PTEN loss is associated with follicular variant of Middle Eastern papillary thyroid carcinoma.

BACKGROUND: PTEN gene at chromosomes 10q23.3 is a tumour suppressor gene that is inactivated in many types of human cancers. The known mechanisms of PTEN inactivation are rendered to mutation, epigenetic silencing by aberrant methylation or gene deletion. Although PTEN role has been documented in many cancers, PTEN alteration in papillary thyroid carcinoma (PTC) has not been fully elucidated. The aim of this study is to comprehensively investigate PTEN alterations in a large cohort of Middle Eastern papillary thyroid cancer by immunohistochemistry and fluorescent in situ hybridisation (FISH). METHODS: PTEN protein expression was analysed by immunohistochemistry in a tissue microarray (TMA) format in a large cohort of more than 1000 patients with papillary thyroid cancer. Copy number changes in PTEN were analysed by FISH and data were correlated with clinicopathological parameters along with survival analysis. RESULTS: PTEN inactivation reflected by complete absence of staining was seen in 24.5% of PTC samples, whereas PTEN deletion was seen only in 4.8% of the tested samples by FISH. No association was seen between PTEN loss of protein expression and PTEN gene deletion. However, interestingly, PTEN loss of expression was significantly associated with the follicular variant subset of papillary thyroid cancer. CONCLUSION: Our study confirmed that PTEN might have a role in pathogenesis in a subset of PTC. PTEN loss of protein expression is a more common event in follicular variant of papillary thyroid cancer. Lack of association between PTEN loss of protein expression and PTEN gene deletion might indicate that gene deletion may not be the sole cause for PTEN loss of expression and these results might raise the possibility of other mechanism such as promoter methylation-mediated gene silencing to be responsible for PTEN inactivation.

ALK gene amplification is associated with poor prognosis in colorectal carcinoma.

BACKGROUND: Recently, the anaplastic lymphoma kinase (ALK) has been found to be altered in several solid and haematological tumours. ALK gene copy number changes and mutations in colorectal cancers (CRCs) are not well characterised. We aimed to study the prevalence of ALK copy number changes, translocations, gene mutations and protein expression in 770 CRC patients, and correlate these findings with molecular and clinico-pathological data. METHODS: ALK gene copy number variations and ALK expression were evaluated by fluorescence in situ hybridisation (FISH) and immunohistochemistry, respectively. RESULTS: Translocations of the ALK gene were not observed; 3.4% (26 out of 756) of the CRC patients tested had an increase in ALK gene copy number either amplification or gain. Interestingly, increased ALK gene copy number alteration was associated with poor prognosis (P=0.0135) and was an independent prognostic marker in multivariate Cox proportional hazards model. The study reveals a significant impact of ALK gene copy number alterations on the outcome of patients with CRC. CONCLUSION: The findings of our study highlight a potential role of targeting ALK in advanced CRCs by using ALK FISH and ALK IHC as a screening tool to detect ALK alterations. Based on these findings, a potential role of ALK inhibitor as a therapeutic agent in a subset of CRC merits further investigation.

S-phase kinase protein 2 is an attractive therapeutic target in a subset of diffuse large B-cell lymphoma.

S-phase kinase protein 2 (SKP2), an F-box protein, targets cell-cycle regulators including cycle-dependent kinase inhibitor p27KiP1 via ubiquitin-mediated degradation. SKP2 is frequently overexpressed in a variety of cancer cells and has been implicated in oncogenesis; however, its role in diffuse large B-cell lymphoma (DLBCL) has not been elucidated. Therefore, we investigated the role of SKP2 and its ubiquitin-proteasome pathway in a large series (301) of DLBCL patient samples and a panel of DLBCL cell lines. Using immunohistochemistry, SKP2 was detected in 41.6% of DLBCL tumours and was inversely associated with p27Kip1 protein level. The DLBCL subset with high SKP2 and low p27Kip1 showed a strong correlation with the proliferating index marker Ki-67 (p < 0.0001) and also with the germinal centre phenotype (p = 0.0147). Treatment of DLBCL cell lines with bortezomib or expression of SKP2-specific siRNA causes down-regulation of SKP2 and accumulation of p27Kip1, leading to suppression of growth by inducing apoptosis. Furthermore, treatment of DLBCL cells with bortezomib causes apoptosis via involving the mitochondrial pathway and activation of caspases. Finally, treatment of DLBCL cells with bortezomib down-regulated the expression of XIAP, cIAP1, and survivin. Altogether, these results suggest that SKP2 and the ubiquitin-proteasome pathway may be a potential target for therapeutic intervention in DLBCL.

RAD52 polymorphisms contribute to the development of papillary thyroid cancer susceptibility in Middle Eastern population.

Genetic polymorphisms of DNA repair genes seem to determine the DNA repair capacity. We hypothesized that polymorphisms of genes responsible for DNA repair may be associated with risk of thyroid cancer. To evaluate the role of genetic polymorphisms of DNA repair genes in thyroid cancer, we conducted a hospital-based case-control study in Saudi population. Two hundred and twenty-three incident papillary thyroid cancer cases and 229 controls recruited from Saudi Arabian population were analyzed for 21 loci in 8 selected DNA repair genes by PCR-restriction fragment length polymorphism including non-homologous end joining pathway genes LIGIV (LIGlV ASP62HIS, PRO231SER, TRP46TER), XRCC4 Splice 33243301G>A and XRCC7 ILE3434THR; homologous recombination pathway genes XRCC3 ARG94HIS and THR241MET, RAD51 UTR 15452658T>C, 15455419A>G, RAD52 2259 and GLN221GLU, conserved DNA damage response gene Tp53 PRO47SER, PRO72ARG, Tp53 UTR 7178189A>C and base excision repair gene XRCC1 ARG194TRP, ARG280HIS, ARG399GLN, ARG559GLN. RAD52 GLN221GLU genotypes CG and variants carrying G allele showed statistical significance and very high risk of developing thyroid cancer compared to wild type [CG vs CC; p<0.001, odds ratio (OR)=15.57, 95% confidence interval (CI)=6.56-36.98, CG+GG vs CC; p<0.001, OR=17.58, 95% CI=7.44-41.58]. Similarly, RAD52 2259 genotypes CT and variant allele T showed a significant difference in terms of risk estimation (CT vs CC; p<0.05, OR=1.53, 95% CI=1.03-2.28, CT+TT vs CC; p<0.001, OR=1.922, 95% CI=1.31-2.82). Remaining loci demonstrated no significance with risk. Of the 21 loci screened, RAD52 2259 and RAD52 GLN221GLU may be of importance to disease process and may be associated with papillary thyroid cancer risk in Saudi Arabian population.

Marriage patterns among HTLV-I seropositive women in Japan.

A significantly higher percentage of asymptomatic HTLV-I seropositive pregnant women in the Kagoshima prefecture were married to men who were also born in that prefecture compared with seronegative women [138/166(83.1%), 221/306 (72.2%); P < 0.01]. A significantly higher percentage of the fathers of the seropositive women were born in the Kagoshima prefecture compared with the fathers of the seronegative women [152/166 (91.6%), 235/306 (76.8%); P < 0.01]. Additionally, a significantly higher seropositivity was found among pregnant women born in the Kagoshima prefecture who were married to men born in that prefecture compared with men born in other prefectures [5.8% (138/2374), 3.4% (28/819); P < 0.01]. Women born in other prefectures had a significantly lower seropositivity irrespective of the birthplace of their spouse [2.9% (12/418); P < 0.05, 3.0% (7/234)]. These findings indicate that HTLV-I seropositive women and their mothers chose their husbands from a smaller geographic region than seronegative women. This marriage pattern within an HTLV-I seropositive group may be one of the factors sustaining the present seroprevalence of HTLV-I.

Risk of HTLV-I infection in Japanese women who are last in birth order.

The percentage of last-born women among pregnant women who were seropositive for human T-lymphotropic virus type I (HTLV-I) significantly exceeded that among HTLV-I seronegative women (119/258 (46.1%): 89/251 (35.4%); P < 0.05). The findings suggest that last-born women are susceptible to HTLV-I infection. At least two possible interpretations of this birth-order effect are: (i) these last-born women were born to mothers who, on the average, were older than those of early-born women and, as a consequence, were more likely to have been seropositive and to have passed on HTLV-I to their daughters; (ii) husband-to-wife transmission of HTLV-I requires time to occur, so last-born women are more likely than early-born women to become infected.

Intrauterine infection, magnesium sulfate exposure and cerebral palsy in infants born between 26 and 30 weeks of gestation.

OBJECTIVE: To identify prenatal events associated with cerebral palsy (CP) in infants born between 26 and 30 weeks of gestation. STUDY DESIGN: Case (n=22)-control (n=170) study was performed using a logistic regression model. RESULTS: Significant association of intrauterine infection with increased risk of CP was found in a logistic regression model that controlled for abnormal FHR patterns, placental infection, fetal acidosis at birth (umbilical artery pH<7. 1), and low Apgar score (<7) (odds ratio (OR) 5.47, 95% confidence interval (CI) 1.46-20.4). Magnesium sulfate exposure was associated with decreased risk (OR 0.13, CI 0.03-0.66) after exclusion of premature rupture of the membranes and abruptio placentae. In the magnesium exposure group, cases were infants born less than 28 weeks of gestation (3/21 vs. 0/61, P=0.015). CONCLUSION: In this case-control study, both intrauterine infection and magnesium sulfate exposure were significant factors related to the occurrence of cerebral palsy.

Treatment for necrotizing enterocolitis perforation in the extremely premature infant (weighing less than 1,000 g).

The frequency of necrotizing enterocolitis (NEC) in the extremely premature infant (less than 1,000 g) is still high and it is very difficult for infants weighing less than 1,000 g with NEC perforation to survive. In our institutes, the management protocol for NEC perforation in infants weighing less than 1,000 g includes peritoneal drainage under local anesthesia, administration of coagulating factor XIII, and the usual treatment for septic shock. During the past 3 years, four infants weighing less than 1,000 g with NEC perforation have survived using this protocol without laparotomy. This management protocol is the treatment of choice in infants in very poor condition or infants weighing less than 1,000 g with NEC perforation.

[The perinatal risk factors and periventricular leukomalacia (PVL) in premature infants--relationship between fetal heart rate decelerations and PVL].

Periventricular leukomalacia (PVL) has recently been recognized as an important risk factor of neurological impairment in premature infants. We studied 29 PVL cases on perinatal risk factors comparing with a non-PVL matched control group retrospectively. Variable decelerations were more frequently observed with statistical significance in the PVL group in the intrapartum period. Then another study was conducted to evaluate the relationship between fetal heart rate (FHR) decelerations and cystic PVL prospectively. Since January 1993 through December 1994 we studied 209 low birth weight infants (31.1 +/- 3.2 weeks, 1,424 +/- 419 g) who had been subjected to intrapartum FHR monitoring and postnatal sonographic intracranial examinations sequentially every 7 days until discharge. Cystic PVL was detected in 6 of 209 cases (2.9%) and occurred only in infants who had revealed severe variable deceleration or prolonged deceleration (6/37, 16%) in intrapartum FHR monitoring. We conclude that in low birth weight infants intrapartum severe variable deceleration or prolonged deceleration might play a causal role in cystic PVL.

In-situ hybridization as a molecular tool in cancer diagnosis and treatment.

In situ hybridization (ISH) is a type of hybridization that uses a labeled complementary DNA or RNA strand to localize a specific DNA or RNA sequence on a chromosome or section of tissue (in-situ) fixed on a slide. Flourescence in-situ hybridization (FISH) technique facilitates the localization of genes to different chromosomal locations. It is extensively applied as a gene mapping tool for identification and validation of cytogenetic aberrations identified through comparative genomic hybridization (CGH) on large cohort of archival samples in a tissue microarray format. The discovery of cytogenetic aberrations in cancer has led to the development of quite a few FDA approved molecularly targeted drugs for the management of patients undergoing cancer treatment. FISH technique is extensively utilized as a predictor of responsiveness to treatment with targeted inhibitors, residual disease monitoring and also in noninvasive methods for the detection of tumor cells. Furthermore detection of circulating tumor cells can be detected which have metastatic potential with poor survival prospects. With the development of high throughput technologies like comparative genomic hybridization CGH and next-generation DNA sequencing, human pathology archival specimens of human tumors in various stages of development, can be utilized in the post-human-genome-sequencing era to obtain diagnostic and therapeutic guidance. This article will discuss the extensive application of FISH in diagnosis, prognosis and therapeutic monitoring of cancer.

Apigenin induces apoptosis via downregulation of S-phase kinase-associated protein 2-mediated induction of p27Kip1 in primary effusion lymphoma cells.

OBJECTIVE: The mechanisms that regulate mitogenic and antiapoptotic signals in primary effusion lymphoma (PEL) are not well known. In efforts to identify novel approaches to block the proliferation of PEL cells, we assessed the effect of apigenin (4',5,7-trihydroxyflavone), a flavonoid on a panel of PEL cell lines. MATERIALS AND METHODS: We studied the effect of apigenin on four PEL cell lines. Apoptosis was measured by annexin V/PI dual staining and DNA laddering. Protein expression was measured by immunoblotting. RESULTS: Apigenin induced apoptosis in PEL cell lines in a dose dependent manner. Such effects of apigenin appeared to result from suppression of constitutively active kinase AKT resulting in down-regulation of SKP2, hypo-phosphorylation of Rb and accumulation of p27Kip1. Apigenin treatment of PEL cells caused dephosphorylation of p-Bad protein leading to down regulation of the anti-apoptotic protein, Bcl-2 and an increase in Bax/Bcl2 ratio. Apigenin treatment also triggered Bax conformational change and subsequently translocation from cytosole to mitochondria causing loss of mitochondrial membrane potential with subsequent release of cytochrome c. Released cytochrome c onto the cytosole activated caspase-9 and caspase-3, followed by polyadenosin-5'-diphosphate-ribose polymerase (PARP) cleavage. Finally, treatment of PEL cells with apigenin down-regulated the expression of inhibitor of apoptosis protein (IAPs). CONCLUSIONS: Altogether, these data suggest a novel function for apigenin, acting as a suppressor of AKT/PKB pathway in PEL cells, and raise the possibility that this agent may have a future therapeutic role in PEL and possibly other malignancies with constitutive activation of the AKT/PKB pathway.

Clinicopathological analysis of colorectal cancers with PIK3CA mutations in Middle Eastern population.

Activation of the phosphatidylinositol 3'-kinase (PI3K)/AKT pathway results in an increase in cell proliferation and survival. Somatic mutations within the PI3K catalytic subunit, PIK3CA are common cause of increasing PI3K activity and are believed to be oncogenic in many cancer types. Few reports addressed the association between PIK3CA mutations and tumor progression specifically in microsatellite instable (MSI) colorectal cancer (CRC). In the present study, we have evaluated PIK3CA mutational status in a series of 410 Middle Eastern CRC and 13 colon cell lines to study the prevalence of PIK3CA mutations in MSI cases, PTEN expression in CRC and possibility of therapeutic targeting of this set of patients. PIK3CA mutations were found in four of the cell lines tested and 51 colorectal carcinomas (12.2%). Three of these four mutated cell lines were MSI. PTEN was inactivated in 66.1% of the CRC. Furthermore, we observed a strong association between PIK3CA mutations and MSI status (P=0.0046) while PTEN loss was more frequent in microsatellite stable (MSS) CRC (P=0.043). A high prevalence of genetic alterations in PI3K/AKT pathway in Saudi cohort of CRC, predominance of PIK3CA mutations in the MSI subgroup and their possible involvement in development/progression of this subset of CRC are some of the significant findings of our study.

Genome-wide expression analysis of Middle Eastern papillary thyroid cancer reveals c-MET as a novel target for cancer therapy.

In an attempt to find genes that may be of importance in malignant progression of papillary thyroid carcinoma (PTC) in the Middle East, which therefore can be targeted in cancer therapy, we screened and validated the global gene expression in PTC using cDNA expression arrays and immunohistochemistry (IHC) on tumour tissue microarrays. Twenty-nine PTC tissue specimens were compared with seven non-cancerous thyroid specimens by use of cDNA microarray. Results for selected genes were confirmed by quantitative real-time PCR. Protein expression of selected genes was further studied using a tissue microarray consisting of 536 PTCs and compared with histologically non-cancerous tissue samples. One hundred and ninety-six genes were overexpressed in PTC tissues relative to non-cancerous thyroid tissues. The genes that were up-regulated in PTC were involved in cell cycle regulation, cell signaling, and oncogenesis. Among these genes, c-MET was identified by immunohistochemical methods as a protein that is overexpressed in 37% of PTCs and was significantly associated with more aggressive behaviour, eg higher stage, nodal involvement, and tall cell variant (p value = 0.01, 0.01 and 0.04, respectively). In this study, 55% of the PTC cases expressed activated AKT (P-AKT), which suggests that activated AKT may play an important role in PTC tumourigenesis. The fact that most of the PTC cases that had activated AKT showed overexpression of c-MET (p = 0.027) leads us to hypothesize that c-MET may be an alternative mechanism of AKT activation in Middle Eastern PTCs. Finally, our data suggest that c-MET dysregulation is associated with aggressive behaviour and may serve as a molecular biomarker and potential therapeutic target in this disease.

Curcumin induces apoptosis via inhibition of PI3'-kinase/AKT pathway in acute T cell leukemias.

Curcumin has been shown to possess variety of biological functions including anti-tumor activity. The mechanism by which curcumin inhibit cell proliferation remains poorly understood. In the present report, we investigated the effect of curcumin on the activation of apoptotic pathway in T-cell acute lymphoblastic leukemia (T-ALL) malignant cells. Our data demonstrate that curcumin causes dose dependent suppression of proliferation in several T cell lines. Curcumin treatment causes the de-phosphorylation/inactivation of constitutively active AKT, FOXO transcription factor and GSK3. Curcumin also induces release of cytochrome c accompanied by activation of caspase-3 and PARP cleavage. In addition, zVAD-fmk, a universal inhibitor of caspases, prevents caspase-3 activation and abrogates cell death induced by curcumin treatment. Finally, treatment of T-ALL cells with curcumin down-regulated the expression of inhibitor of apoptosis protein (IAPs). Taken together, our finding suggest that curcumin suppresses constitutively activated targets of PI3'-kinase (AKT, FOXO and GSK3) in T cells leading to the inhibition of proliferation and induction of caspase-dependent apoptosis.

Vertical transmission of HTLV-I from a seronegative mother using the PA method.

The child was born when his mother was 23 years old. Her serum was negative for human T cell lymphotropic virus type I (HTLV-I) using the gelatin particle agglutination (PA) method when she was 26.5 years. Three years later, she became seropositive for HTLV-I using both the PA method and ELISA. She had breast-fed her child for 3 years. The child was positive for anti-HTLV-I antibody at the age of 4.5 years. Vertical transmission of HTLV-I can occur in women with negative results from the PA method.

Relationship between granulocyte elastase levels and perinatal infections.

This study was conducted in order to investigate the usefulness of granulocyte elastase levels as predictive factors in the onset of perinatal infections. The subjects were 41 patients who delivered within 48 h after amniocentesis after giving their informed consent. The relationship between cervical granulocyte elastase (Cx-E), serum C-reactive protein (CRP) and amniotic fluid granulocyte elastase (Af-E), and placental infections and neonatal infections was comparatively investigated. In some cases, gastric juice granulocyte elastase in neonates (Gj-E) was measured, and the correlation by site was investigated. Elastase levels were not used as a management protocol. In predicting neonatal infections, diagnostic efficacy (sensitivity x specificity) of placental infections (0.97) and abnormal Af-E (0.79) were superior to those of abnormal Cx-E (0.40) and abnormal CRP (0.49). There was no correlation between Cx-E and Af-E or between Cx-E and Gj-E; however, a very close correlation was noted between Af-E and Gj-E. In predicting abnormal amniotic fluids, Cx-E (> or = 1.2 micrograms/ml) + CRP (> or = 1.0 mg/dl) had the highest diagnostic efficacy with 0.58. These findings demonstrate that Af-E is a good index for predicting the onset of neonatal infections. In predicting abnormal amniotic fluid, it might be advisable to consider amniocentesis in order to diagnose intrauterine infections, when both Cx-E and CRP show abnormal levels.

Transmission routes of HTLV-I: an analysis of 66 families.

HTLV-I transmission routes were found for 66 carrier pregnant women by studying sera, from the carrier pregnant women, their mothers, and their husbands, and by obtaining detailed family histories at interview. Forty-one cases (62.1%) were considered to be instances of vertical transmission, 15 (22.8%) of sexual transmission, 6 (9.1%) of blood transfusion, and 4 (6.1%) undecided. To date, most cases of adult T-cell leukemia (ATL) have been considered to result from vertical transmission. Our results therefore imply that about 30% (22.8% + 9.1%) of the carrier pregnant women are at minimal risk of ATL. Moreover, in case of presumed husband-to-wife transmission, more than half (6/11) were infected between one year and four years after marriage.

[The efficacy of prophylactic antibiotic and tocolytic therapy for premature rupture of the membranes--a prospective randomized study].

A comparative study in patients with premature rupture of the membranes (PROM) from 25 to 34 weeks of gestation was carried out, prospectively. Group 1 (34 patients) was given aggressively intrauterine therapy including the administration of tocolytic agents (ritodrine and/or magnesium sulfate) and prophylactic antibiotics (AB-PC 2g/day). Group 2 (41 patients) was managed conservatively with bed rest only. At the time of admission to the study, there were no clinical signs of infection, fetal distress, or active labor in either group. All patients were delivered if the pregnancy had reached 35 weeks of gestation or later, had established labor, or developed evidence of chorioamnionitis or fetal distress. Prolongation for more than 72 hours was greater in group 1 than in group 2. There was no difference in the incidence of chorioamnionitis, postpartum endometritis, or placental infection in the groups. However, the incidence of a low Apgar score (7 < at 5 min), requiring artificial ventilation, and infection was more common in group 1. It is concluded that the use of antibiotics and tocolytics might make the management of PROM more complicated.