Tumor-infiltrating lymphocytes predict survival outcomes in patients with cervical cancer treated with concurrent chemoradiotherapy.

OBJECTIVES: To (i) identify correlations between selected immunogenic factors and clinicopathological characteristics, (ii) determine whether intratumoral abundance of various specific tumor-infiltrating lymphocytes (TILs) is a prognostic indicator in women with Stage II and III cervical cancer who undergo treatment with cisplatin-based concurrent chemoradiotherapy (CCRT), and (iii) investigate subtypes of FOXP3+ T cells in 15 fresh samples of cervical cancer. METHODS: In this retrospective study, intratumoral lesions in colposcopic biopsies from 55 women with advanced cervical cancer who subsequently underwent CCRT at our institution were subjected to automatic immunological staining using the following six mouse monoclonal antibodies: anti-CD3, anti-CD4, anti-CD8, anti-CD20, anti-CD206, and anti-FOXP3. Associations between the findings on automatic scoring of the number of each type of TIL in each specimen and various clinicopathological characteristics were analyzed, as were associations between the abundance of various specific types of TIL and survival. Subtypes of FOXP3+ TILs in 15 additional fresh tumor samples were also investigated using flow cytometry. RESULTS: Infiltration with CD8+ TILs was associated with pelvic lymph node metastasis. Abundant infiltration by CD3+, CD4+, CD8+, CD206+, and FOXP3+ TILs were statistically significant indicators of better progression-free and overall survival. Regarding subtypes of FOXP3+ TILs, non-Tregs (Fr-III) were found in all samples tested for this. CONCLUSIONS: The abundance of various specific intratumoral TILs may be prognostic indicators in patients with advanced cervical cancer undergoing CCRT.

Establishment of an evaluation method to detect drug distribution in hair follicles.

Development of an appropriate method to evaluate drug disposition or targeting ability in hair follicles (HFs) is urgently needed in order to develop useful pharmaceutical products with pharmacological effects in HFs. In the present study, a cyanoacrylate biopsy (CB) method was used to measure drug disposition in HFs using a model hydrophilic drug, caffeine (CAF), and a lipophilic drug, 4-butylresorcinol (BR), in excised porcine skin. As a result, the height of HF replicas and the recovery ratio decreased with an increase in the application times of the CB method. HF replicas with a length of approximately 175 µm were obtained using a single application of the CB method. Drug distribution in the HF was detected even 5 min after topical application regardless of the lipophilicity of the drugs, although no drug disposition was observed in the deeper layers of the stratum corneum at the same time (5 min). Furthermore, significantly higher amounts of BR were observed in the stratum corneum and HF, compared with those of CAF. These results suggested that the CB method could be useful to evaluate the safety and efficacy as well as the disposition of topically applied chemicals, especially for HF-targeting drugs.

Determination of poor prognostic immune features of tumour microenvironment in non-smoking patients with lung adenocarcinoma.

We have previously demonstrated that the prognostic significance of tumour-infiltrating CD8+ T cells significantly differs according to histological type and patient smoking habits in non-small cell lung cancer (NSCLC). This work suggested that infiltrating CD8+ T cells may not be activated sufficiently in the immunosuppressive microenvironment in non-smokers with adenocarcinoma. To understand the immunogenic microenvironment in NSCLC, we characterised immune cells comprehensively by performing an immunohistochemical evaluation using an alternative counting method and multicolour staining method (n = 234), and assessed immune-related gene expression by using genetic analytical approaches (n = 58). We found that high infiltration of activated CD8+ T cells expressing interferon gamma (IFN-γ) and granzyme was correlated with postoperative survival in patients with non-adenocarcinoma. On the contrary, CD8+ T-cell accumulation was identified as a worse prognostic factor in patients with adenocarcinoma, particularly in non-smokers. Infiltrating CD8+ T cells were significantly less activated in this microenvironment with high expression of various immunoregulation genes. Potentially immunoregulatory CD8+ FOXP3+ T cells and immunodysfunctional CD8+ GATA3+ T cells were increased in adenocarcinoma of non-smokers. CD4+ FOXP3+ regulatory T cells expressing chemokine receptor-4 (CCR4)- and chemokine ligand (CCL17)-expressing CD163+ M2-like macrophages also accumulated correlatively and significantly in adenocarcinoma of non-smokers. These characteristic immune cells may promote tumour progression possibly by creating an immunosuppressive microenvironment in non-smoking patients with lung adenocarcinoma. Our findings may be helpful for refining the current strategy of personalised immunotherapy including immune-checkpoint blockade therapy for NSCLC.

Comparison of methods using paraffin-embedded tissues and exfoliated cervical cells to evaluate human papillomavirus genotype attribution.

Monitoring the attribution of human papillomavirus (HPV) genotypes to cervical precancerous lesions is essential in assessing the efficacy of HPV vaccines. To resolve the lack of studies comparing the HPV genotyping procedures used to estimate HPV genotype attribution, we undertook a retrospective cross-sectional study to determine the appropriate genotyping procedures for evaluating the potential efficacy of HPV vaccines. Three procedures, including two different genotyping methods, Clinichip HPV test (C-Chip) and modified GP5+/6+ PCR coupled to fluorescent bead sorter detection (MGP), using exfoliated cervical cells (C-Chip and C-MGP, respectively) or formalin-fixed paraffin-embedded tissues (F-MGP), were compared. The overall agreement in detecting high-risk HPV was 88.5-92.1% among the three procedures, and genotype-specific agreement was 83.9-100% for all pairwise comparisons. In cervical intraepithelial neoplasia grade 2/3 specimens, HPV16/18 attribution estimated with the hierarchical attribution method was consistent among the procedures: 52.3% (45/86) for C-Chip, 54.7% (47/86) for C-MGP, and 52.3% (45/86) for F-MGP (P = 0.81). HPV16/18/31/33/45/52/58 hierarchical attribution was 88.4% (76/86) with C-Chip, 86.0% (74/86) with C-MGP, and 83.7% (72/86) with F-MGP (P = 0.49). In cervical intraepithelial neoplasia grade 3 specimens, the corresponding hierarchical attribution was 96.4% (53/55) with C-Chip, 89.1% (49/55) with C-MGP, and 94.5% (52/55) with F-MGP (P = 0.27). Although F-MGP is theoretically a reliable method for determining HPV genotype attribution, it is acceptable to use C-Chip or C-MGP, coupled to the hierarchical attribution formula to correct the bias of multiple infections. These approaches using exfoliated cervical cells are practical for monitoring the efficacy of HPV vaccines.

Performance of p16INK4a/Ki-67 immunocytochemistry for identifying CIN2+ in atypical squamous cells of undetermined significance and low-grade squamous intraepithelial lesion specimens: a Japanese Gynecologic Oncology Group study.

BACKGROUND: p16(INK4a) immunohistochemistry has revealed a high rate of positivity in cervical intraepithelial neoplasia grade 2 (CIN2) and more severe conditions (CIN2+). The Lower Anogenital Squamous Terminology Standardization project proposed p16(INK4a) immunohistochemistry as an ancillary test for CIN. Immunocytochemistry involving dual staining for p16(INK4a) and Ki-67 in the triage of atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesions (LSIL) is reported to be useful in the identification of CIN2+. However, it is unclear whether p16(INK4a)/Ki-67 immunocytochemistry is of practical relevance for the triage of ASCUS and LSIL in the Japanese screening system. METHODS: From 427 women fulfilling the eligibility criteria, 188 ASCUS and 239 LSIL specimens were analyzed. The accuracy of p16(INK4a)/Ki-67 immunocytochemistry and genotyping of high-risk human papillomaviruses (HPVs) in detecting CIN2+ were compared. RESULTS: p16(INK4a)/Ki-67 immunocytochemistry was positive in 33.5 % (63/188) of ASCUS, and 36.8 % (88/239) of LSIL specimens. The sensitivity and specificity of p16(INK4a)/Ki-67 immunocytochemistry was 87.3 % (95 % confidence interval 78.0-93.8 %) and 76.4 % (71.6-80.8 %), respectively. The positive and negative predictive values were 45.7 % (37.6-54.0 %) and 96.4 % (93.4-98.3 %), respectively; positive and negative likelihood ratios were 3.71 and 0.17, respectively. Using the McNemar test, p16(INK4a)/Ki-67 immunocytochemistry showed equivalent sensitivity but higher specificity than the HPV genotyping test CONCLUSIONS: Compared with high-risk HPV genotyping, p16(INK4a)/Ki-67 immunocytochemistry was a more accurate triage test for identifying CIN2+ in ASCUS and LSIL specimens.

Cytokine profile in cervical mucosa of Japanese patients with cervical intraepithelial neoplasia.

BACKGROUND: Immune responses in the uterine cervix are considered to play an important role in persistent human papillomavirus (HPV) infection and carcinogenesis, but many aspects of the mechanism are still unclear. The goal of this study was to measure cytokines to analyze immune responses in patients with cervical intraepithelial neoplasia (CIN). MATERIALS AND METHODS: The levels of 17 cytokines (IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, G-CSF, GM-CSF, INF-γ, MCP-1, MIP-1ß, and TNFα) in cervical mucus were simultaneously measured using a multiplex immunoassay in 52 high-grade squamous intraepithelial lesion (HSIL) cases and overproduction of IL-1ß, IL-8, and MIP-1ß was identified. The levels of these 3 cytokines were measured in 130 patients with or without CIN lesions using enzyme-linked immunosorbent assay. The associations of the cytokine levels with the cytology, infecting HPV type, and status of cigarette smoking were investigated. RESULTS: IL-1ß and IL-8 levels were associated with the cytology, and these levels were higher in HSIL cases than in NILM (negative for intraepithelial lesion and malignancy) and LSIL (low-grade squamous intraepithelial lesion) cases (P = 0.005, P = 0.001, respectively). The MIP-1ß level was significantly lower in smokers (P = 0.018) and high-risk (HR)-HPV-infected patients (P = 0.021). CONCLUSIONS: Enhanced expression of IL-1ß and IL-8 indicates that Th2 inflammatory responses become stronger in the local uterine cervical region with the progression of CIN lesions, and a decrease in the MIP-1ß level may be advantageous for immunoescape of HPV. Cigarette smoking may further facilitate persistent HPV infection.

p16(INK4a) immunohistochemistry is a promising biomarker to predict the outcome of low grade cervical intraepithelial neoplasia: comparison study with HPV genotyping.

OBJECTIVE: In cervical intraepithelial neoplasia (CIN), p16(INK4a) immunohistochemistry has been reported to be a useful diagnostic biomarker. However, limited information is available about the association between the p16(INK4a) immunohistochemistry and the outcomes of CIN. Here, we report p16(INK4a) immunohistochemistry as an effective biomarker to predict the outcomes of CIN. METHODS: p16(INK4a) immunohistochemistry was performed in patients with CIN from January 2000 to August 2009. Among these patients, we have performed a retrospective analysis of the medical records to evaluate the outcome of CIN 1-2 and performed statistical analysis to determine the correlation between p16(INK4a) expression and the outcomes. We also performed HPV genotyping and analyzed the relation between the infecting human papillomavirus (HPV) genotype and the outcomes. RESULTS: A total of 244 patients, including 82 with CIN 1, 60 with CIN 2, and 102 with CIN 3, were examined. The rate of p16(INK4a) overexpression increased with increasing CIN grade, 20.7% for CIN 1, 80.0% for CIN 2, and 89.2% for CIN 3, with significant differences between CIN 1 and CIN 2-3 group. In the 131 CIN 1-2 patients, the progression rate was significantly higher for the patients showing p16(INK4a) overexpression than for those not showing p16(INK4a) overexpression (p=0.005); the regression rate was also found to be significantly lower for the patients showing p16(INK4a) overexpression (p=0.003). High-risk HPV genotypes were detected in 73 patients (73.7%). Both progression and regression rates were not significantly different between the high-risk HPV-positive and HPV-negative groups (p=0.401 and p=0.381, respectively). CONCLUSION: p16(INK4a) overexpression was correlated with the outcome of CIN 1-2, and p16(INK4a) is considered to be a superior biomarker for predicting the outcome of CIN 1-2 compared with HPV genotyping.

Methylation of human papillomavirus-52 and -58 is a candidate biomarker in cervical neoplasia.

BACKGROUND: Previous studies of human papillomavirus (HPV)16/18 genome methylation have concluded that methylation status of the L1 gene might act as a biomarker for cervical intraepithelial neoplasia (CIN). OBJECTIVES: We investigated the correlation between methylation status in the L1 gene and the long control region (LCR) of HPV52/58 and CIN. STUDY DESIGN: Exfoliated cervical cells were taken from 54 HPV52-positive and 41 HPV58-positive women. The HPV genome was examined using bisulfite modification, polymerase chain reaction amplification, and sequencing. RESULTS: The CpGs were unmethylated or hypomethylated in the HPV52/58 LCR. In contrast, the methylation status of the HPV52 L1 gene was correlated with the severity of cervical neoplasia, with average percentages of 15%, 34%, and 52% for cervicitis/CIN1, CIN2, and CIN3, respectively (P<0.05). Methylation status of the HPV52 L1 gene was also correlated with the prognosis of CIN1/2, with median percentages of 15% and 35% for regression and persistence/progression, respectively (P<0.05). The methylation status of the HPV58 L1 gene was correlated with the severity of cervical neoplasia, with average percentages of 12%, 38%, and 61% for cervicitis/CIN1, CIN2, and CIN3, respectively (P<0.05). CONCLUSIONS: The increased methylation at the CpG sites in the HPV52/58 L1 gene was correlated with the severity of cervical neoplasia, similar to HPV16/18 in previous studies. These data suggest that HPV methylation status of the L1 gene is a candidate biomarker of CIN for detecting CIN2 and CIN3.

Tumor volume and lymphovascular space invasion as a prognostic factor in early invasive adenocarcinoma of the cervix.

OBJECTIVE: The aim of this study was to investigate the risk and recurrence of early invasive adenocarcinoma of the cervix, and to determine whether non-radical methods of management could be performed. METHODS: The medical and histopathological records of 50 patients with early invasive adenocarcinoma of the cervix treated at Keio University Hospital between 1993 and 2005 were reviewed, and compared with the literature. RESULTS: The median follow-up period was 64.3 months. The depth of stromal invasion was ≤3 mm in 33 cases and >3 mm, but ≤5 mm in 17 cases. The horizontal spread was ≤7 mm in 25 cases and >7 mm in 25 cases. One of the 33 cases that had tumor volumes of ≤500 mm(3), and three of the 17 cases with tumor volumes of >500 mm(3) were positive for lymph node metastasis. When our data were combined with previously reported results, statistically significant differences were observed between the tumor volume and the frequency of pelvic lymph node metastasis/the rate of recurrence (p<0.0001). The frequency of pelvic lymph node metastases was significantly higher in the lymphovascular space invasion (LVSI)-positive group than in the LVSI-negative group (p=0.02). No adnexal metastasis or parametrial involvement was noted. CONCLUSION: Assessment of the depth of stromal invasion, tumor volume, and LVSI is critical for selecting an appropriate therapeutic modality. Non-radical methods of management are considered suitable for patients with LVSI-negative adenocarcinoma of the cervix exhibiting a stromal invasion depth of ≤5 mm and a tumor volume of ≤500 mm(3).

Genetic variation and haplotype structures of the glutathione S-transferase genes GSTA1 and GSTA2 in Japanese colorectal cancer patients.

Glutathione S-transferases (GSTs) play a vital role in the phase II biotransformation of many chemicals, including anticancer drugs. In this study, to elucidate the haplotype structures of the two closely related alpha-class genes GSTA1 and GSTA2, we screened for genetic variation in 214 Japanese colorectal cancer patients who received oxaliplatin-based chemotherapy. By direct resequencing of the 5'-flanking region, all the exons, and their flanking introns for 107 patients, 29 and 27 variants were identified in GSTA1 and GSTA2, respectively. The known functional single nucleotide polymorphisms (SNPs) -567T>G, -69C>T, and -52G>A in GSTA1*B were found at allele frequencies of 0.140. Of the four major GSTA2 allelic variants reported previously (GSTA2*A, *B, *C, and *E), only GSTA2*B (frequency = 0.154), *C (0.706), and *E (0.140) were detected. Following linkage disequilibrium analysis, haplotypes of both genes were separately estimated. Then, rapid genotyping methods for 7 and 6 SNPs tagging common haplotypes of GSTA1 and GSTA2, respectively, were developed using the single-base extension assay, and an additional 107 patients were genotyped. Finally, haplotype combinations of both genes were classified into 3 major types: GSTA1*A-GSTA2*C, GSTA1*A-GSTA2*B, and GSTA1*B-GSTA2*E. These findings will be useful in pharmacogenomic studies on xenobiotics including anticancer drugs.

Digital colposcopy for the diagnosis of cervical adenocarcinoma using a narrow band imaging system.

INTRODUCTION: Although the colposcopic features of cervical glandular disease and cervical adenocarcinoma are not widely well known, unique microvascular patterns are reportedly useful for identifying such diseases. The narrow band imaging (NBI) system used in endoscopy can be used to obtain high-contrast vascular images. Therefore, we examined the utility of NBI colposcopy and compared the results with those of conventional colposcopy. METHODS: Twenty-one patients with adenocarcinoma in situ or early invasive adenocarcinomas were examined using digital NBI colposcopy, and the photo records were compared with those of conventional colposcopy. The histological examination and immunohistochemistry with anti-CD31 antibody confirmed the microvascular pattern. RESULTS: Digital NBI colposcopy depicted the fine vascular texture on the surface of the cervix more clearly than conventional colposcopy. The vascular pattern was depicted in 86% (18/21) of glandular disease cases. The characteristic fine vascular patterns were critical for identifying cervical glandular diseases. CONCLUSIONS: Digital NBI colposcopy was useful for identifying early cervical adenocarcinoma as well as adenocarcinoma in situ. This system yields cervical glandular disease-related colposcopic findings that may be useful for both clinical and educational purposes.

Genetic polymorphisms of copper- and platinum drug-efflux transporters ATP7A and ATP7B in Japanese cancer patients.

ATP7A and ATP7B are involved in cellular resistance to platinum compounds such as cisplatin. By sequencing ATP7A, 38 genetic variations, including 30 novel ones were detected from 203 Japanese cancer patients. Of these, seven nonsynonymous variations were found: novel 1030A>G (R344G), 2111A>G (Q704R), 2200C>A (Q734K), 2948C>T (T983M) and 3112G>A (V1038I) at 0.004 frequencies and known 2299G>C (V767L) and 4390A>G (I1464V) at 0.351 and 0.075 frequencies, respectively. Regarding ATP7B, 28 novel and 33 known genetic variations were detected including 13 nonsynonymous ones: novel 1258A>G (M420V), 1426G>A (A476T), and 2401A>C (T801P) were found at 0.002, 0.005, and 0.002, respectively and known 1216G>T (A406S), 1366G>C (V456L), 2495A>G (K832R), 2785A>G (I929V), 2855G>A (R952K), 2871delC (P957PfsX9), 3419T>C (V1140A), 3836A>G (D1279G), 3886G>A (D1296N) and 3889G>A (V1297I) at 0.483, 0.463, 0.387, 0.005, 0.384, 0.005, 0.387, 0.002, 0.012, and 0.015 frequencies, respectively. Linkage disequilibrium between detected variations was also analyzed. Our results would provide fundamental and useful information for genotyping ATP7A and ATP7B in the Japanese and probably other Asian populations.

Ancillary testing of liquid-based cytology specimens for identification of patients at high risk of cervical cancer.

Integration of human papillomavirus DNAs into the host genome is crucial to the development of cervical cancer. Overexpression of the P16 protein has been reported in cervical intraepithelial neoplasia (CIN) as well as cervical cancer. Such molecular biomarkers have been utilized for ancillary testing of liquid-based cytology specimens; however, their clinical application remains controversial. To detect CIN 2 or more advanced lesions, 153 liquid-based cytology (LBC) specimens were investigated to determine the physical status of the human papillomavirus (HPV) DNA by in situ hybridization (ISH) and to detect overexpression of the P16 protein by immunocytochemistry combined with HPV genotyping by polymerase chain reaction. The combination of ISH, P16 immunocytochemistry, and LBC showed high sensitivity (89.3%) as well as high specificity (92.6%). We confirmed the usefulness of P16 immunocytochemistry combined with ISH and HPV genotyping as ancillary molecular-biological tests of LBC specimens for identifying patients at high risk of cervical cancer.

Intratumor injection of small interfering RNA-targeting human papillomavirus 18 E6 and E7 successfully inhibits the growth of cervical cancer.

Human papillomavirus (HPV) 18 is related not only to squamous cell carcinoma of the cervix, but also to adenocarcinoma and small cell carcinoma of the cervix, in which prognosis is known to be poor. Small interfering RNA (siRNA) that targets HPV18 E6 and E7 was tested in HPV18-positive cell lines to investigate its effect and investigate its mechanism of action. Nude mice were also tested in a combination of siRNA and atelocollagen to determine whether it might be useful as a new molecule-targeting therapy for cervical cancer. siRNAs targeting HPV18 E6 and E7 were transfected into cervical cancer cells in vitro and they were investigated for cell growth inhibition, expression of E6 and E7 mRNA, expression of retinoblastoma protein, and senescence-associated beta-galactosidase staining. Sequence-specific siRNA inhibited cell growth. Decreased expression of E6 and E7 mRNA followed with E7 protein was observed in the transfected cells, but the expression of retinoblastoma protein and the beta-galactosidase staining increased, suggesting cell growth inhibitory effect through senescence. Treatment of xenografts established from SKG-II cells with siRNA specific for E6 and E7 obviously suppressed tumor growth in vivo. These results indicate that atelocollagen-mediated delivery of siRNA HPV18 E6 and E7 can be used as a novel therapeutic approach for cervical cancer.

Soft and hard keratin expression in Epstein-Barr-virus-associated gastric carcinoma.

BACKGROUND: Epstein-Barr-virus-associated gastric carcinoma (EBVaGC) is a distinct subset of gastric carcinoma (GC). The expressions of cytokeratins (CK)7, 8, 18, 19 and 20 and truncated basic hair keratin 1 (hHb1-deltaN) were investigated in GC to clarify the characteristics of EBVaGC. MATERIALS AND METHODS: For immunohistochemical evaluation, 173 GC tissues were examined and 31 GC tissues and 5 GC cell lines were used for quantitative real-time RT-PCR (qrt-RT-PCR) and RT-PCR. RESULTS: EBVaGC showed significantly lower immunohistochemical positivity of CK7 (-/(+/-)/+/+ +/+ + +; 27/15/4/1/1) compared to EBV-negative GC (12/29/27/44/13), even after stratification by histological types. The qrt-RT-PCR test demonstrated decreased amounts of CK7, 18 and 19 mRNAs in EBVaGC. Two among 5 GC cell lines showed a decrease of CK7 mRNA level after recombinant EBV infection. hHb1-deltaN expression was not specific to EBVaGC. CONCLUSION: Abnormalities of CK7, 18 and 19 expressions, especially a decreased amount of CK7 expression, are characteristics of EBV-associated epithelial malignancies and might be important in carcinogenesis.

Comparison between in situ hybridization and real-time PCR technique as a means of detecting the integrated form of human papillomavirus 16 in cervical neoplasia.

Integration of the human papillomavirus (HPV) genome is thought to be one of the causes of cancer progression. However, there is controversy concerning the physical status of HPV 16 in premalignant cervical lesions, and there have been no reports on the concordance between detection of the integrated form of HPV16 by real-time PCR and by in situ hybridization. We investigated specimens of cervical intraepithelial neoplasia (CIN) and invasive carcinomas for the physical status of HPV 16 by real-time PCR and in situ hybridization. The presence of the integrated form was detected by both real-time PCR and in situ hybridization in zero of four cases of CIN1, three of six cases of CIN2, nine of 27 cases of CIN3, and two of six cases of invasive carcinomas. Integrated HPV 16 was present in some premalignant lesions but was not always present in carcinomas. The concordance rate between the two methods for the detection of the presence of the integrated form was 37 of 43 (86%) cases. Real-time PCR and in situ hybridization were found to be complementary and convenient techniques for determining the physical status of the HPV genome. We conclude that a combination of both methods is a more reliable means of assessing the physical status of the HPV genome in cervical neoplasia.

Solution structure of the PWWP domain of the hepatoma-derived growth factor family.

Among the many PWWP-containing proteins, the largest group of homologous proteins is related to hepatoma-derived growth factor (HDGF). Within a well-conserved region at the extreme N-terminus, HDGF and five HDGF-related proteins (HRPs) always have a PWWP domain, which is a module found in many chromatin-associated proteins. In this study, we determined the solution structure of the PWWP domain of HDGF-related protein-3 (HRP-3) by NMR spectroscopy. The structure consists of a five-stranded beta-barrel with a PWWP-specific long loop connecting beta2 and beta3 (PR-loop), followed by a helical region including two alpha-helices. Its structure was found to have a characteristic solvent-exposed hydrophobic cavity, which is composed of an abundance of aromatic residues in the beta1/beta2 loop (beta-beta arch) and the beta3/beta4 loop. A similar ligand binding cavity occurs at the corresponding position in the Tudor, chromo, and MBT domains, which have structural and probable evolutionary relationships with PWWP domains. These findings suggest that the PWWP domains of the HDGF family bind to some component of chromatin via the cavity.

P16 overexpression and human papillomavirus infection in small cell carcinoma of the uterine cervix.

Carcinogenesis of cervical cancer has been investigated, and p16(INK4a) overexpression in squamous cell carcinoma of the cervix has been reported as a result of infection by human papillomavirus (HPV) (eg, HPV 16), and the consequence of the retinoblastoma (Rb) protein inactivation by HPV E7 protein. However, to our knowledge, there have been no studies on the relation between p16(INK4a) overexpression associated with HPV and small cell carcinoma of the cervix, which behaves more aggressively clinically than squamous cell carcinoma. The purpose of this study was to determine whether p16(INK4a) is overexpressed in small cell carcinoma, and if p16(INK4a) is overexpressed, the types of HPV that are related to this cancer. We reviewed 10 cases of small cell carcinoma and examined them for p16(INK4a) overexpression by immunohistochemistry. We also performed HPV typing with polymerase chain reaction (PCR)-sequencing analysis and in situ hybridization and found that p16(INK4a) was overexpressed in every case. PCR-sequencing analyses revealed that all cases were HPV-positive and that 9 cases were positive for HPV 18. Five of the 9 cases positive for HPV 18 were also positive by in situ hybridization and yielded a punctate signal, considered to represent the integrated form. In conclusion, p16(INK4a) was overexpressed and HPV 18 was frequently detected in an integrated form in small cell carcinoma. Therefore, inactivation of Rb protein by HPV 18 E7 protein may be associated with carcinogenesis of small cell carcinoma the same as inactivation of Rb protein by HPV 16 E7 protein is associated with carcinogenesis of squamous cell carcinoma.

Correlation of p16INK4A overexpression with human papillomavirus infection in cervical adenocarcinomas.

SUMMARY: As human papillomavirus (HPV) infection is the main risk factor for squamous cell carcinoma of the cervix and overexpression of p16INK4a occurs when retinoblastoma protein is inactivated by high-risk HPV, the authors studied the association of HPV infection and expression of p16INK4a in cervical adenocarcinomas. Specimens of cervical glandular neoplasias were immunostained with a p16INK4a-specific monoclonal antibody (clone E6H4). Approximately 80% of glandular neoplasms showed overexpression of p16INK4a. Exfoliated cells from 14 adenocarcinomas were further examined by p16INK4a-specific immunocytochemistry, and 12 cases showed overexpression of p16INK4a, suggesting that immunostaining for p16INK4a may be a useful diagnostic tool for cervical adenocarcinomas. The authors further examined HPV DNA in cervical adenocarcinomas with the polymerase chain reaction method. Overexpression of p16INK4a was positive in 94% of cases in which HPV16 or 18DNA was positive, a finding suggesting that HPV16 or 18 may play an important role in cervical adenocarcinomas. Overexpression of p16INK4a may be an indicator of pathogenic activity of high-risk HPVs.

Frequent co-localization of Cox-2 and laminin-5 gamma2 chain at the invasive front of early-stage lung adenocarcinomas.

Laminin-5 is an extracellular matrix protein that plays a key role in cell migration and tumor invasion. Cox-2 is an induced isoform of cyclooxygenases that plays an important role in carcinogenesis, suppression of apoptosis, angiogenesis, and metastasis of colon cancer. We report frequent co-expression of cox-2 and laminin-5 at the invasive front of early-stage lung adenocarcinomas. We investigated the expression of cox-2 and laminin-5 immunohistochemically in 102 cases of small-sized lung adenocarcinoma (maximum dimension, 2 cm or less). Cox-2 and laminin-5 were expressed in 97 (95.1%) and 82 (80.4%) cases, respectively. Both were preferentially localized in cancer cells at the cancer-stroma interface, although cox-2 tended to show a diffuse staining pattern in some cases. A comparison of their staining patterns revealed a striking similarity in their distribution in 24 cases, and a partial overlap between their localization in another 20 cases. Moreover, an overall correlation was found between the expression levels of cox-2 and laminin-5 (P = 0.018). To gain insight into the mechanisms that regulate the expression of these proteins, we additionally studied their expression in 58 cases of stage I lung adenocarcinoma, in which p53 status was determined by immunohistochemistry, polymerase chain reaction-single strand conformation polymorphism analysis, and direct sequencing. The results showed that tumors with mutant p53 tended to express more cox-2 than those with wild-type p53 (P = 0.080). Also, tumors that overexpressed p53 had higher levels of cox-2 and laminin-5 than those without p53 overexpression (P = 0.032 and 0.047, respectively). Further immunohistochemical analysis showed that tumors that overexpressed both epidermal growth factor receptor (EGFR) and erbB-2 had higher levels of cox-2 and laminin-5 than those without concomitant overexpression of these proteins (P = 0.014 and P = 0.018, respectively). To see whether EGFR signaling is involved in cox-2 and laminin-5 expression, we further conducted in vitro analyses using six lung adenocarcinoma cell lines (A549, HLC-1, ABC-1, LC-2/ad, VMRC-LCD, and L27). Western blot analyses showed that cox-2 mRNA levels, and to a lesser extent laminin-5 gamma2 mRNA levels, correlated with the expression levels of erbB-2 and the phosphorylated form of MAPK/ERK-1/2 protein. The addition of transforming growth factor-alpha increased both cox-2 and laminin-5 gamma2 mRNA levels in A549, ABC-1, and L27 with different kinetics; the induction of cox-2 occurred earlier than that of laminin-5 gamma2. Finally, the migration of ABC-1 cells was inhibited by MAP kinase kinase inhibitor PD98059 and a selective cox-2 inhibitor NS-398. In contrast, the migration of A549 cells was inhibited by PD98059, but much less effectively by NS-398. These results suggest that co-stimulatory mechanisms may exist that increase the expression of cox-2 and laminin-5 at the invasive front of lung adenocarcinomas and that EGFR signaling could be one of the mechanisms. Further investigations are warranted concerning the role of cox-2 and laminin-5 in cancer cell invasion and the significance of p53 and EGFR signaling in the regulation of cox-2 and laminin-5 expression.