Clinical predominance of whole-exome sequencing to evaluate microsatellite instability status.

The microsatellite instability (MSI)/mismatch repair (MMR) status is one of the critical genomic biomarkers for predicting patient response to immune checkpoint inhibitors (ICIs). In this study, we aimed to investigate the concordance among the MSIsensor score obtained from whole-exome sequencing (WES), which could be a futuristic clinical cancer sequencing method, using only tumor tissues, MSI-PCR results, and immunohistochemistry (IHC) results to analyze various solid cancer types. We first endeavored to set the cut-off value of MSIsensor to determine functional deficient mismatch repair (f-dMMR) status. The MSI status of 1054 patients analyzed using WES was evaluated using MSIsensor. In addition, 87 of these patients were further analyzed using MSI-PCR and MMR IHC to calculate the sensitivity and specificity of the MSIsensor cut-off score. Our results showed that score 12.5 was an adequate cut-off score equivalent to PCR-confirmed MSS/MSI-low and MSI-high statuses, with sensitivity, specificity, and area under the curve values of 95.2%, 100%, and 0.998, respectively. Moreover, we identified false-positive cases of tumors with high mutational burden with an MSIsensor score <12.5, and optional IHC examination could rescue these cases. In conclusion, the MSIsensor score obtained using WES with tumor tissue showed a high clinical validity, with a cut-off value of 12.5 for f-dMMR detection, in combination with optional IHC analysis for MMR. Our novel algorithm will provide insights into the development of ICIs for cancer treatment, particularly when WES becomes a more common cancer genomic test in the near future.

Recommendations related to the analytical equivalence assessment of gene panel testing.

Advances in cancer genome care over the past few years have included the development of gene panel testing for various biomarkers. This article summarizes issues and provides recommendations related to analytical performance evaluations for new oncology gene panels. The scope of these recommendations includes comprehensive genomic profiling assays related to gene panel testing that uses histological or serum specimens to detect gene mutations. As a research project of the Japan Agency for Medical Research and Development Research on Regulatory Science of Pharmaceuticals and Medical Devices, we convened the working group committee that consisted of more than 30 experts from academia, industry, and government. We have discussed the points that should be considered to allow maximal simplification of assessments using clinical specimens in evaluating accuracy and limit of detection in equivalence and analytical performance for 3 years. We provide recommendations specific to each type of gene mutation as well as to reference standards or specimens used for evaluations. In addition, in order to facilitate the discussion on the analytical performance of gene panel tests by multidisciplinary tumor boards of hospitals, the present recommendations also describe the items that companies are expected to provide information on in their packaging inserts and reports, and the items that are expected to be discussed by multidisciplinary tumor boards. Our working group document will be important for participants in multidisciplinary tumor boards, including medical oncologists and genome scientists, and developers of gene panels not only in Japan but also in other countries.

Genetic Profiling of Malignant Melanoma Arising from an Ovarian Mature Cystic Teratoma: A Case Report.

Ovarian mature cystic teratomas comprise tissues derived from all three germ layers. In rare cases, malignant tumors arise from ovarian mature cystic teratoma. A variety of tumors can arise from mature cystic teratoma, among which primary malignant melanoma (MM), for which no molecular analyses such as genomic sequencing have been reported to date, is exceedingly rare, thereby limiting possible therapeutic options using precision medicine. We used targeted gene sequencing to analyze the status of 160 cancer-related genes in a patient with MM arising from an ovarian mature cystic teratoma (MM-MCT). KRAS amplification and homozygous deletion in PTEN and RB1 were detected in tumor samples collected from the patient. No KRAS amplification has been previously reported in cutaneous MM, indicating that the carcinogenesis of MM-MCT differs from that of primary cutaneous melanomas. A better understanding of the underlying genetic mechanisms will help clarify the carcinogenesis of MM-MCT. In turn, this will enable treatment with novel targeting agents as well as the initial exploration of gene-based precision oncological therapies, which aim to improve treatment outcomes for patients with this disease.

Heat shock protein 22 (HSPB8) reduces the migration of hepatocellular carcinoma cells through the suppression of the phosphoinositide 3-kinase (PI3K)/AKT pathway.

Small heat shock proteins (HSPs) regulate a variety of cell functions. Among them, HSP22 and HSP20 are recognized to be ubiquitously expressed in various tissues. With regard to hepatocellular carcinoma (HCC) cells, we previously reported that phosphorylated HSP20 plays a suppressive role in transforming growth factor (TGF)-α-induced cell migration and invasion. In the present study, we investigated whether or not HSP22 is implicated in HCC cell migration. We detected HSP22 protein expression both in human HCC tumor (189.9±68.4ng/mg protein) and the adjacent non-tumor liver tissues (167.9±94.6ng/mg protein). The cases of low-quantity HSP22 protein level group (88.3≧ng/mg protein, the optimum cut-off value of HSP22) were increased in tumor tissues compared with the adjacent non-tumor tissues. The migration of human HCC-derived HuH-7 cells stimulated by TGF-α or hepatocyte growth factor (HGF) was significantly enhanced by the knockdown of HSP22 expression. Down-regulation of HSP22 protein in the cells markedly strengthened the AKT phosphorylation induced by TGF-α or HGF. Inhibitors of the phosphoinositide 3-kinase (PI3K)/AKT pathway, which suppressed the TGF-α-induced migration, significantly reduced the amplification by HSP22 knockdown. PI3K but not AKT was coimmunoprecipitated with HSP22 in HuH-7 cells. In addition, in human HCC tissues, a significantly lower HSP22 protein level in tumor tissues than in adjacent non-tumor tissues was observed more frequently in cases of moderately or poorly differentiated HCC than well-differentiated HCC. Taken together, our results strongly suggest that HSP22 represses HCC progression, especially HCC cell migration, by the down-regulation of the PI3K/AKT signaling pathway.

Cervical cytology and human papillomavirus among asymptomatic healthy volunteers in Vientiane, Lao PDR.

BACKGROUND: Cervical cancer is the most common cancer in women living in Vientiane, Lao People's Democratic Republic (PDR). This study examines cervical cytology using a liquid-based cytology (LBC) method and reports the presence of high-risk (HR) human papillomavirus (HPV). METHODS: We collected cervical samples from 1475 asymptomatic and healthy volunteers from six hospitals in Lao PDR. A total of 1422 volunteers (mean age 39.1 ± 6.4 years, range 30-54 years) were included in the final analysis. We performed HPV typing using the polymerase chain reaction technique to detect HR-HPV samples with abnormal cytology. RESULTS: The overall rates of abnormal cytology and HR-HPV-positive in the samples were 9.3% (132/1422) and 47.7% (63/132), respectively. The samples with abnormal cytology included 13 high-grade squamous intraepithelial lesions and one squamous cell carcinoma case. The results showed that the most common type of HPV was HPV16 (20.5%) followed by HPV58 (9.1%). CONCLUSIONS: Healthy women in Vientiane, the capital of Lao PDR, have high rates of abnormal cervical cytology and are likely to be HR-HPV-positive. A system for detection and prevention of cervical cancer in these women should be developed in the near future.

Regulation by heat shock protein 22 (HSPB8) of transforming growth factor-α-induced ovary cancer cell migration.

Accumulating evidence suggests that heat shock proteins (HSPs) are implicated in progression of cancer. HSP22 (HSPB8), a small HSP, is recognized to be ubiquitously expressed in various tissues. However, the expression and the role of HSP22 in ovarian cancer remain to be clarified. In the present study, we investigated the involvement of HSP22 in transforming growth factor (TGF)-α-induced migration of ovarian cancer cells. The expression of HSP22 was detected in a serous ovarian cancer cell line, SKOV3.ip1. The migration was reduced by down-regulation of HSP22 expression. The TGF-α-induced migration was reduced by SB203580 (a p38 MAP kinase inhibitor), SP600125 (a SAPK/JNK inhibitor) and Y27632 (a Rho-kinase inhibitor). However, down-regulation of HSP22 had little effect on the TGF-α-induced phosphorylation of p38 MAP kinase, SAPK/JNK and MYPT, a target protein of Rho-kinase. The HSP22 expression was further analyzed in 20 resected specimens of human ovarian serous carcinoma. The expression of HSP22 was detected in all the twenty tissues (8.24-109.22 pg/mg protein), and the cases with highly expression of HSP22 showed a tendency to acquire the progressive ability. Our results strongly suggest that HSP22 acts as a positive regulator in TGF-α-induced migration of ovarian cancer cells, subsequently directing ovarian cancer toward progression.

Next­generation sequencing to identify genetic mutations in pancreatic cancer using intraoperative peritoneal washing fluid.

The efficacy of next-generation sequencing (NGS) of tumor-derived DNA from intraoperative peritoneal washing fluid (IPWF) of patients with pancreatic ductal adenocarcinoma (PDAC) who intend to undergo curative resection remains unclear. The aim of the present study was to evaluate whether genomic mutations in tumor-derived DNA from IPWF samples of patients with PDAC who intend to undergo curative resection could be detected using NGS. A total of 12 such patients were included in this study. Cytology of IPWF (CY) was assessed and NGS of genomic tumor-derived DNA from the IPWF was performed to determine whether genomic mutations could be detected in these patient samples. A total of 2 patients (16.7%) had a CY(+) status and 1 patient (8.3%) showed intraoperative macro-peritoneal dissemination; 11 patients underwent radical surgery. Actionable gene alterations were detected in 8 (80.0%) out of the 10 patients with CY(-) status based on NGS of IPWF samples, and 3 (37.5%) patients among those with actionable gene mutations identified from IPWF samples underwent peritoneal dissemination after surgery within ~12 months. The most common genomic mutation was in KRAS (9 patients, 75.0%), followed by TP53 (3 patients, 25.0%), SMAD4 (1 patient, 8.3%) and CDKN2A (1 patient, 8.3%). These findings indicated that the genomic mutations identified in tumor-derived DNA from IPWF samples of patients with PDAC with a CY(-) status who intend to undergo curative resection are potential biomarkers for predicting the recurrence of early peritoneal dissemination.

Role of androgen signaling in androgen receptor-positive extramammary Paget's disease: Establishment of organoids and their biological analysis as a novel therapeutic target.

BACKGROUND: Extramammary Paget's disease (EMPD) is a rare intraepithelial adenocarcinoma that mainly affects the anogenital and axillary regions. Although its etiology has not been fully elucidated, there is evidence that androgen receptors (AR) are expressed in most cases of EMPD. However, the role of androgen signaling in the pathogenesis of EMPD remains unclear. OBJECTIVE: To evaluate the role of androgen signaling in tumor growth of AR-positive EMPD. METHODS: Patient-derived organoids were established and cultured from two AR-positive EMPD patients: one man and one woman. Cultured organoids were treated with androgen agonists and/or antagonists, then subjected to analysis of changes in organoid proliferation, as well as changes in androgen signaling pathway-specific genes. RESULTS: Organoid cultures were established from each EMPD sample. These organoids were immunohistologically and genetically identical to the original tumor. For each organoid sample, viable cell number increased in response to androgen exposure. The mRNA level of Fkbp5, a known AR target gene, increased in a concentration-dependent manner in organoids exposed to the synthetic androgen R1881. Conversely, the AR inhibitor darolutamide suppressed the viable cell number in a concentration-dependent manner. The mRNA expression levels of MKI67 and Fkbp5 were also suppressed by darolutamide. CONCLUSION: Our results indicate that androgen signaling is a key pathway involved in the growth of AR-positive EMPD. Therefore, androgen signaling inhibition may be a novel treatment option for EMPD patients who require systemic therapy.

Identification of preneoplastic lesions as mucin-depleted foci in patients with sporadic colorectal cancer.

In experimental models, mucin-depleted foci (MDF), formed by dysplastic crypts devoid of mucin production have been recognized to be correlated with colorectal carcinogenesis and to serve as preneoplastic lesions of colorectal cancer (CRC). In humans, there is only one report of identification of MDF in patients with familial adenomatous polyposis and CRC; however, the histological characteristics of human MDF are not discussed extensively in the report. In the present study, colonic samples from 53 patients with sporadic CRC were stained with Alcian blue and examined for the presence of MDF. Subsequently, the samples were examined for the presence of aberrant crypt foci (ACF) by methylene blue staining. We classified MDF into two categories: flat-MDF and protruded-MDF (having the characteristics of both ACF and MDF). We found a total of 354, 41 and 19 colonic mucosal lesions with a mean multiplicity of 44, 38.9 and 66.9 crypts (ACF, flat-MDF and protruded-MDF, respectively). The density of MDF was 0.0082 lesions/cm(2) . The ACF identified in sporadic CRC patients corresponded to hyperplastic or non-dysplasic lesions. However, MDF identified in these patients corresponded to low-grade dysplasia. In addition, we found that Paneth cell metaplasia and inflammatory cell infiltration were specific histological features of MDF. These histological characteristics are reported to be associated with the development of CRC. Therefore, our results indicate that MDF might represent preneoplastic lesions in human colorectal carcinogenesis.

Aberrant expression of the transcription factor Twist in adult T-cell leukemia.

Adult T-cell leukemia (ATL) is a T-cell malignancy etiologically associated with human T-cell leukemia virus type 1 (HTLV-1). Twist, a highly conserved basic helix-loop-helix transcription factor, is a newly identified oncogene. However, there are no reports on Twist expression in ATL. To define the role of Twist in leukemogenesis of ATL, we examined its expression in T-cell lines and PBMC. HTLV-1-infected T-cell lines and ATL cells expressed high levels of Twist compared with uninfected T-cell lines and normal PBMC. Immunohistochemistry showed immunostaining for Twist in ATL cells in ATL lymph nodes and skin lesions. Infection of normal PBMC with HTLV-1 induced Twist expression. Induction of the viral protein Tax in a human T-cell line led to upregulation of Twist. Tax-induced Twist expression involved the NF-kappaB and CREB signaling pathways. Twist augmented Tax-mediated HTLV-1 LTR and NF-kappaB activation. Short interfering RNA against Twist inhibited cell growth of HTLV-1-infected T-cell lines and downregulation of Twist expression in an HTLV-1-infected T-cell line inhibited the expression of Akt1, interleukin-2 receptor alpha chain, and Tax as well as the known target genes of Twist, YB-1 and Akt2. In conclusion, the results suggest that Tax-induced induction of Twist contributes to leukemogenesis of ATL.

Inhibition of Akt/GSK3beta signalling pathway by Legionella pneumophila is involved in induction of T-cell apoptosis.

Legionella pneumophila is the causative agent of human Legionnaires' disease. L. pneumophila has been shown to induce apoptosis of T-cells and this may be important pathologically and clinically. The present study has determined the molecular mechanisms underlying L. pneumophila-induced apoptosis, which were unclear. Wild-type L. pneumophila and flagellin-deficient Legionella, but not L. pneumophila lacking a functional type IV secretion system Dot/Icm, replicated in T-cells. However, apoptosis was efficiently induced in T-cells only by wild-type L. pneumophila, and not flagellin-deficient or Dot/Icm-deficient Legionella. Induction of apoptosis involved activation of the initiator caspase 9 and effector caspase 3. Infection with L. pneumophila inhibited phosphorylation of Akt (also known as protein kinase B) and the Akt substrate GSK3beta (glycogen synthase kinase 3beta), and reduced the levels of beta-catenin, a transcriptional activator regulated by GSK3beta. It also caused the activation of the pro-apoptotic protein Bax and inhibited the expression of the anti-apoptotic protein XIAP (X-linked inhibitor of apoptosis) via inhibition of the Akt pathway. In conclusion, L. pneumophila induces mitochondria-mediated T-cell apoptosis through inhibition of the Akt/GSK3beta signalling pathway.

RT-PCR Screening Tests for SARS-CoV-2 with Saliva Samples in Asymptomatic People: Strategy to Maintain Social and Economic Activities while Reducing the Risk of Spreading the Virus.

The year 2020 will be remembered for the coronavirus disease 2019 (COVID-19) pandemic, which continues to affect the whole world. Early and accurate identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is fundamental to combat the disease. Among the current diagnostic tests, real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) is the most reliable and frequently used method. Herein, we discuss the interpretation of RT-qPCR results relative to viral infectivity. Although nasopharyngeal swab samples are often used for RT-qPCR testing, they require collection by trained medical staff. Saliva samples are emerging as an inexpensive and efficient alternative for large-scale screening. Pooled-sample testing of saliva has been applied for mass screening of SARS-CoV-2 infection. Current policies recommend isolating people with borderline cycle threshold (Ct) values (35

Spiradenoma of the breast: a rare diagnostic pitfall in the evaluation of solid-basaloid breast lesions with a dual cell population.

Breast spiradenoma is extremely rare, with only 4 cases reported previously. We describe an instructive case of breast spiradenoma resembling adenoid cystic carcinoma (AdCC). A 71-year-old woman underwent excisional biopsy of a breast mass after a conclusive diagnosis was unable to be obtained from core needle biopsy showing an AdCC-like pattern. Histopathologically, the lesion demonstrated solid and cribriform foci comprising basaloid cells, luminal cells, and eosinophilic hyaline material, reminiscent of solid-basaloid AdCC, alongside convoluted lumens, stromal edema, lymphocytic infiltration, and c-kit negativity. On molecular analysis, neither MYB fusion genes nor CYLD gene abnormalities were identified. These results were supportive of spiradenoma. Salivary gland- and skin adnexal-type tumors are challenging to diagnose due to morphological overlaps. This case, highlighting histopathological and molecular features, shows that breast spiradenoma can be a diagnostic pitfall among the differential diagnoses of AdCC.

Molecular characterization of Legionella pneumophila-induced interleukin-8 expression in T cells.

BACKGROUND: Legionella pneumophila is the causative agent of human Legionnaire's disease. During infection, the bacterium invades macrophages and lung epithelial cells, and replicates intracellularly. However, little is known about its interaction with T cells. We investigated the ability of L. pneumophila to infect and stimulate the production of interleukin-8 (IL-8) in T cells. The objective of this study was to assess whether L. pneumophila interferes with the immune system by interacting and infecting T cells. RESULTS: Wild-type L. pneumophila and flagellin-deficient Legionella, but not L. pneumophila lacking a functional type IV secretion system Dot/Icm, replicated in T cells. On the other hand, wild-type L. pneumophila and Dot/Icm-deficient Legionella, but not flagellin-deficient Legionella or heat-killed Legionella induced IL-8 expression. L. pneumophila activated an IL-8 promoter through the NF-kappaB and AP-1 binding regions. Wild-type L. pneumophila but not flagellin-deficient Legionella activated NF-kappaB, p38 mitogen-activated protein kinase (MAPK), Jun N-terminal kinase (JNK), and transforming growth factor beta-associated kinase 1 (TAK1). Transfection of dominant negative mutants of IkappaBalpha, IkappaB kinase, NF-kappaB-inducing kinase, TAK1, MyD88, and p38 MAPK inhibited L. pneumophila-induced IL-8 activation. Inhibitors of NF-kappaB, p38 MAPK, and JNK blocked L. pneumophila-induced IL-8 expression. In addition, c-Jun, JunD, cyclic AMP response element binding protein, and activating transcription factor 1, which are substrates of p38 MAPK and JNK, bound to the AP-1 site of the IL-8 promoter. CONCLUSIONS: Taken together, L. pneumophila induced a flagellin-dependent activation of TAK1, p38 MAPK, and JNK, as well as NF-kappaB and AP-1, which resulted in IL-8 production in human T cells, presumably contributing to the immune response in Legionnaire's disease.

Quantitative digital image analysis of tumor-infiltrating lymphocytes in HER2-positive breast cancer.

As visual quantification of the density of tumor-infiltrating lymphocytes (TILs) lacks in precision, digital image analysis (DIA) approach has been applied in order to improve. In several studies, TIL density has been examined on hematoxylin and eosin (HE)-stained sections using DIA. The aim of the present study was to quantify TIL density on HE sections of core needle biopsies using DIA and investigate its association with clinicopathological parameters and pathological response to neoadjuvant chemotherapy in human epidermal growth factor receptor 2 (HER2)-positive breast cancer. The study cohort comprised of patients with HER2-positive breast cancer, all treated with neoadjuvant anti-HER2 therapy. DIA software applying machine learning-based classification of epithelial and stromal elements was used to count TILs. TIL density was determined as the number of TILs per square millimeter of stromal tissue. Median TIL density was 1287/mm2 (range, 123-8101/mm2). A high TIL density was associated with higher histological grade (P = 0.02), estrogen receptor negativity (P = 0.036), and pathological complete response (pCR) (P < 0.0001). In analyses using receiver operating characteristic curves, a threshold TIL density of 2420/mm2 best discriminated pCR from non-pCR. In multivariate analysis, high TIL density (> 2420/mm2) was significantly associated with pCR (P < 0.0001). Our results indicate that DIA can assess TIL density quantitatively, machine learning-based classification algorithm allowing determination of TIL density as the number of TILs per unit area, and TIL density established by this method appears to be an independent predictor of pCR in HER2-positive breast cancer.

Helicobacter pylori VacA activates NF-kappaB in T cells via the classical but not alternative pathway.

BACKGROUND: Helicobacter pylori secretes vacuolating cytotoxin (VacA) that damages the gastric epithelium by erosion and loosening of tight junctions. VacA has also immunosuppressive effects, inhibiting interleukin (IL)-2 secretion by interference with the T cell receptor/IL-2 signaling pathway. This study investigated the effect of VacA on gene expression of T cells. MATERIALS AND METHODS: Gene expression profile of a T cell line, Jurkat, was analyzed by the cDNA microarray technique after VacA challenge. The expression of specific mRNAs was assessed by reverse transcription-polymerase chain reaction. Interleukin (IL)-8 concentrations in culture supernatants and cell surface expression of CD69 were measured by enzyme-linked immunosorbent assay and flow cytometry, respectively. We evaluated nuclear factor-kappaB (NF-kappaB) activation in Jurkat cells challenged with VacA by luciferase assay, electrophoretic mobility shift assay, and Western blot analysis. RESULTS: VacA produced two or greater fold up-regulation of expression of 60 genes. Most of these genes were associated with signal transduction, regulation of gene expression, apoptosis, and inflammation. Up-regulation of four genes (IL8, IL2RA, ICAM1, and CD69) was confirmed. The supernatants of cells incubated with VacA showed significantly higher secretion levels of IL-8 than those incubated without VacA. VacA also induced the cell surface expression of CD69. Since microarray analysis indicated NF-kappaB was involved in the transcriptional activation of the above genes, we examined NF-kappaB signaling pathway. VacA activated NF-kappaB via classical but not alternative pathway. CONCLUSIONS: VacA has two paradoxical effects on T cells, immunosuppression, and proinflammatory effects. The latter is mediated by NF-kappaB activation.

Comparison of GLUT-1, SGLT-1, and SGLT-2 expression in false-negative and true-positive lymph nodes during the 18F-FDG PET/CT mediastinal nodal staging of non-small cell lung cancer.

INTRODUCTION: Although positron emission tomography (PET) with 2-deoxy-2-[fluorine-18]fluoro-d-glucose integrated with computed tomography (CT), (18F-FDG PET/CT), has recently improved the mediastinal nodal staging of non-small cell lung cancer (NSCLC), this method can show false negativity. We immunohistochemically investigated the expression of glucose transporters (GLUT-1, SGLT-1, and SGLT-2) in false negative and true positive mediastinal nodes via 18F-FDG PET/CT. METHODS: We investigated patients with clinically-diagnosed N0/pathological N2 diseases and patients with clinically-diagnosed N2/pathological N2 disease. The patients who were included in this study were evaluated using 18F-FDG PET/CT followed by surgical resection between January 2004 and December 2015. The expression of GLUT-1, SGLT-1, and SGLT-2 in the metastatic mediastinal lymph nodes, and clinicopathological variables such as primary tumor size, lymph node size, histological type, and SUVmax of the primary lesion, were compared between false negative nodes and true positive nodes. RESULTS: The total number of PET false negative metastatic mediastinal lymph nodes was 22 in the 17 patients who were clinical N0/pathological N2, and the number of PET true positives was 15 in the 11 patients who were clinical N2/pathological N2. GLUT-1 expression was positive in five false negative nodes and 10 true positive nodes. SGLT-2 expression was positive in 12 false negative nodes and one true positive node, whereas both false negative and true positive nodes showed no SGLT-1 staining. Univariate analysis showed that the reduced expression of GLUT-1 (P = 0.015), and overexpression of SGLT-2 (P = 0.004) were the significant causative factors for false negative nodes. Multivariate analysis also showed that the reduced expression of GLUT-1 (P = 0.012) and overexpression of SGLT-2 (P = 0.006) were the significant causative factors for false negative nodes. CONCLUSION: It suggests that the reduced expression of GLUT-1 and overexpression of SGLT-2 are associated with false-negative lymph node metastases in NSCLC.

Growth inhibitory activities of crude extracts obtained from herbal plants in the Ryukyu Islands on several human colon carcinoma cell lines.

There is increasing interest in the use of herbs for the treatment of human diseases including cancer. Therefore, the purpose of this study was to determine whether crude extracts obtained from 44 herbal plants in the Ryukyu Islands might contain components capable of inhibiting the growth of a variety of human colon carcinoma cell lines. Leaves, roots and other parts of the plants were extracted with chloroform, and the crude extracts were dissolved in dimethylsulfoxide and used for the experiments. Extracts of Hemerocallis fulva, Ipomoea batatas, Curcuma longa, and Nasturium officinale caused marked dose-dependent growth inhibition, with IC(50) values in the range of 10-80 mug/ml. With the HCT116 cell line, the extracts of Hemerocallis fulva and Ipomoea batatas induced G1 cell cycle arrest after 48 h of treatment. In addition, we found that extracts of Curcuma longa, and Nasturium officinale induced apoptosis in these cells after 48 h of treatment. The present studies are the first systematic examination of the growth inhibitory effects of crude extracts obtained from herbal plants in the Ryukyu Islands. The findings provide evidence that several plants in the Ryukyu Islands contain components that may have anticancer activity.

Pre-neoplastic lesion, mucin-depleted foci, reveals de novo high-grade dysplasia in rat colon carcinogenesis.

Aberrant crypt foci (ACF) and mucin-depleted foci (MDF) have recently been recognized as pre-neoplastic lesions in the colon of carcinogen-treated rodents. In the present study, we analyzed the sequential development of ACF and MDF histopathologically in the colon of rats from 5 to 40 weeks after DMH treatment. The numbers of ACF per colon increased over time during the experiment, and were much higher than the number in tumors, while the number of MDF per colon remained unchanged from the early stage (the 5th week after carcinogen exposure), and approximate to those in tumors. The incidence of ACF, which was much higher than that of tumors, also increased gradually in a time-dependent manner. The incidence of MDF, however, was similar to that of tumors and did not change significantly during the whole experiment. No lesion as dysplasia with high-grade (DHG) or adenocarcinoma (AC) were found in any large ACF from the 5th to 40th week histopathologically, whereas all of the large MDF showed DHG or AC features. Even at 5 weeks, MDF showed features of DHG. We classified these into two forms of MDF: flat and protruded MDF. At 40 weeks, the number of flat MDF per colon decreased significantly compared with that at 20 weeks (p<0.05), however, the number of protruded MDF per colon increased (p<0.01), and the percentage of DHG in a protruded MDF lesion decreased but that of AC increased remarkably. In conclusion, MDF may develop into cancer through the so-called 'de novo cancer' pathway.

First trial of cervical cytology in healthy women of urban Laos using by self-sampling instrument.

Cervical cancer is the most common cancer in Laos women and a screening programme, even with the PAP smear test (PAP test), has yet to be established for routine use. The Pap test is accepted as the most appropriate for cervical cancer screening in some settings but it is not commonly available in Laos hospitals, because there are few cytopathologists and gynecologists have little experience. As a pilot program, seminars for the PAP test were given in 2007 and 2008, and then PAP tests were carried out using self-sampling instrument (Kato's device) with 200 healthy volunteers in Setthathirath hospital, Laos, in 2008. The actual examination number was 196, divided into class I 104 (53.1%), class II 85 (43.3%), class IIIa 4 (2.0%), class IIIb 1 (0.5%), and class V 1 (0.5%) by modified Papanicolau classification. Four cases had menstruation. There were 6 cases with epithelial cell abnormalities including malignancy. There were 7 cases with fungus and 2 cases with trichomonas in Class II. More than 70% volunteers felt comfortable with the Kato's device and wanted to use it next time, because of the avoidance of the embarrassment and a low cost as compared with pelvic examination by gynecologists. This first trial for PAP test for healthy Laos women related to a hospital found three percent to have abnormal cervical epithelial cells. Therefore, this appraoch using a self-sampling device suggests that it should be planned for cervical cancer prevention in Laos.