an active form of PKCζ/λ. However, p-PKCζ/λ expression and its clinicopathologic implication in colorectal adenocarcinoma (CRAC) are unclear. Methods: Seven whole-tissue sections of malignant polyps containing both non-neoplastic and neoplastic mucosa, 11 adenomas with low-grade dysplasia, and 173 CRACs were examined by immunohistochemistry and western blot assay for p-PKCζ/λ protein expression. The association of p-PKCζ/λ expression with clinicopathologic factors including patient survival was studied. Results: In non-neoplastic epithelia, p-PKCζ/λ showed a weak cytoplasmic immunostaining. Adenomas and CRACs demonstrated up-regulated p-PKCζ/λ detection. Cytoplasmic p-PKCζ/λ expression was higher in CRAC than in adenoma. In CRACs, p-PKCζ/λ expression was inversely correlated with pathologic TNM stage (I-II versus III-IV) and poor differentiation. Statistical correlations between low expression of p-PKCζ/λ with shortened overall survival and disease-free survival were seen (p=0.004 and p=0.034, respectively). Conclusions: P-PKCζ/λ overexpression is implicated in tumorigenesis but down-regulation was a poor prognostic factor in CRAC.

Association of PD-L1 expression and PD-L1 gene polymorphism with poor prognosis in lung adenocarcinoma and squamous cell carcinoma.

Programmed cell death 1 receptor (PD-1)/programmed death-1 ligand-1 (PD-L1) interaction has been linked to tumor immune evasion. PD-L1 expression has been indicated in identifying non-small cell lung carcinoma (NSCLC) patients for treatment with anti-PD-1 or anti-PD-L1 therapy. The goal of this study was to evaluate the clinicopathologic values of PD-L1 expression and single-nucleotide polymorphisms (SNPs) in the PD-L1 gene in lung adenocarcinoma (ADC) and squamous cell carcinoma (SqCC). The 147 NSCLC tissues consisted of 84 samples of ADC and 63 samples of SqCC. All tissue microarray paraffin blocks were used for PD-L1 immunohistochemical assays with 22C3, SP263, and SP142 clones. Three SNPs in the PD-L1 gene, rs4143815, rs822336, and rs822337, were genotyped using SNP pyrosequencing. The PD-L1 expression was significantly higher in SqCC than in ADC. Among ADCs, PD-L1 expression was significantly higher in papillary and solid types than in lepidic and acinar types. Statistical associations of the PD-L1 expression with a shorter disease-free survival outcome and lymph node metastasis in the ADCs were found but no associations in SqCCs. Among the three SNPs, the rs4143815 genotype CC was statistically associated with positive 22C3 PD-L1 labeling in NSCLCs. The rs4143815 genotype GG instead showed a trend of shorter survival outcomes but did not reach statistical significance in the ADCs. Our results showed a significantly higher prevalence of positive PD-L1 expression in lung SqCC than in ADC. However, the PD-L1 expression and rs4143815 genotype GG might be useful for the prediction of poor prognosis in lung ADC cases.

Up-regulation of Cytoplasmic CD24 Expression Is Associated with Malignant Transformation but Favorable Prognosis of Colorectal Adenocarcinoma.

AIM: Cluster of differentiation 24 (CD24) is known to be a putative marker of stem cell and tumor metastasis. This study aimed to verify the clinicopathological value of CD24 expression in colorectal adenocarcinoma (CRAC). MATERIALS AND METHODS: A total of seven whole-tissue sections of malignant polyps including the sequence non-neoplastic colorectal tissue-adenoma-CRAC, 48 adenomas and 161 CRACs arranged as tissue microarray were examined by immunohistochemistry for CD24 protein expression. Association of CD24 expression with clinicopathological parameters were also studied. RESULTS: CD24 was not detected in normal mucosal epithelia. Cytoplasmic CD24 expression was higher in CRAC than in adenoma (p<0.001). In CRACs, cytoplasmic CD24 expression was inversely correlated with poor differentiation (grades 1 to 3), tumor size, and pathological TNM stage (I to III versus IV) (p=0.005, p=0.034, and p=0.006, respectively). Statistical correlations between high CD24 expression and longer overall and disease-free survival were found (p=0.023 and p=0.033, respectively). CONCLUSION: Our findings suggest that up-regulation of CD24 expression in CRAC occurs at malignant transformation but is a marker of good prognosis, being down-regulated in pathological TNM stage IV. CD24 expression may be a challenging diagnostic marker in differentiating early invasive CRAC from adenoma and may serve as a prognostic marker in patients with CRAC.

No Detection of Episomal or Integrated High-Risk Human Papillomavirus in Nonsmall Cell Lung Carcinomas among Korean Population.

OBJECTIVES: High-risk human papillomavirus (hrHPV) is known to be a representative cancer-causing agent in the genital and head and neck regions. Many studies have detected hrHPV DNA in nonsmall cell lung carcinoma. However, hrHPV-etiologic correlation in nonsmall cell lung carcinoma remains unclear. This study is designed to determine the prevalence of episomal or integrated hrHPV DNA in nonsmall cell lung carcinoma among the Korean population. METHODS: Surgically resected nonsmall cell lung carcinoma tissues, including 134 cases of squamous cell carcinoma (SqCC) and 99 cases of adenocarcinoma (ADC), were examined. In situ hybridization (ISH) for detecting episomal or integrated hrHPV DNA was performed using the INFORM HPV III Family 16 Probe (B) in the Ventana-validated assay. Anyplex II HPV28 detection kit based on real-time polymerase chain reaction was used for HPV DNA detection and genotyping. RESULTS: All members of the study population were of Korean ethnicity. Episomal or integrated hrHPV DNA ISH analysis result was negative in all 233 cases. One SqCC of 89 samples (42 SqCCs and 47 ADCs) was positive for an hrHPV genotype by Anyplex II HPV28 detection kit. CONCLUSION: Our finding did not demonstrate hrHPV-etiologic correlation in primary lung SqCC and ADC in the Korean population.

Differential microscopic finding and glucose transporter 3 expression in terminal chorionic villi among birth weight-discordant twin placentas.

OBJECTIVE: To evaluate differences in microscopic findings and glucose transporter 3 (GLUT3) expression in terminal chorionic villi (TV) among birth weight-discordant twin (BWDT) placentas compared with the birth weight-concordant twin (BWCT) placentas. METHODS: We retrospectively studied a cohort of 26 BWDT, 10 BWCT, 10 pre-eclampsia singleton and 10 normal singleton pregnancies. Placentas were scored for the percentage of TV, the percentage of TV with syncytial knots, the presence of capillary branching patterns of TV, the capillary to terminal villous ratios, the membranous expression of GLUT3 and the nuclear expression of HIF-1α in trophoblasts and capillary endothelial cells of TV using immunohistochemistry. The clinical characteristics and microscopic findings were analyzed and compared. RESULTS: BWDT placentas exhibited differential percentages of TV, percentages of TV with syncytial knots, capillary to terminal villous ratios, expression of HIF-1α in capillary endothelial cells and expression of GLUT3 in trophoblasts and capillary endothelial cells of TV among each twin pair compared with BWCT placentas (P=0.003, P=0.022, P=0.037, P=0.007, P=0.046 and P=0.002, respectively). Pre-eclampsia singleton placentas exhibited higher GLUT3 expression in trophoblasts, higher HIF-1α expression in capillary endothelial cells of TV and high capillary to terminal villous ratios compared with normal singleton placentas (P=0.001, P<0.001 and P=0.001, respectively). CONCLUSIONS: We observed a strong relationship between characteristics of adaptive change to hypoxia (GLUT3 expression, TV and syncytial knotting and higher capillary to terminal villous ratios) and BWDT pregnancy but not BWCT pregnancy.

Differential expression of Yes-associated protein and phosphorylated Yes-associated protein is correlated with expression of Ki-67 and phospho-ERK in colorectal adenocarcinoma.

Yes-associated protein (YAP) is a transcriptional co-activator and functions as a nuclear downstream effector of the Hippo pathway. Differential expression of YAP and phosphorylated Yes-associated protein (pYAP), which are involved in the expression of Ki-67 and phosphorylated extracellular signal-regulated kinase (pERK) in colorectal adenocarcinoma (CRAC), is not clear. Herein, we hypothesized that nuclear expression of YAP could predict cell proliferation and poor prognosis, while cytoplasmic expression of pYAP would show a reverse correlation with cell proliferation. Paraffin-embedded samples from 144 CRAC patients were studied using immunohistochemistry for YAP, pYAP, Ki-67 and pERK. Frozen samples from 20 CRAC patients were examined for YAP mRNA in tumor and non-tumor tissues, using quantitative real-time PCR. High nuclear YAP expression coincided with high Ki-67 expression (P=0.002). The high nuclear YAP expression group tended to display a poor overall and disease-free survival (P=0.089 and P=0.089, respectively), but YAP mRNA levels in the 20 CRAC tissues were not significantly different in comparison with the 20 non-tumor tissues (P=0.929). We observed an inverse correlation between high cytoplasmic pYAP expression and high Ki-67 expression (P=0.001). Nuclear pERK expression was positively correlated with nuclear YAP expression, but negatively correlated with cytoplasmic pYAP expression (P=0.017 and P=0.020, respectively). Activated nuclear YAP and inactivated cytoplasmic pYAP in CRAC showed a positive correlation with Ki-67 and nuclear pERK expression, suggesting that the expression of YAP and pYAP is a possible predictor of tumor cell proliferation and prognosis in CRAC.

Expression of sonic hedgehog signaling molecules in normal, hyperplastic and carcinomatous endometrium.

The aim of the present study was to determine the expression profile of the hedgehog (Hh) signaling molecules in normal, hyperplastic, and carcinomatous uterine endometrium. For this purpose, 271 endometrial tissue samples, (62 of normal endometrium, 127 of endometrial hyperplasias, and 82 endometrial adenocarcinomas) were studied using antibodies recognizing Hh-related signaling proteins, such as, sonic hedgehog (Shh), Patched (PTCH), Smoothened (Smo), Suppressor of fused [Su(Fu)], Gli-1, Gli-2, and Gli-3 by immunohistochemistry. The mRNA expression of these molecules was also assessed on reverse transcription-polymerase chain reaction. In the normal endometrium, the expression of Hh signaling molecules was generally downregulated except for Su(Fu), Gli-2, and Shh. In particular, the expression of both PTCH and Smo was very low or almost absent. Overall expression of Hh signaling molecules increased in hyperplastic endometrium; in particular, PTCH and Smo were significantly highly expressed in complex and atypical hyperplasia. In carcinoma samples extensive alterations were observed in the expression pattern of the signaling molecules. Nuclear Gli-2, cytoplasmic Gli-3, and Su(Fu) were overexpressed, whereas Shh, PTCH, and Smo expression were significantly reduced compared with the hyperplastic endometrium. The results suggest that the alteration of Hh signaling may be implicated in tumorigenesis of the endometrium.

Enhanced CD24 expression in endometrial carcinoma and its expression pattern in normal and hyperplastic endometrium.

CD24 is known to be an important diagnostic and prognostic marker of several major cancers affecting females. We aimed to determine CD24 expression in normal, hyperplastic, and carcinomatous endometrium and its correlation with estrogen and progesterone receptor expression. A total of 271 cases including 62 normal/atrophic endometrium cases (47/15), 127 endometrial hyperplasia cases (51/52/24, simple/complex/atypical hyperplasia), and 82 endometrial carcinoma cases were immunohistochemically analyzed by using anti-CD24, ER, and PR antibodies that were embedded on paraffin blocks. Next, we assessed the CD24 mRNA expression in these tissues by using RT-PCR. In the normal endometrium, cyclic expression of membranous CD24 was detected during the regular menstrual cycle, i.e., down-regulation in the proliferative phase and up-regulation in the secretory phase. CD24 expression was very infrequent and weak in the atrophic endometrium. In hyperplasias and carcinomas, the expression of both membranous and cytoplasmic CD24 was found to be sharply reduced in the hyperplastic lesions and significantly enhanced in the carcinomas. In the case of carcinomas, high CD24 expression showed significant correlation with high-grade (G2 and 3) (P<0.05). In addition, an inverse correlation was apparent between CD24 and the estrogen and progesterone receptor expressions in normal and diseased endometrium. In conclusion, we demonstrated that CD24 was expressed in a cyclic pattern in the normal endometrium, and its expression was enhanced in case of endometrial carcinoma. These results suggest that CD24 may be involved in tumor progression and can be a useful diagnostic biomarker.

Mutations of the BRAF gene in papillary thyroid carcinoma in a Korean population.

The B-type Raf kinase (BRAF) protein is a serine/threonine kinase that has an important role in cellular proliferation, differentiation, and programmed cell death. The BRAF gene has been recently found to be mutated in human carcinomas, predominantly in malignant melanoma. The aim of this study was to investigate the frequency of the BRAF mutation in papillary thyroid carcinoma (PTC) of Koreans through direct DNA sequencing of the polymerase chain reaction (PCR)- amplified exon 15 with clinicopathological features. Seventy paraffin-embedded conventional papillary carcinomas in the thyroid gland were evaluated. The BRAF missense mutation at V599E was found in 58 of 70 PTCs (83%). The frequency of our series was much higher than the frequencies of other PTC series (36 - 69%). The frequency of nodal metastasis was also significantly higher in the BRAF mutation group (p= 0.048). These results suggest that the BRAF mutation is involved in the carcinogenesis in most conventional PTCs, especially those occurring in Koreans, and this is a potentially valuable marker for the evaluation of prognosis of patients with PTC. These findings support the specific inhibitors of BRAF being promising targets for the disease outcome.