Uncovering the complex relationship between pre-eclampsia, preterm birth and cerebral palsy.

Pre-eclampsia is a leading cause of preterm birth, which is strongly associated with cerebral palsy (CP). However, there is controversy about whether pre-eclampsia is associated with increased risk of CP. We evaluated the association between pre-eclampsia and CP in 122,476 mother-child pairs insured by the South Carolina Medicaid programme, with births between 1996 and 2002. Prenatal billing records were linked to the children's Medicaid billing records after birth until December 2008. The odds of CP were modelled using logistic regression with generalised estimating equations. There were 337 children (0.28%) diagnosed with CP by at least two different health care providers, and 4226 (3.5%) women were diagnosed with pre-eclampsia at least twice during pregnancy. Children whose mothers had pre-eclampsia were almost twice as likely to have CP compared with children of mothers without pre-eclampsia [odds ratio (OR)=1.94, 95% confidence interval (CI) 1.25, 2.97]. The association was only significant for pre-eclampsia diagnosed prior to 37 weeks' gestation. Full term (gestational age ≥ 37 weeks) infants whose mothers were diagnosed with pre-eclampsia prior to 37 weeks had increased odds of CP compared with full term children whose mothers did not have pre-eclampsia (OR=3.41, 95% CI 1.40, 8.31). Preterm infants whose mothers had pre-eclampsia were at significantly increased risk of CP compared with full term infants whose mothers did not have pre-eclampsia (OR=5.88, 95% CI 3.40, 10.17). The greatest risk for CP was in preterm infants whose mothers did not have pre-eclampsia (OR=8.12, 95% CI 6.49, 10.17 compared with full term infants without exposure to pre-eclampsia). We conclude that pre-eclampsia with onset before 37 weeks' gestation is a significant risk factor for CP. Some of the association is probably attributable to high risk of preterm birth because of early pre-eclampsia, while a 'direct' effect of pre-eclampsia on fetal brain development also seems likely.

Variability in the identification of lymphovascular space invasion for early stage cervical cancer.

OBJECTIVE: To evaluate the inter- and intra-rater variability of lymphovascular space invasion (LVSI) in early stage cervical cancer. METHODS: We identified invasive cervical cancer tissue samples from radical hysterectomies in our institutional pathology database. The cases were stained with Hematoxylin & Eosin (H&E) and immunostains (CD-31 and D2-40). They were evaluated for the presence of LVSI by 6 pathologists on 3 separate occasions: with H&E staining only, then with H&E and immunostained specimens, and finally using a shared written criterion for diagnosis of LVSI. With 80 cases, a two-sided 95% confidence interval for the Kappa of 0.7 with a precision of 0.1 on each side was estimated. RESULTS: Stage distribution was: IA 10%, IB 85%, and IIA 5%. The majority of cases were squamous cell carcinoma (55%), followed by adenocarcinoma (39%) and adenosquamous or other histology (6%). The mean inter-rater Kappa was 0.41 (95% CI: 0.37-0.45) for H&E. Usage of immunohistochemistry made a statistically significant improvement in the mean Kappa, but it still remained low: 0.52 (p = 0.02). Adding evaluation criteria for LVSI did not significantly increase the mean Kappa: 0.49 (p = 0.16). The mean intra-rater variability of H&E staining alone compared with H&E staining plus immunostaining was 0.53 (range: 0.43-0.64). The mean Kappa comparing H&E staining and H&E staining with criteria was 0.50 (range: 0.40-0.59). CONCLUSIONS: We noted high inter- and intra-rater variability in the diagnosis of LVSI underscoring the challenges of LVSI diagnosis. Considering the significance assigned to LVSI and its implication for treatment, comprehensive guidelines with regards to determination of LVSI status are of paramount importance.

In vivo and in vitro propagation of intraductal papillary mucinous neoplasms.

Intraductal papillary mucinous neoplasms (IPMNs) are one of the three known curable precursor lesions of invasive pancreatic ductal adenocarcinoma, an almost uniformly fatal disease. Cell lines from IPMNs and their invasive counterparts should be valuable to identify gene mutations critical to IPMN carcinogenesis, and permit high-throughput screening to identify drugs that cause regression of these lesions. To advance the study of the biological features of IPMNs, we attempted in vivo and in vitro growth of selected IPMNs based on the hypothesis that IPMNs could be grown in the most severely immunodeficient mice. We examined 14 cases by implanting them into nude, severe combined immunodeficient (SCID), and NOD/SCID/IL2Rgamma(null) (NOG) mice, in addition to direct culture, to generate tumor xenografts and cell lines. One sample was directly cultured only. Thirteen tumors were implanted into the three types of mice, including 10 tumors implanted into the triple immunodeficient NOG mice, in which the majority (8 of 10) grew. This included five IPMNs lacking an invasive component. One of the explanted IPMNs, with an associated invasive carcinoma, was successfully established as a cell line. Tumorigenicity was confirmed by growth in soft agar, growth in immunodeficient mice, and the homozygous deletion of p16/cdkn2a. Epithelial differentiation of the cell line was documented by cytokeratin expression. Patient origin was confirmed using DNA fingerprinting. Most non-invasive IPMNs grow in NOG mice. We successfully established one IPMN cell line, and plan to use it to clarify the molecular pathogenesis of IPMNs.

Appropriate citation of placenta cell lines 3A(tPA-30-1) and 3A-sub E [post crisis of 3A(tPA-30-1)] in medical literature.

INTRODUCTION: To determine how often placenta cell lines 3A (tPA-30-1) and 3A-sub E [post crisis of 3A (tPA-30-1)] are appropriately cited, or identified, as "term"-gestation placental cell lines in medical literature. METHODS: We performed a literature search on two databases, PubMed and One Search, using the terms "3A (tPA-30-1)," "3Asub-E," "3AsubE," "tPA-30-1," "tPA30-1," and "3A AND (placenta OR placental OR trophoblast OR trophoblastic) AND (cell OR line OR cell line)." Of the 218 citations retrieved, 181 were excluded due to duplication, article content irrelevance or lack of access to a full manuscript. The remaining 37 citations were thoroughly reviewed for 1)the presence of a full citation as designated by the supplier, and 2)the identification of the placental lines as "term." RESULTS: Of the 37 eligible citations included in the study, five demonstrated complete identifications of the placental cell lines of interest, while 32 demonstrated partial identifications that failed to match the designations provided by the manufacturer. Furthermore, of the 37 citations, eight accurately identified the cell lines as "term," while 27 lacked any description of gestational age, and two incorrectly identified them as "first trimester" cell lines. Overall, only three citations contained both a full citation and correct identification as a "term" placenta cell line. DISCUSSION: Only 5 of the 37 (13.5%) publications demonstrated a complete citation and only 8 publications accurately identified the gestational age of the placenta cell line as "term". Such findings confirm the need for a representative set of standards for the documentation of cell lines to improve the quality of publications in the scientific community.

Intracranial Pressure Monitoring via External Ventricular Drain: Are We Waiting Long Enough Before Recording the Real Value?

BACKGROUND: External ventricular drain (EVD) is a standard approach for both monitoring intracranial pressure (ICP) and draining cerebrospinal fluid (CSF) for patients with subarachnoid hemorrhage. Documenting an accurate ICP value is important to assess the status of the brain, which would require the EVD system to be leveled properly and closed to CSF drainage for an adequate period. It is suggested that a minimum period of 5-minute EVD closure is needed before documenting a true ICP; however, there is no commonly agreed upon standard for documenting ICP. To obtain an insight into how well the intermittent EVD clamping procedure is performed for ICP documentation, we conducted a retrospective analysis of ICP recordings obtained through EVD from 107 patients with subarachnoid hemorrhage. METHODS: The EVD was kept open for continuous CSF drainage and then intermittently closed for ICP documentation. For each EVD closure, mean ICP, standard deviation of ICP, duration of EVD closure, and time interval between 2 adjacent EVD closures were studied. The total number of EVD closures was calculated for each patient. We developed an algorithm to evaluate whether ICP reached a new equilibrium before the EVD was reopened to drainage. The percentage of EVD closures that reach the equilibrium was calculated. RESULTS: The 107 patients had 32 755 EVD closures in total, among which 65.9% instances lasted less than 1 minute and only 16.3% of all the EVD closure episodes lasted longer than 5 minutes. The median duration of each EVD closure was 25 seconds (interquartile range, 10.2 seconds to 2.33 minutes). Only 22.9% of the EVD closures reached ICP equilibrium before EVD reopening. CONCLUSION: A standard guideline and proper training are needed for bedside nurses, and a potential tool that can render ICP trend at a proper scale at bedside would help clinicians correctly document ICP.

Absence of deleterious palladin mutations in patients with familial pancreatic cancer.

It has been reported that germline mutations in the palladin gene (PALLD) cause the familial aggregation of pancreatic cancer, but the evidence is weak and controversial. We sequenced the coding regions of PALLD in 48 individuals with familial pancreatic cancer. We did not find any deleterious mutations and find no evidence to implicate mutations in PALLD as a cause of familial pancreatic cancer.

Genetic and epigenetic alterations of familial pancreatic cancers.

BACKGROUND: Little is known about the genetic and epigenetic changes that contribute to familial pancreatic cancers. The aim of this study was to compare the prevalence of common genetic and epigenetic alterations in sporadic and familial pancreatic ductal adenocarcinomas. METHODS: DNA was isolated from the microdissected cancers of 39 patients with familial and 36 patients with sporadic pancreatic adenocarcinoma. KRAS2 mutations were detected by BstN1 digestion and/or cycle sequencing. TP53 and SMAD4 status were determined by immunohistochemistry on tissue microarrays of 23 archival familial pancreatic adenocarcinomas and in selected cases by cycle sequencing to identify TP53 gene mutations. Methylation-specific PCR analysis of seven genes (FoxE1, NPTX2, CLDN5, P16, TFPI-2, SPARC, ppENK) was done on a subset of fresh-frozen familial pancreatic adenocarcinomas. RESULTS: KRAS2 mutations were identified in 31 of 39 (80%) of the familial versus 28 of 36 (78%) of the sporadic pancreatic cancers. Positive immunolabeling for p53 was observed in 57% of the familial pancreatic cancers and loss of SMAD4 labeling was observed in 61% of the familial pancreatic cancers, rates similar to those observed in sporadic pancreatic cancers. The mean prevalence of aberrant methylation in the familial pancreatic cancers was 68.4%, which was not significantly different from that observed in sporadic pancreatic cancers. CONCLUSION: The prevalence of mutant KRAS2, inactivation of TP53 and SMAD4, and aberrant DNA methylation of a seven-gene panel is similar in familial pancreatic adenocarcinomas as in sporadic pancreatic adenocarcinomas. These findings support the use of markers of sporadic pancreatic adenocarcinomas to detect familial pancreatic adenocarcinomas.

Multiple genes are hypermethylated in intraductal papillary mucinous neoplasms of the pancreas.

Ductal adenocarcinoma of the pancreas is the fourth leading cause of cancer death and is usually diagnosed late. Intraductal papillary mucinous neoplasms are an increasingly recognized precursor to invasive ductal adenocarcinoma of the pancreas. Identifying the alterations in DNA methylation that arise during intraductal papillary mucinous neoplasm development may facilitate the development of markers that could be used to differentiate intraductal papillary mucinous neoplasms from non-neoplastic pancreatic cystic lesions. Surgically resected intraductal papillary mucinous neoplasms and adjacent ductal adenocarcinomas were microdissected from 50 patients. Normal pancreas was also obtained from 27 patients with intraductal papillary mucinous neoplasms or pancreatic adenocarcinomas and 10 patients with well-differentiated pancreatic endocrine neoplasms. Methylation-specific PCR was performed on isolated DNA for seven genes (SPARC, SARP2, TSLC1, RELN, TFPI2, CLDN5, UCHL1) known to be commonly aberrantly methylated in pancreatic ductal adenocarcinomas. The mean percentage of genes methylated in invasive ductal adenocarcinomas arising in association with an intraductal papillary mucinous neoplasm (mean+/-s.d., 81+/-17%) was significantly higher than that in noninvasive-intraductal papillary mucinous neoplasms (57+/-26%, P=0.007) or peritumoral normal epithelial cells (22+/-17%, P<0.0001). Carcinomas (intraductal papillary mucinous neoplasms with carcinoma in situ or their associated infiltrating adenocarcinoma) had significantly more methylated genes (71+/-19%) than low-grade (low and moderate dysplasia) intraductal papillary mucinous neoplasms (44+/-26%, P<0.0001). The mean percentage of genes methylated in histologically normal pancreatic ductal cells from patients with ductal neoplasia (22+/-17%) was significantly higher than in normal ductal cells from patients with well-differentiated pancreatic endocrine neoplasms (4+/-7%, P=0.002). Thus, aberrant DNA methylation increases with histologic grades of intraductal papillary mucinous neoplasm. Low-level aberrant methylation in the normal ductal cells is more prevalent in patients with ductal neoplasia than in controls without ductal neoplasms and may contribute to carcinogenesis. The detection of aberrant methylation in pancreatic cystic lesions could facilitate the diagnosis of intraductal papillary mucinous neoplasms.

Ascertainment and verification of end-stage renal disease and end-stage liver disease in the north american AIDS cohort collaboration on research and design.

The burden of HIV disease has shifted from traditional AIDS-defining illnesses to serious non-AIDS-defining comorbid conditions. Research aimed at improving HIV-related comorbid disease outcomes requires well-defined, verified clinical endpoints. We developed methods to ascertain and verify end-stage renal disease (ESRD) and end-stage liver disease (ESLD) and validated screening algorithms within the largest HIV cohort collaboration in North America (NA-ACCORD). Individuals who screened positive among all participants in twelve cohorts enrolled between January 1996 and December 2009 underwent medical record review to verify incident ESRD or ESLD using standardized protocols. We randomly sampled 6% of contributing cohorts to determine the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of ESLD and ESRD screening algorithms in a validation subcohort. Among 43,433 patients screened for ESRD, 822 screened positive of which 620 met clinical criteria for ESRD. The algorithm had 100% sensitivity, 99% specificity, 82% PPV, and 100% NPV for ESRD. Among 41,463 patients screened for ESLD, 2,024 screened positive of which 645 met diagnostic criteria for ESLD. The algorithm had 100% sensitivity, 95% specificity, 27% PPV, and 100% NPV for ESLD. Our methods proved robust for ascertainment of ESRD and ESLD in persons infected with HIV.

Epigenetic silencing of EYA2 in pancreatic adenocarcinomas promotes tumor growth.

To identify potentially important genes dysregulated in pancreatic cancer, we analyzed genome-wide transcriptional analysis of pancreatic cancers and normal pancreatic duct samples and identified the transcriptional coactivator, EYA2 (Drosophila Eyes Absent Homologue-2) as silenced in the majority of pancreatic cancers. We investigated the role of epigenetic mechanisms of EYA2 gene silencing in pancreatic cancers, performed in vitro and in vivo proliferation and migration assays to assess the effect of EYA2 silencing on tumor cell growth and metastasis formation, and expression analysis to identify genes transcriptionally regulated by EYA2. We found loss of tumoral Eya2 expression in 63% of pancreatic cancers (120/189 cases). Silencing of EYA2 expression in pancreatic cancer cell lines correlated with promoter methylation and histone deacetylation and was reversible with DNA methyltransferase and HDAC inhibitors. EYA2 knockdown in pancreatic cancer cell lines increased cell proliferation. Compared to parental pancreatic cancer cells, pancreatic cancers stably-expressing EYA2 grew more slowly and had fewer metastases in orthotopic models. The transcriptional changes after stable expression of EYA2 in pancreatic cancer cells included induction of genes in the TGFbeta pathway. Epigenetic silencing of EYA2 is a common event in pancreatic cancers and stable expression EYA2 limits the growth and metastases of pancreatic adenocarcinoma.

The risk of intellectual disability in children born to mothers with preeclampsia or eclampsia with partial mediation by low birth weight.

OBJECTIVE: Preeclampsia and eclampsia (PE) are potentially modifiable risk factors for maternofetal complications. Owing to a paucity of research connecting PE to the risk of intellectual disability (ID) in the offspring, this study examined this relationship. Furthermore, we explored how low birth weight (LBW) mediates the effect of PE on ID. METHODS: Data related to South Carolina Medicaid births from 1996 to 2002 were comprised of linked data from maternal Medicaid records, delivery records, birth certificates, Department of Education (DOE), and the Department of Disabilities and Special Needs (DDSNs). After exclusions such as nonidiopathic etiologies of ID, multiple gestations, subsequent siblings in the cohort, pregnancy losses, births under 20 weeks' gestation, and children neither in DOE nor DDSN records, 80,866 maternal-child dyads remained. After adjusting for five covariates of maternal age, race, and education as well as the child's birth year and sex, the effect of PE on ID was examined. RESULTS: The rates of PE and ID were 6.4 and 2.0%, respectively. The rates of ID among children exposed and not exposed to PE were 3.0 and 2.0%, respectively. The crude odds ratio (OR) was 1.549 (95% CI 1.310, 1.832) and the adjusted OR was 1.58 (95% CI 1.334, 1.870). LBW was a significant mediator of the relationship accounting for approximately half of the association. CONCLUSION: Because of the association of PE, ID, and LBW, additional research is needed to explain mechanisms and to investigate possible impacts of different PE treatment.

Overexpression of smoothened activates the sonic hedgehog signaling pathway in pancreatic cancer-associated fibroblasts.

PURPOSE: Accumulating evidence suggests that cancer-associated stromal fibroblasts (CAF) contribute to tumor growth by actively communicating with cancer cells. Our aim is to identify signaling pathways involved in tumor-stromal cell interactions in human pancreatic cancer. EXPERIMENTAL DESIGN: We established primary fibroblast cultures from human pancreatic adenocarcinomas and nonneoplastic pancreas tissues. To identify differentially expressed genes in CAFs, we did gene expression profiling of human pancreatic CAFs and nonneoplastic pancreatic fibroblasts. RESULTS: The Hedgehog receptor Smoothened (SMO) was upregulated in CAFs relative to control fibroblasts. CAFs expressing SMO could transduce the Sonic hedgehog signal to activate Gli1 expression, and small interfering RNA knockdown of SMO blocked the induction of Gli1 in these cells. Stromal fibroblasts of human primary pancreatic adenocarcinomas overexpressed Smo compared with normal pancreatic fibroblasts. CONCLUSIONS: These findings implicate overexpression of Smo as a mechanism for the activation of Hedgehog signaling in human pancreatic CAFs and suggest that stromal cells may be a therapeutic target for Smo antagonists in pancreatic cancer.

Pancreatic cancers epigenetically silence SIP1 and hypomethylate and overexpress miR-200a/200b in association with elevated circulating miR-200a and miR-200b levels.

Aberrant DNA methylation and microRNA expression play important roles in the pathogenesis of pancreatic cancer. While interrogating differentially methylated CpG islands in pancreatic cancer, we identified two members of miR-200 family, miR-200a and miR-200b, that were hypomethylated and overexpressed in pancreatic cancer. We also identified prevalent hypermethylation and silencing of one of their downstream targets, SIP1 (ZFHX1B, ZEB2), whose protein product suppresses E-cadherin expression and contributes to epithelial mesenchymal transition. In a panel of 23 pancreatic cell lines, we observed a reciprocal correlation between miR-200, SIP1, and E-cadherin expression, with pancreatic cancer-associated fibroblasts showing the opposite expression pattern to most pancreatic cancers. In Panc-1 cells, which express SIP1, have low E-cadherin expression, and do not express miR-200a or miR-200b, treatment with miR-200a and miR-200b downregulated SIP1 mRNA and increased E-cadherin expression. However, most pancreatic cancers express miR-200a and miR-200b, but this expression does not affect SIP1 expression, as the SIP1 promoter is silenced by hypermethylation and in these cancers E-cadherin is generally expressed. Both miR-200a and miR-200b were significantly elevated in the sera of pancreatic cancer and chronic pancreatitis patients compared with healthy controls (P < 0.0001), yielding receiver operating characteristic curve areas of 0.861 and 0.85, respectively. In conclusion, most pancreatic cancers display hypomethylation and overexpression of miR-200a and miR-200b, silencing of SIP1 by promoter methylation, and retention of E-cadherin expression. The elevated serum levels of miR-200a and miR-200b in most patients with pancreatic cancer could have diagnostic utility.

Cyclooxygenase-deficient pancreatic cancer cells use exogenous sources of prostaglandins.

Genes that are differentially expressed in pancreatic cancers and under epigenetic regulation are of considerable biological and therapeutic interest. We used global gene expression profiling and epigenetic treatment of pancreatic cell lines including pancreatic cancer cell lines, pancreatic cancer-associated fibroblasts, and cell lines derived from nonneoplastic pancreata. We examined expression and epigenetic alterations of cyclooxygenase-1 (COX-1) and COX-2 in pancreatic cancers and normal pancreas and performed proliferation, knockdown, and coculture experiments to understand the role of stromal sources of prostaglandins for pancreatic cancers. We identify COX-1 as a gene under epigenetic regulation in pancreatic cancers. We find that COX-1 expression is absent in many pancreatic cancer cells and some of these cancers also lack COX-2 expression. Suspecting that such cancers must rely on exogenous sources of prostaglandins, we show that pancreatic cancer stromal cells, such as fibroblasts expressing COX-1 and COX-2, are a likely source of prostaglandins for pancreatic cancer cells deficient in COX. Knocking down the prostaglandin transporter multidrug resistance-associated protein-4 in fibroblasts suppresses the proliferation of cocultured pancreatic cancer cells lacking COX. Pancreatic cancers that lack COX can use exogenous sources of prostaglandins. Blocking multidrug resistance-associated protein-4 may be a useful therapeutic strategy to deplete COX-deficient pancreatic cancers of prostaglandins.

Serum fatty acid synthase as a marker of pancreatic neoplasia.

Markers of early pancreatic cancer and its precursors are needed to improve the uniformly poor prognosis of this disease. Fatty acid synthase (FAS) catalyzes the synthesis of long-chain fatty acids and is overexpressed in most human solid tumors. We therefore evaluated serum FAS as a marker of pancreatic adenocarcinoma. FAS expression patterns in primary pancreatic adenocarcinomas, intraductal papillary mucinous neoplasms (IPMN), and chronic pancreatitis tissues were analyzed by immunohistochemistry. Serum FAS levels were determined by ELISA in 102 patients with pancreatic adenocarcinomas, in 42 patients with IPMNs, in 27 patients with chronic pancreatitis, and in 39 healthy control subjects. FAS protein was overexpressed in the ductal epithelium of 343 of 399 primary pancreatic adenocarcinomas (86.0%) and 28 of 30 IPMNs (93.3%), and in the islet and ductal cells in 3 of 54 chronic pancreatitis tissues (5.6%), whereas normal ductal epithelium lacked FAS expression. Serum FAS levels were significantly higher in patients with pancreatic ductal adenocarcinoma (first quartile median, 22.0; 4.5 ng/mL), in patients with IPMNs (20.7; 9.4 ng/mL), and in patients with chronic pancreatitis (31.1; 11.9 ng/mL) than in healthy controls (0; 0 ng/mL). FAS levels declined postoperatively in 8 of 9 patients with pancreatic adenocarcinoma and elevations of their preoperative serum FAS. In conclusion, serum FAS levels are elevated in patients with pancreatic cancer and IPMNs and are associated with neoplastic overexpression of FAS.

Pancreatic cancer associated fibroblasts display normal allelotypes.

BACKGROUND: Recent studies have reported widespread copy number alterations and p53 mutations arising in cancer associated stromal cells. The aim of this study was to determine if pancreatic cancer associated fibroblasts display similar genetic alterations. DESIGN: Cancer-associated fibroblast cultures were established from 7 primary pancreatic adenocarcinomas. These fibroblasts and corresponding normal tissues when available were analyzed for genome-wide copy number changes using Affymetrix 250K SNP microarrays. Evidence of p53 protein expression, an indicator of p53 mutation was determined by immunohistochemical labeling of tissue microarrays containing 117 pancreatic ductal adenocarcinomas. RESULTS: Pancreatic cancer associated fibroblasts did not show any evidence of somatic copy number gains or losses. p53 protein expression was confined to invasive pancreatic adenocarcinoma cells and was not expressed in cancer-associated fibroblasts. CONCLUSIONS: We find no evidence that pancreatic cancer associated fibroblasts harbor somatic copy number changes or immunohistochemical evidence of p53 mutations.