Shin Nieh

Viral load of high-risk human papillomavirus in cervical squamous intraepithelial lesions.

Objectives: This case‐control study was conducted to investigate the role of viral load of high‐risk human papillomaviruses (HPVs) in the development of cervical squamous intraepithelial lesions (SILs) and invasive cancers. Methods: A total of 30 female cases who had histological evidence of low‐grade SIL (n=10) or high‐grade SIL and above (n=20) were identified as the case group at the Tri‐Service General Hospital, Taipei between September 1998 and March 1999. In addition, 80 female controls who had normal cervical cytology were enrolled and individually matched on age (±5 years) and date of recruitment to each case. Cervical swabs collected from study subjects were tested for the positivity and viral load of high‐risk HPVs by Hybrid Capture II assay. Additionally, subjects completed a risk factor questionnaire. Results: Among sex behavioral factors studied, younger age at first intercourse was associated with a significantly elevated risk of cervical SIL and invasive cancers. With respect to HPV infection, high‐risk HPV DNA was present in 70% (21/30) of case and 21% (17/80) of control subjects, resulting in an odds ratio (OR) of 6.6 [95% confidence interval (C.I.)=2.6–17.0]. Moreover, women who had a high viral load were at significantly greater risk for cervical SIL and invasive cancers than those who were infected with a low viral load (OR=18.0, 95% C.I.=3.0–108.5). Conclusions: Among the variables tested, infection with a high viral load of high‐risk HPVs is the strongest determinant for cervical SIL and cervical cancers in Taiwan.

Nonadhesive Culture System as a Model of Rapid Sphere Formation with Cancer Stem Cell Properties

Background Cancer stem cells (CSCs) play an important role in tumor initiation, progression, and metastasis and are responsible for high therapeutic failure rates. Identification and characterization of CSC are crucial for facilitating the monitoring, therapy, or prevention of cancer. Great efforts have been paid to develop a more effective methodology. Nevertheless, the ideal model for CSC research is still evolving. In this study, we created a nonadhesive culture system to enrich CSCs from human oral squamous cell carcinoma cell lines with sphere formation and to characterize their CSC properties further. Methods A nonadhesive culture system was designed to generate spheres from the SAS and OECM-1 cell lines. A subsequent investigation of their CSC properties, including stemness, self-renewal, and chemo- and radioresistance in vitro, as well as tumor initiation capacity in vivo, was also performed. Results Spheres were formed cost-effectively and time-efficiently within 5 to 7 days. Moreover, we proved that these spheres expressed putative stem cell markers and exhibited chemoradiotherapeutic resistance, in addition to tumor-initiating and self-renewal capabilities. Conclusions Using this nonadhesive culture system, we successfully established a rapid and cost-effective model that exhibits the characteristics of CSCs and can be used in cancer research.

The paracrine effect of cancer-associated fibroblast-induced interleukin-33 regulates the invasiveness of head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is one of the leading causes of cancer‐related death worldwide. The prognosis of HNSCC is usually poor because of its propensity for extensive invasion, local recurrence and frequent regional lymph node metastasis, even at initial diagnosis. Carcinoma‐associated fibroblasts (CAFs), a major type of tumour‐surrounding stromal cell, generate mediators through which they interact with tumours and contribute to cancer progression. The orchestration between CAFs and cancer cells is complex. Despite recent studies demonstrating the paracrine effect of stromal cells in the tumour microenvironment on initiation and progression of cancer cells, the major mediator related to CAFs and its underlying mechanism remain unknown. In the present study, we used organotypic culture to investigate CAFs that promote aggressive behaviour of HNSCC cells. Using microarray analysis, we detected abundant expression of interleukin‐33 (IL‐33) in CAFs and identified IL‐33 as a critical mediator in CAF‐induced invasiveness. Counteracting IL‐33 activity diminished the aggressive phenotype of cancer cells induced by CAFs. Administration of IL‐33 promoted cancer cell migration and invasion through induction of epithelial‐to‐mesenchymal transdifferentiation and increased IL‐33 gene expression in cancer cells. In 40 patients with HNSCC, IL‐33 expression in CAFs correlated with IL‐33 expression in cancer cells. Most cases with a low invasion pattern grading score (IPGS) showed low or no expression of IL‐33, whereas most HNSCC cases with high IPGS displayed over‐expression of IL‐33 in CAFs and cancer cells. High IL‐33 expression associated with poor prognosis in terms of nodal metastasis‐free survival. These results indicate that CAFs promote cancer invasiveness via paracrine and autocrine effects on microenvironmental IL‐33 signalling, and suggest that IL‐33 is a potential prognostic biomarker that could be considered in therapeutic strategies for the treatment of patients with HNSCC. Copyright

Quercetin Suppresses Drug-Resistant Spheres via the p38 MAPK–Hsp27 Apoptotic Pathway in Oral Cancer Cells

Background Treatment failure in oral squamous cell carcinoma (OSCC) leading to local recurrence(s) and metastases is mainly due to drug resistance. Cancer stem cells (CSCs) are thought be responsible for the development of drug resistance. However, the correlations between CSCs, drug resistance, and new strategy against drug resistance in OSCC remain elusive. Methods A drug-resistant sphere (DRSP) model was generated by using a nonadhesive culture system to induce drug-resistant cells from SCC25 oral cancer cells. A comparative analysis was performed between the parent control cells and DRSPs with a related treatment strategy focusing on the expression of epithelial–mesenchymal transition (EMT)-associated markers, drug-resistance-related genes, and CSC properties in vitro, as well as tumorigenicity and the regimen for tumor regression in vivo. Results Our data show the presence of a phenomenon of EMT with gradual cellular transition from an epithelioid to mesenchymal-like spheroid morphology during induction of drug resistance. The characterization of DRSPs revealed the upregulation of the drug-resistance-related genes ABCG2 and MDR-1 and of CSC-representative markers, suggesting that DRSPs have greater resistance to cisplatin (Cis) and stronger CSC properties compared with the control. Moreover, overexpression of phosphorylated heat-shock protein 27 (p-Hsp27) via the activation of p38 MAPK signaling was observed in DRSPs. Knockdown of Hsp27 decreased Cis resistance and induced apoptosis in DRSPs. Furthermore, an inhibitor of Hsp27, quercetin (Qu), suppressed p-Hsp27 expression, with alterations of the EMT signature, leading to the promotion of apoptosis in DRSPs. A xenographic study also confirmed the increase of tumorigenicity in DRSPs. The combination of Qu and Cis can reduce tumor growth and decrease drug resistance in OSCC. Conclusions The p38 MAPK–Hsp27 axis plays an important role in CSCs-mediated drug resistance in OSCC. Targeting this axis using Qu combined with Cis may be a treatment strategy to improve prognosis in patients with OSCC.

Genetic polymorphism of the interferon‐γ gene in cervical carcinogenesis

Beyond human papillomavirus (HPV) infection, host genetic factors may contribute to cervical carcinogenesis. This study aims to test the hypothesis that CA‐dinucleotide repeat polymorphism in the first intron of the interferon‐gamma (IFN‐γ) gene is associated with HPV‐initiated cervical carcinogenesis. A hospital‐based case‐control study including patients with low‐grade squamous intraepithelial lesions (LSILs; n = 93), high‐grade squamous intraepithelial lesions (HSILs; n = 123) and invasive carcinomas (n = 153) of the uterine cervix, as well as 1:1 age‐matched controls, was conducted. The IFN‐γ genotype was determined by PCR and capillary electrophoresis with internal standards. HPV genotype was determined by consensus PCR and reverse line blot hybridization. Genotypes containing the 12 or 14 allele (12 or 14 CA repeats) were significantly more common in patients with HSILs than in controls (46% vs. 22%; OR = 3.0; 95% CI = 1.7–5.2; p < 0.0001). In contrast, genotypes containing 13 and 18 were significantly more common in controls than in patients with HSILs (76% vs. 53%; OR = 0.3; 95% CI = 0.2–0.6; p = 0.0001) or squamous cell carcinomas (74% vs. 63%; OR = 0.6; 95% CI = 0.4–1.0; p = 0.037). The frequency of the 12 and 14 genotypes increased significantly in accordance with the severity of cervical carcinogenesis (ptest for trend = 0.0002), whereas the 13 and 18 genotypes showed the opposite trend (ptest for trend = 0.007). Comparing IFN‐γ genotype and HPV status, 18‐containing genotypes were more frequently found in HPV+ LSILs, and 12‐containing genotypes were less frequently found in HPV+ HSILs. Compared with non‐13 genotypes, 13 genotype HSILs were more frequently infected with HPV58 (70% vs. 45%) and less frequently infected with HPV18 (0% vs. 16%; p= 0.007). Genetic polymorphism of the IFN‐γ gene is associated with individual susceptibility to cervical carcinogenesis. This polymorphism correlates with HPV infection in a disease‐ and type‐specific manner.

Expression of p16INK4A in Pap smears containing atypical glandular cells from the uterine cervix.

OBJECTIVE To verify one of the diagnostic dilemmas concerning atypical glandular cells (AGC) by immunocytochemical detection of p16INK4A (p16) applied to routine Pap smears with correlation of follow-up biopsies for improvement of cytologic diagnoses. STUDY DESIGN The study included 36 Pap smears in AGC diagnostic categories, all of which were correlated histologically. The cytologic diagnoses of AGC were further classified according to the 2001 Bethesda System. All Pap smears were decolorized and immunostained with the primary anti-p16 antibody, clone E6H4. Immunoreactivity for p16 was correlated with histologic sections in a semiblind fashion. RESULTS Of the 36 smears containing AGC, 22 (61%) were reclassified as general AGC and 14 (39%) as AGC--favor neoplasia. Follow-up biopsies revealed that 15 (42%) cervixes had no obvious abnormalities and that 21 (58%) cases had different cervical lesions. More than half the cases (19/36, 53%) of follow-up biopsies concerning AGC-containing smears represented significant lesions. There was a much higher proportion of significant lesions (13/14, 93%) in AGC--favor neoplasia than those (6/22, 27%) in general AGC cases. Fifteen of 36 (36%) AGC-containing cases were actually squamous abnormalities on follow-up biopsies. p16 Immunocytochemical stain was reactive in 22 (61%) of 36 smears, either weakly/sporadically (2 cases, 6%) or strongly positively (20 cases, 55%). Conversely, 14 (39%) of the smears were negative for p16 and displayed predominantly reactive changes. However, there was 1 case of high grade squamous intraepithelial lesion showing negative immunostaining for p16. From the view-point of clinical significance, this analysis was highly sensitive (sensitivity, 95%) and specific (specificity, 88%) and had favorable positive (90%) and negative (94%) predictive values. CONCLUSION On the basis of both morphologic and immunostaining patterns, there was a clear association between strong p16 immunostaining of atypical cells in smears and the presence of significant lesions in the cervix except in 1 patient. Similarly, there was a clear association between lack of p16 expression and absence of cervical lesions. p16 Immunocytochemical stain can be applied successfully to conventional Pap smears and may serve as a useful biomarker in diagnoses of AGC-containing smears. This may offer a more objective parameter to help clarify this ambiguous area of gynecologic cytopathology.

Expression pattern, ethanol-metabolizing activities, and cellular localization of alcohol and aldehyde dehydrogenases in human large bowel: association of the functional polymorphisms of ADH and ALDH genes with hemorrhoids and colorectal cancer

Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are principal enzymes responsible for metabolism of ethanol. Functional polymorphisms of ADH1B, ADH1C, and ALDH2 genes occur among racial populations. The goal of this study was to systematically determine the functional expressions and cellular localization of ADHs and ALDHs in human rectal mucosa, the lesions of adenocarcinoma and hemorrhoid, and the genetic association of allelic variations of ADH and ALDH with large bowel disorders. Twenty-one surgical specimens of rectal adenocarcinoma and the adjacent normal mucosa, including 16 paired tissues of rectal tumor, normal mucosae of rectum and sigmoid colon from the same individuals, and 18 surgical mixed hemorrhoid specimens and leukocyte DNA samples from 103 colorectal cancer patients, 67 hemorrhoid patients, and 545 control subjects recruited in previous study, were investigated. The isozyme/allozyme expression patterns of ADH and ALDH were identified by isoelectric focusing and the activities were assayed spectrophotometrically. The protein contents of ADH/ALDH isozymes were determined by immunoblotting using the corresponding purified class-specific antibodies; the cellular activity and protein localizations were detected by immunohistochemistry and histochemistry, respectively. Genotypes of ADH1B, ADH1C, and ALDH2 were determined by polymerase chain reaction-restriction fragment length polymorphisms. At 33mM ethanol, pH 7.5, the activity of ADH1C*1/1 phenotypes exhibited 87% higher than that of the ADH1C*1/*2 phenotypes in normal rectal mucosa. The activity of ALDH2-active phenotypes of rectal mucosa was 33% greater than ALDH2-inactive phenotypes at 200μM acetaldehyde. The protein contents in normal rectal mucosa were in the following order: ADH1>ALDH2>ADH3≈ALDH1A1, whereas those of ADH2, ADH4, and ALDH3A1 were fairly low. Both activity and content of ADH1 were significantly decreased in rectal tumors, whereas the ALDH activity remained unchanged. The ADH activity was also significantly reduced in hemorrhoids. ADH4 and ALDH3A1 were uniquely expressed in the squamous epithelium of anus at anorectal junctions. The allele frequencies of ADH1C*1 and ALDH2*2 were significantly higher in colorectal cancer and that of ALDH2*2 also significantly greater in hemorrhoids. In conclusion, ADH and ALDH isozymes are differentially expressed in mucosal cells of rectum and anus. The results suggest that acetaldehyde, an immediate metabolite of ethanol, may play an etiological role in pathogenesis of large bowel diseases.

Expression of survivin and cortactin in colorectal adenocarcinoma: association with clinicopathological parameters.

Objective: Survivin and cortactin are factors that promote tumor progression. We tested the hypothesis that survivin and cortactin expressions correlate with the clinico-pathological parameters of colorectal adenocarcinomas and survival time. Methods: Immunohistochemical analysis of survivin and cortactin were performed using tissue microarrays of 119 specimens from 18 well, 50 moderately, and 27 poorly differentiated colorectal adenocarcinomas and 24 colorectal adenomas with dysplasia. As control, 10 specimens of normal colorectal epithelia were included. Results: The percentage of cells immunostained and the immunostaining scores for survivin and cortactin were all significantly higher in well-, moderately, and poorly differentiated colorectal adenocarcinomas than in normal colorectal epithelia. The survivin immunostaining score was significantly correlated with T, M, and AJCC/TNM stages (p `0.05). For cortactin, the score was significantly correlated with T and M stages (p ` 0.05). Higher survivin immunostaining score was associated with higher mortality. Conclusions: Higher expression of survivin and cortactin correlates significantly with tumor stages and shorter survival time. Survivin and cortactin may be good biomarkers of aggressiveness of colorectal adenocarcinomas. Our findings require validation in independent cohorts and these data support the potential targeting of survivin and cortactin for the development of novel therapeutic strategies.

Association of EMMPRIN and fascin expression in renal cell carcinoma: correlation with clinicopathological parameters

EMMPRIN and fascin are important factors in tumor invasion and progression. We tested the hypothesis that expression of EMMPRIN and fascin correlate with clinicopathological parameters of renal cell carcinoma (RCC). Immunohistochemical analysis of EMMPRIN and fascin were performed in tissue microarrays of 100 surgical specimens, including 35 clear-cell RCC (CRCC), 21 clear-cell RCC with granular differentiation (GRCC), 12 chromophobe RCC (ChRCC), 8 papillary RCC (PRCC), 9 carcinoma of the collecting duct of Bellini (CDC), 10 clear-cell RCC with sarcomatoid differentiation (SRCC), and 6 metastatic RCC. Average immunoscores of EMMPRIN were 100.8 in CRCC, 195.2 in GRCC, 298.4 in ChRCC, 219.2 in PRCC, 186.1 in CDC, 226.9 in SRCC, and 151.7 in metastatic RCC. Among all included cases, average EMMPRIN immunoscores were 84.6 in grade I, 130.4 in grade II, 184.3 in grade III, and 223.5 in grade IV. Additionally, average immunostaining scores of fascin were 53.6 in CRCC, 289.3 in GRCC, 193.3 in ChRCC, 151.8 in PRCC, 181.3 in CDC, 275.4 in SRCC, and 131.7 in metastatic RCC. Average fascin immunoscores were 59.3 in grade I, 91.6 in grade II, 130.2 in grade III, and 194.7 in grade IV. Higher EMMPRIN and fascin immunoscores also correlated significantly with TNM stages and survival rates in RCC. Significant correlation was found between EMMPRIN and fascin expression. In conclusion, higher expression of EMMPRIN and fascin correlate significantly with histological grades, clinical stages, and survival rates of RCC

Overexpression of Fascin-1 in Advanced Colorectal Adenocarcinoma: Tissue Microarray Analysis of Immunostaining Scores with Clinicopathological Parameters

Objective: Fascin-1 is an actin-binding protein that promotes cell proliferation, adhesion and motility. We tested the hypothesis that fascin-1 expression correlates with clinicopathological parameters of colorectal adenocarcinomas. Methods: Immunohistochemical analysis of fascin-1 was performed in tissue microarrays of 91 surgical specimens, including 32 well, 33 moderately, and 26 poorly differentiated colorectal adenocarcinomas; and in 22 specimens from colorectal adenomas with dysplasia. Results: Scattered fascin-1 expression was demonstrated in 9 control specimens of normal colonic glandular epithelia. Higher fascin-1 immunostaining scores were significantly associated with advanced dysplasia in colorectal adenomas (mild 4.2 ± 1.3, moderate 13.5 ± 5.3, and severe 22.5 ± 6.7) and high-grade histopathological differentiation of colorectal adenocarcinomas (grade I 88.6 ± 9, grade II 101 ± 11, and grade III 144 ± 13). Higher immunostaining scores of fascin-1 were also significantly associated with advanced T stage (T1: 42 ± 10, T2: 60 ± 12, T3: 108 ± 12, and T4: 142 ± 15). Higher fascin-1 scores were related with more advanced M and N stages of colorectal carcinomas, but not significant correlation. Conclusions: Higher expression of fascin-1 correlates significantly with tumor grades and TNM stages in colorectal adenocarcinomas and also with levels of dysplastic change in colorectal adenomas.